R E V I E W
Inhibin at 90: From Discovery to Clinical Application,
a Historical Review
Yogeshwar Makanji, Jie Zhu, Rama Mishra, Chris Holmquist, Winifred P. S. Wong,
Neena B. Schwartz, Kelly E. Mayo, and Teresa K. Woodruff
Department of Obstetrics and Gynecology (Y.M., J.Z., C.H., W.P.S.W., T.K.W.), Feinberg School of Medicine,
Northwestern University, Chicago, Illinois 60610; Center for Molecular Innovation and Drug Discovery (R.M., C.H.),
Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208; and Department of Molecular
Biosciences (N.B.S., K.E.M., T.K.W.), Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208
When it was initially discovered in 1923, inhibin was characterized as a hypophysiotropic hormone that acts on
pituitary cells to regulate pituitary hormone secretion. Ninety years later, what we know about inhibin stretches
far beyond its well-established capacity to inhibit activin signaling and suppress pituitary FSH production.
Inhibin is one of the major reproductive hormones involved in the regulation of folliculogenesis and steroidogenesis. Although the physiological role of inhibin as an activin antagonist in other organ systems is not as well
defined as it is in the pituitary-gonadal axis, inhibin also modulates biological processes in other organs through
paracrine, autocrine, and/or endocrine mechanisms. Inhibin and components of its signaling pathway are
expressed in many organs. Diagnostically, inhibin is used for prenatal screening of Down syndrome as part of
the quadruple test and as a biochemical marker in the assessment of ovarian reserve. In this review, we provide
a comprehensive summary of our current understanding of the biological role of inhibin, its relationship with
activin, its signaling mechanisms, and its potential value as a diagnostic marker for reproductive function and
pregnancy-associated conditions. (Endocrine Reviews 35: 747–794, 2014)
I. Historical Context
A. Introduction
B. Assay development and molecular cloning—crucial
parallel efforts driving inhibin research
II. Structure, Function, and Mechanism of Action of Inhibins and Activins
A. Inhibin and activin subunits
B. Molecular mechanisms controlling inhibin and activin transcription, and physiological regulation
C. Structural insights from the TGF␤ superfamily
D. Activin signaling
E. Mechanisms of inhibin action and antagonism
F. Soluble inhibin binding proteins
III. Physiological Roles of Inhibin
A. Dissecting the roles of inhibins using knockout and
transgenic mouse models
B. Sites of inhibin expression
C. Pituitary-gonadal axis
D. Bone metabolism
E. Adrenal gland growth and function
F. Retinal development and vision
G. Hematopoiesis
H. Placenta
I. Branching morphogenesis
IV. Clinical Applications of Inhibin
ISSN Print 0163-769X ISSN Online 1945-7189
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received January 15, 2014. Accepted June 17, 2014.
First Published Online July 22, 2014
doi: 10.1210/er.2014-1003
A. Early pregnancy viability
B. Ectopic pregnancy
C. Down syndrome
D. Pre-eclampsia
E. Ovarian reserve
F. Polycystic ovarian syndrome (PCOS)
G. Premature ovarian failure
H. Ovarian cancer
I. Breast cancer
J. Menopause
K. Male reproductive function
V. Conclusion
I. Historical Context
A. Introduction
he word “inhibin” was first introduced into the literature in 1932 by D. Roy McCullagh (1). He postulated that a hormone in testicular extracts, urine, and
T
Abbreviations: ActRII, activin type II receptor; AFP, ␣-fetoprotein; ALK4, activin receptorlike kinase 4; AMH, anti-Müllerian hormone; AP, activator protein; ART, assisted reproductive technology; BFU-E, erythroid burst-forming units; BMP, bone morphogenetic protein; CFU, colony-forming unit; CRE, cAMP responsive element; CREB, cAMP binding
protein; E, gestation day; FLRG, follistatin-related gene; FSHR, FSH receptor; GDF, growth
and differentiation factor; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte;
GM, granulocyte-macrophage; GnRHR, GnRH receptor; hCG, human chorionic gonadotropin; hpg, hypogonadal; IGFBP, IGF binding protein; IVF, in vitro fertilization; LRH-1, liver
receptor homolog 1; P, postnatal day; PCOS, polycystic ovarian syndrome; POF, premature
ovarian failure; SF-1, steroidogenic factor-1; TRE, 12-O-tetradecanoyl phorbol 13-acetate
responsive element; uE3, unconjugated estriol.
Endocrine Reviews, October 2014, 35(5):747–794
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Inhibin at 90, a Historical Review
blood prevented pituitary hypertrophy and hyperplasia in
rats, whereas the destruction of the seminiferous tubule
walls had the opposite effect (1). But it was Mottram and
Cramer (2) (1923) who first discovered a soluble factor
secreted by the testes that regulated pituitary function;
they showed that rats developed pituitary hypertrophy
and obesity after irradiation of the testes. Nearly 60 years
later, the protein inhibin was identified in follicular fluid
by Professor Neena Schwartz and Cornelia Channing (3)
in the United States and by David de Kretser (4) in Australia. It was ultimately isolated and characterized in 1985
by teams led by Dr. de Kretser (5), Wylie Vale (6), Roger
Guillemin (7), and Hisayuki Matsuo (8). Cloning of the
subunit cDNAs and genes was done in laboratories
throughout the world (9 –13) and created a field of collaborators working on the mechanisms of peptide hormone control of reproduction (for an in-depth description
of the race to identify and clone inhibin, the reader is directed to several excellent reviews [14 –27]). Today, inhibin is described as a gonadal hormone that down-regulates FSH production by anterior pituitary gonadotropes
(28, 29) and a paracrine factor that regulates ovarian folliculogenesis (30) and steroidogenesis (31). Advancement
in our understanding of the physiological role of inhibin in
reproductive biology, and more recently in bone metabolism and adrenal gland growth, has been facilitated by
the development of ELISA systems that are both sensitive
and specific for the various inhibin subunits (32–35). A
great deal of research is under way to assess the clinical use
of serum inhibin level as a biochemical marker in the diagnosis and/or monitoring of reproduction-associated
and pregnancy-related conditions and outcomes. Serum
inhibin now has clinical applications as a diagnostic
marker in the prenatal screening for Down syndrome and
as a prognostic marker of ovarian reserve in assisted reproductive technologies. On the occasion of the 90th
birthday of inhibin, we review its structure and its biology
within and beyond the reproductive tract, illustrating the
critical role of this powerful gonadal hormone in reproduction and, therefore, life itself.
B. Assay development and molecular cloning— crucial
parallel efforts driving inhibin research
1. Development of in vitro and in vivo bioassays for inhibin
The development of in vitro and in vivo inhibin bioassays was a crucial part of inhibin research that led to future
discoveries about the molecule’s structure and function.
Without the concomitant development of sensitive and
specific assays, we would know much less about inhibin.
This period began when inhibins were being isolated from
the follicular fluid of various species. The earliest inhibin
Endocrine Reviews, October 2014, 35(5):747–794
in vivo bioassay, from Chari et al (36), was based on the
dose-dependent suppression of ovarian weight increase
after 24 hours in human chorionic gonadotropin (hCG)stimulated female rats. Similarly, in orchidectomized immature male rats, administration of crude ovine testicular
extract resulted in a dose-dependent suppression of
plasma FSH within 3– 6 hours, without an effect on LH
levels (37). Ramasharma et al (38) further refined the
Chari inhibin bioassay to measure the dose-dependent inhibition of hCG-induced increments in uterine weight and
serum FSH in immature mice after 24 hours. However,
these assays were largely insensitive and required large
amounts of inhibin to see a measurable effect. The development of an in vitro anterior pituitary cell bioassay to
measure inhibin activity by the dose-dependent suppression of FSH was an important step forward and was crucial to the subsequent isolation of inhibins (39 – 42).
2. Purification and characterization of inhibin A and B
The development of in vivo and in vitro bioassays not
only provided early insight into inhibin function but was
also crucial for the isolation and purification of inhibin. In
1985, inhibin was isolated from bovine (5) and porcine
(6 – 8) follicular fluid, based on its ability to suppress FSH.
Inhibin was subsequently isolated from ovine follicular
fluid (43). Inhibins were defined as heterodimers of a common ␣-subunit and a ␤A- or ␤B-subunit; the inhibins are
produced as precursor molecules that undergo further
processing into mature subunits that assemble into active
inhibin dimers. Several groups isolated a number of forms
of inhibin (ranging from 27–120 kDa) from the follicular
fluid and serum/plasma of various species (ovine [43], porcine [6 – 8], bovine [5, 44 – 48], equine [49], rat [50 –52],
nonhuman primate [53, 54], and human [55, 56]). Heterodimers of the inhibin ␣C-subunit and ␤-subunit are
regarded as the mature inhibin forms. Heterodimers of the
full-length and cleavage products of the ␣-subunit and
␤-subunit are classified as high molecular weight inhibins
and have also been shown to be biologically active in pituitary bioassays (5, 48, 56). It is widely accepted that the
mature 31kDa inhibin A and B are of biological relevance.
Studies have been unable to show that the high molecular
weight inhibins are proteolytically cleaved to mature 31k
just prior to mediating its action; however, immunoneutralization studies in sheep using antibodies against the ␣N
region of inhibin resulted in reduced fertility in ewes (57).
Therefore, the high molecular weight inhibins may have
roles in fertility. The inhibin isoforms present in bovine
follicular fluid closely resemble those in in vitro fertilization (IVF) serum, with the exception of the 29k form, and
all isoforms were found to be bioactive (5, 47, 58).
doi: 10.1210/er.2014-1003
3. Cloning of inhibin cDNAs
The discovery and isolation of inhibin coincided with
the advent of molecular biology in the 1980s, and by 1985,
several groups were actively trying to clone the cDNAs of
inhibin subunits from gonadal tissues from various species. Porcine (11), bovine (10), human (12), and rat (9, 13)
inhibin cDNAs were ultimately cloned, revealing some
important characteristics of the molecule.
4. Development of immunoassays for inhibin A and B
Cloning of the inhibin subunits led the way to the next
phase of the inhibin research: the development of various
immunoassays capable of detecting inhibin in the circulation. These assays would further reveal the functional
role of inhibin as an endocrine hormone with clinical relevance beyond the reproductive system.
The development of the Monash RIA in particular was
a major advance for inhibin research. The RIA utilized a
rabbit polyclonal antiserum (1989b) against purified bovine 58-kDa inhibin A and cross-reacted extremely well
with the human inhibin ␣-subunit (59). Studies using the
Monash RIA were the first to define the secretion pattern
of inhibin across the human menstrual cycle, which was
found to be largely inversely correlated to FSH levels (60).
However, the utility of the assay was limited because it
exclusively recognized the inhibin ␣-subunit and thus was
unable to distinguish between free ␣-subunit and dimeric
inhibins or distinguish between inhibin A and B.
Once monoclonal antibodies against the inhibin ␣- and
␤-subunits became available, specific inhibin A and B
ELISAs were developed and greatly enhanced our understanding of the distribution of each inhibin isoform in the
circulation of both men and women. The inhibin A and B
ELISAs are sandwich assays in which the monoclonal antibodies E4 (anti-␤A-subunit) and C5 (anti-␤B-subunit)
are utilized as capture antibodies and alkaline phosphatase conjugated R1 monoclonal antibodies are used as the
detection antibody. R1 was raised against a synthetic peptide corresponding to AA1–32 of the mature inhibin
␣-subunit (61– 63). The inhibin A-specific antibody, E4,
was raised against AA82–114 of the mature ␤A-subunit
(64), and the inhibin B-specific antibody, C5, was raised
against AA82–114 of the mature ␤B-subunit (63). The E4
antibody also serves as the capture and detection antibody
in the activin A ELISA (65), although the C5 antibody
could not be utilized in the same manner for an activin B
ELISA. To improve the sensitivity of the inhibin A and B
ELISAs, samples are subjected to hydrogen peroxide treatment that oxidizes the Met residues in the consensus sequence Met-Ser-Met on the ␤A- and ␤B-subunits. In these
assays, the serum samples require sodium dodecyl sulfate
treatment and boiling to improve sensitivity to 7 pg/mL
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(66). Recently, the 46A/F antibody raised specifically
against an as-yet uncharacterized region of the mature
human ␤B-subunit was utilized in activin B and inhibin B
ELISAs. Serum samples in these assays do not require hydrogen peroxide, sodium dodecyl sulfate, or heat treatment of samples and have improved sensitivity to 4 pg/mL
(67).
The total inhibin ELISA was primarily developed to
detect all inhibin forms, including the free ␣-subunit, in
order to monitor various ovarian cancers (68). This ELISA
replaced a two-site total inhibin ␣C immunofluorometric
assay (55) that utilized polyclonal antibodies (no. 41 was
detection antibody and 128 was capture antibody) raised
against a human ␣C-subunit fusion protein. In contrast,
the broadly specific total inhibin ELISA utilizes monoclonal antibodies raised against the inhibin ␣C-subunit and
alkaline phosphatase fused-R1 (as detection antibodies)
and a combination of PO#14 and PO#23 (as capture antibodies) (68). The total inhibin ELISA has improved specificity and sensitivity in detecting the various molecular
mass forms of inhibins in postmenopausal women and
women with various types of ovarian cancers (69). The set
of available inhibin assays has become an essential tool in
the understanding of the functional roles of inhibin A and
inhibin B in various physiological and disease processes.
II. Structure, Function, and Mechanism of
Action of Inhibins and Activins
A. Inhibin and activin subunits
Inhibins are glycoprotein hormones that belong to the
TGF␤ superfamily. Inhibins are composed of two subunits, an ␣-subunit (20 kDa) and a ␤-subunit (13 kDa),
linked by a disulfide bridge (Figure 1). There are two main
isoforms of the ␤-subunit, ␤A and ␤B (7), resulting in two
isoforms of the mature 32-kDa inhibin protein, inhibin A
(␣␤A) and inhibin B (␣␤B). The closely related activins are
dimers of two ␤-subunits. Activins were initially isolated
from porcine follicular fluid during the purification of the
related peptide, inhibin (7). Fractions that eluted before
inhibin A and inhibin B were found to stimulate FSH secretion in pituitary cell cultures. The eluted proteins were
identified as heterodimers of the inhibin ␤A- and ␤B-subunits (70). Homodimers of the ␤A- (71, 72) and ␤B- (73)
subunits were subsequently found to be capable of stimulating FSH release. These proteins were termed activin A
(␤A␤A), activin AB (␤A␤B), and activin B (␤B␤B). To
date, four activin ␤-subunit genes (␤A, ␤B, ␤C [74], and
␤E [75]) have been isolated and characterized in humans;
however, the physiological functions of the ␤C- and ␤Esubunits, and whether they form functional dimers, is not
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Endocrine Reviews, October 2014, 35(5):747–794
Figure 1.
Figure 1. Precursor and mature forms of inhibins and activins. The inhibin ␣- and ␤-subunits are produced as larger precursor proteins that are
cleaved by proconvertases to form the mature inhibins and activins. Inhibins are heterodimers of inhibin ␣- and ␤-subunits that assemble via
disulfide bridges. Activins are homodimers of two ␤-subunits. The molecular masses of the subunits are indicated in kilodaltons. The cleavage sites
are denoted by a carat (⵩). Asterisks (*) represent glycosylation sites; red asterisk denotes differential glycosylation sites.
yet known. The activin ␤C-subunit knockout mouse has
no observable abnormalities (76). Interestingly, activin C
can antagonize activin A biological activity, and activin C
transgenic mice have testis, liver, and prostate pathologies
(77). That inhibin and activin share ␤-subunits underlies
the complex functional relationship between the two hormones. This review discusses activin A and activin B as
they relate to inhibin biology. Mature inhibins are cleared
from circulation rapidly with half-lives of approximately
3– 6 minutes (inhibin A) (78, 79) and approximately 3
minutes (inhibin B) (78). In a whole-body autoradiography rat study, radiolabeled inhibin A accumulated in the
spleen, adrenal, bone marrow, and ovary (80). Interestingly, radiolabeled activin A only accumulated in the pituitary and ovary, with some accumulation in the bone
marrow (80). The precursor ␣-subunit is synthesized as an
inactive propeptide comprised of three domains: the 43amino acid prodomain, the 171-amino acid ␣N domain,
and the 134-amino acid ␣C domain, which is separated by
two polyarginine cleavage sites (Figure 1) (81, 82). Cleavage of the ␣-subunit precursor produces a mature protein
of 134 amino acids with a molecular mass of 18 kDa. The
␤A- and ␤B-subunits are also produced as precursor molecules of 425 and 407 amino acids, respectively, with corresponding molecular masses of 47 and 45 kDa (83, 84).
The precursor ␤-subunits consist of a prodomain at the N
terminus and a mature ␤A or ␤B domain at the C terminus,
separated by a polyarginine cleavage site (Arg-XX-Arg or
RXXR). The prodomain of the ␤A-subunit plays an important role in activin A folding, dimerization, and secretion (85). The membrane-associated, calcium-dependent
serine peptidase furin recognizes the RXXR consensus sequence and is involved in the proteolytic processing of
inhibin and activin (86). The 59-kDa ␤A- and ␤B-subunits
undergo proteolytic cleavage giving rise to mature forms
of 116 and 115 amino acids, respectively, with corresponding molecular masses of 13 kDa (Figure 1). The ma-
doi: 10.1210/er.2014-1003
ture ␤A- and ␤B-subunits are approximately 64% identical, with a difference of 42 amino acids.
Interestingly, activin A simultaneously up-regulates
mRNA transcripts of the ␣-subunit, ␤B-subunits, and furin (86). A related proconvertase, PCSK5 (PC5/6), can also
cleave the ␣- and ␤B-subunits into their mature forms. In
ovarian follicles, during the transition from a two-layer
secondary to preantral stage, the PCS5K transcript is upregulated, and this may be important for ovarian inhibin
and activin bioavailability (87). Mason et al (81) showed
that noncleavable 55-kDa and 64-kDa inhibin A molecules are fully functional and inhibit FSH release in a rat
pituitary bioassay despite mutation of the proteolytic
cleavage sites. In contrast, a noncleavable 110-kDa activin
A molecule failed to exhibit FSH-releasing activity in a
pituitary bioassay. This suggests that proteolytic cleavage
of the precursor ␣-subunit (pro-␣N-␣C) is not essential for
the biological activity of inhibin A, but processing of the
precursor pro-␤A-subunit is necessary for the biological
response of activin A (81). Research efforts are focusing on
possible independent functions of inhibin subunit precursors and cleavage products, as is observed with other
prodomains of TGF␤ superfamily ligands (88 –91).
Both mature ␣- and ␤A-subunits contain cysteine residues that are important for intramolecular and intermolecular disulfide bonding necessary for protein stability
and folding. The C terminus of the mature ␤A- and ␤Bsubunits form an intersubunit disulfide bond via Cys79
and Cys80, respectively, leading to the formation of a
dimer with a molecular mass of 26 kDa (11, 69, 84). An
interchain disulfide bond between Cys80 of the ␤A-subunit (Cys79 of the ␤B-subunit) and Cys95 of the ␣-subunit
covalently connects the two chains within a heterodimer.
The human inhibin ␣-subunit has two N-linked glycosylation sites at Asn268 and Asn302, which are responsible for the observed molecular mass heterogeneity between inhibin A and B. Asn268 is always glycosylated
(31-kDa inhibin A or B), whereas Asn302 is differentially
glycosylated (34-kDa inhibin A or B) (78, 81, 92) (Figure
1). Site-directed mutagenesis studies have demonstrated
that N-linked glycosylation of the ␣-subunit is required for
inhibin assembly and secretion (93). Antenos et al (93)
showed that elimination of these ␣-subunit glycosylation
sites dramatically reduces inhibin secretion; specifically,
an asparagine residue at position 268 was identified as a
major site involved in protein folding. The hydrophobic
residues at positions Leu30, Phe37, and Leu41 in the proregion of the ␣-subunit also play an important role in
dimeric inhibin assembly (94). When these residues are
mutated to alanine, heterodimer assembly and secretion
are disrupted (94). Walton et al also demonstrated that
mutation of Ile62, Leu66, Phe329, and Pro341 in the
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pro-␤A precursor prevents inhibin A and activin A production, suggesting that these hydrophobic residues are
also essential for structural assembly of functional inhibin
and activin dimers (94).
B. Molecular mechanisms controlling inhibin and activin
transcription, and physiological regulation
Dynamic regulation of inhibin and activin subunit
mRNA and proteins is highly dependent on developmental
age and endocrine status in both women and men. There are
considerable species differences in the expression of activin
and inhibin subunits in females. The rodent model has been
well characterized by groups around the globe.
1. Gene structure of inhibin subunits
In humans, the inhibin ␣-subunit is located on chromosome 2 (2q33-q36) (95) and is highly conserved across
species (approximately 85% sequence homology in bovine, porcine, and human) (84). The human inhibin ␣-subunit gene is composed of two exons separated by a 1.7-kb
intron (96). The 5⬘ noncoding region of the inhibin ␣-subunit contains tightly regulated and highly conserved promoter elements—12-O-tetradecanoyl phorbol 13-acetate
responsive element (TRE) and cAMP responsive element
(CRE), GATA binding sites, steroidogenic factor (SF)-1
binding sites, and Smad-binding elements (97, 98). These
promoter sites are regulated by activator protein (AP)-1,
AP-2, GATA, Smad3/4, and cAMP binding protein
(CREB) (for review, see Ref. 99) (Figure 2). In the mouse,
both INHA (100) and INHBB (95) reside on chromosome
1, whereas INHBA is found on chromosome 13 (101). In
humans, the ␤A- and ␤B-subunit genes are located on
chromosomes 7 (7p15-p13) and 2 (2cen-q13), respectively (84). The human ␤A-subunit gene consists of three
exons and a 2.6-kb intron region. The 5⬘ noncoding region
encodes for conserved promoter regions; TRE and CRE
binding sites and multiple enhancer sites are essential for
gene regulation (Figure 2) (102). The promoter region
contains the TATA box and several potential specificity
protein 1 binding sites (103). The human ␤B-subunit consists of two exons separated by a 2.5-kb intron (73). Based
on DNA sequence analysis, no TATA or CAAT-like elements have been identified; however, the promoter region
is GC-rich, with multiple specificity protein 1 binding sites
and three CRE sequences (73). Two 3.8-kb and 4.8-kb
␤B-subunit mRNAs are present in human tissues (104),
and in the rat, the origin maps to an alternative transcriptional initiation site (105).
Inhibin ␣- and ␤A-subunit expression is initiated by
RNA polymerases at conventional TATA boxes (103,
106). The ␤B-subunit lacks the TATA element (and
CAAT-like sequences), and transcription is possibly ini-
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Endocrine Reviews, October 2014, 35(5):747–794
Figure 2.
tiated via a different mechanism.
Transcription of the ␤B-subunit
likely involves the GC-rich regions of
the promoter, SP-1 and AP-2 (73,
105). SP-1 can also modulate inhibin ␣- and ␤A-subunit expression.
GATA binding sites have also been
identified in the inhibin ␣- and ␤Asubunit promoter regions (Figure 2)
and may act as transcriptional activators (107, 108).
2. Transcriptional regulation of
inhibin subunits
Figure 2. Schematic inhibin ␣-subunit (A), ␤A-subunit (B), and ␤B-subunit (C) genes. The mRNA
transcripts for each of the subunits are mapped (second line), and important regulatory elements
within the 50 untranslated regions (UTRs) are noted, including GATA binding sites, Smad-binding
elements (SBEs), CREs, and TREs. Transcriptional activators that act as these sites are also marked;
CREB, SF-1, LRH-1, AP-1, and AP-2. [Adapted from Ref. 238. Reprinted from K. L. Walton et al:
The synthesis and secretion of inhibins. Vitam Horm. 2011;85:149 –184 (99), with permission.
© Elsevier.]
Transcriptional activation of inhibins in the ovary and testis is modulated by gonadotropins (13, 109,
110). FSH and LH increase cAMP
levels through G protein-coupled
membrane receptors and modulate
many downstream target genes—
gonadotropin receptors, steroidogenic enzymes, and inhibin and activin subunits (for reviews, see Refs.
99, 111, and 112).
The inhibin ␣-subunit promoter
contains functional CRE and steroidogenic factor-1 (SF-1) binding
sites and has been shown to bind
CREB, inducible cAMP early repressor, and SF-1, respectively, with direct interactions with SF-1 and
CREB leading to synergism between
cAMP and SF-1 pathways (113). Activated G protein-coupled receptors
increase intracellular cAMP levels
via activation of adenylyl cyclase.
This results in increased cAMP and
subsequent activation of the protein
kinase A pathway, leading to the
phosphorylation of CREB (114,
115). Phosphorylation of CREB
stimulates inhibin transcription via a
CREB-mediated interaction with the
CRE in the promoter (102, 115,
116). Novel coactivators like half
LIM domain 2 (FHL2) interact with
nuclear receptors, liver receptor homolog 1 (LRH-1) and SF-1, and enhance CREB binding and cAMP signaling, thereby augmenting inhibin
gene expression (117). It is postu-
doi: 10.1210/er.2014-1003
Figure 3.
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repression in the corpus luteum
(123). DNA methylation and histone
modifications of the inhibin ␣-subunit proximal promoter is low in preovulatory and ovulatory follicles but
is elevated in the corpus luteum. Increased methylation within the CRE
of ␣-subunit promoter during luteinization prevents CREB from binding
this site (123).
The transcription regulation of
inhibin subunits is complex and is
mediated by various transcription
factors, cofactors, and coactivators.
The physiological demands of the
cell/tissue type are achieved by modulating these factors to get an appropriate response.
C. Structural insights from the TGF␤
superfamily
The ␤A- and ␤B-subunits are
structurally homologous to subunits
of other members of the TGF␤ superfamily, and the structures of activin A and other related TGF␤ suFigure 3. Conserved cysteine residues in TGF␤ superfamily ligands. Sequence alignment of the
perfamily members have been solved
mature domains of selected members of the TGF␤ superfamily in humans. Cysteine residues are
either alone or as ligand/receptor
shown in blue. The highly conserved amino acids are indicated with an asterisk (*).
complexes: TGF␤2 (127), TGF␤3
(128, 129), activin A (130 –132),
lated that during low levels of inhibin ␣-subunit gene
bone
morphogenetic
protein
(BMP)2 (133, 134), BMP3 (135),
expression, SF-1 and CREB occupy SF-1 and CRE bindBMP6
(135,
136),
BMP7
(137),
BMP9 (138), and growth and
ing sites, respectively. However, during long periods of
increased cAMP levels, there is a preferential switch to differentiation factor (GDF)8 (myostatin) (139). The deLRH-1 at the SF-1 binding site—mediated by MAPK fining feature of the TGF␤ superfamily proteins is the
six to nine highly conserved cysteine residues in the
and phosphatidylinositol-3-kinase pathways (118).
Many genes—inhibin ␤A- and ␤B-subunits (119), FSH mature domain, which form intra- and intermolecular
and LH receptors (120), cytochrome p450 side chain disulfide bonds (Figure 3) (127, 140). The intersubunit
cleavage (121), and steroidogenic acute regulatory protein disulfide bridge is responsible for the formation of ho(122)— undergo dramatic changes in expression during mo- and heterodimers. The characteristic cysteine knot
ovulation and luteinization; these can be controlled by scaffold is highly conserved in the mature domains of
epigenetic changes like DNA methylation and histone both ␤-subunits, and the nine cysteine residues of the
␤A-subunit are essential for synthesis, structural stabilmodification (123).
In rats, sheep, cows, and pigs, inhibin ␣-subunit ex- ity, and biological activity of activin A dimers (141).
pression remains repressed in the corpus luteum. In the rat, The cysteine knot directs members of the superfamily to
LRH-1, SF-1, and CREB maintain this repression. How- adopt a “butterfly-shaped” or “open hand” configuraever, in humans, the corpus luteum secretes inhibin during tion, with the ␣-helix defining the “wrist” and ␤-sheets
the luteal phase of the menstrual cycle (124). Inducible forming the “fingers” of the molecule (Figure 4). Each
cAMP early repressor (125), CCAAT/enhancer binding monomer includes two pairs of antiparallel ␤-sheets,
protein ␤ (126), and NR4A orphan nuclear receptors have forming a short and a long finger. These slightly curved
been implicated in maintaining this repression in response finger-like projections stretch out like wings from the
to the LH surge; however, the transient expression of these cysteine-knot core of the molecule, creating concave
transcription factors does not account for the sustained and convex surfaces for receptor and monomer inter-
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Figure 4.
activin receptors. The “wrist” region of the ␣-subunit helix is present only in mammalian inhibin.
This motif may be important for interaction with the inhibin coreceptor, betaglycan, suggesting that the
insertion of the wrist region into
the ␣-subunit structure introduced
a new mechanism of antagonism
within the pituitary-gonadal axis in
mammals.
D. Activin signaling
1. Activin receptors
Like other members of the TGF␤
superfamily, activins signal through
a hetero-oligomeric serine/threonine
receptor complex and intracellular
Smad proteins (145). Activins bind
to one of two type II receptors, ActRII or ActRIIB. Upon ligand binding, these type II receptors phosphorylate the activin type IB receptor,
activin receptor-like kinase 4 (ALK4).
Activated ALK4 phosphorylates the intracellular signaling proteins, Smad2 and Smad3, which then dissociate
from the receptor complex and bind to the co-Smad,
Smad4. The activated Smad complexes translocate to the
nucleus, where they interact with cofactor proteins to
stimulate or repress target gene transcription.
To date, only the crystal structure of activin A with
ActRIIB has been solved (Figure 6, A and B). In the ActRIIBactivin A complex, one type II receptor docks on the outer
convex surface (knuckles) of each activin ␤A-subunit, resulting in a 2:1 binding ratio between receptors and the activin
ligand. The binding interface involves hydrophobic (Phe17,
Ile30, Ala31, Pro32, Pro88, Leu92, Tyr94, and Ile100) as
well as ionic/polar residues (Arg87, Ser90, Lys102, Asp104,
and Glu111). The corresponding hydrophobic (Tyr60,
Trp78, Leu79, Phe82, Val 99, Phe101) and ionic/polar residues (Glu39, Lys55, Tyr60, Val73, Lys74, Cys77, Leu79,
and Asp80) on the ActRIIB concave surface were also identified (Figure 7) (130). Before the crystal structure of the ActRIIB-activin A complex was solved, mutagenesis studies of
ActRII identified Tyr60, Trp78, and Phe101 as the key residues of the activin/inhibin binding epitope (146, 147). Other
mutagenesis studies identified Lys102 on activin A as the key
residue of the ActRII binding epitope essential for biological
activity (148). The findings of these mutagenesis studies are
consistent with the solved crystal structure of activin and its
type II receptor.
Figure 4. Common structural elements of TGF␤ superfamily ligands. “Open hand” or
“butterfly” configuration of the BMP3 dimer depicting the structural elements that are
conserved across the TGF␤ superfamily. A, The “fingers” formed by two pairs of antiparallel
␤-sheets stretch out from the cysteine-knot core of the molecule (B) and the “wrist” region
(C), formed by the ␣-helix of one monomer and the contralateral concave surface created by
the fingers of the second monomer. Type II receptors bind to the outer convex surface of the
fingers of TGF␤ superfamily ligands, whereas the wrist is the interface for type I receptor
binding. [Adapted from G. P. Allendorph et al: BMP-3 and BMP-6 structures illuminate the
nature of binding specificity with receptors. Biochemistry. 2007;46:12238 –12247 (135),
with permission. © American Chemical Society.]
actions. The ␣-helix (wrist region) of the monomers sits
in the contralateral concave surfaces created by the finger-like projections of the ␤-sheets (137). Several members of the family (eg, GDF-9, BMP-15, GDF-3, lefty-1,
and lefty-2) have a serine substitution for the cysteine
normally involved in intermolecular disulfide bond formation; as a result, these dimers are expected to be noncovalently associated and possibly more labile.
Although crystal structures of the ␣-subunit and inhibin dimers have not been solved, it is assumed that the
␤-subunits would retain a similar conformation to that
observed in the activin A dimer (142, 143). It is therefore possible to model a hypothetical structure of the
inhibin dimer (Figure 5). Based on the crystal structures
of related ligands (128, 142), it is predicted that the
␣-subunit would also consist of two “fingers” projecting outward from the cysteine knot core. However, the
high proline content (eight of 24 residues) through the
wrist region of the ␣-subunit would ensure that this
region remains disordered (ie, no ␣- helix structure).
A recent study found that the ␤-subunit underwent a
series of changes to evolve into a functional ␣-subunit
(144). During the evolution of vertebrates, the loss of the
␤-subunit helix region and simultaneous extension of the
N-terminal region is believed to mark the shift of the ligand from an agonist to an antagonist because these regions are required for binding of the ␤-subunit to the
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Figure 5.
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tified key residues on the concave
surface of the ␤A-subunit fingers
(Met91, Ile105, and Met108) and
several residues through the ␣-helix
(wrist region) (Ser60-Ser72). The
residues on the concave surface of
the “fingers” of one monomer and
residues of the “wrist” ␣-helix on the
other monomer form a hydrophobic pocket that facilitates interaction with hydrophobic elements on
ALK4. This proposed binding configuration between activin and
ALK4 is similar to that seen between TGF␤ and its type I receptor,
ALK5. Interestingly, this similarity
does not extend to the interaction
of the activin and TGF␤ type II reFigure 5. Inhibin structure model. The structure of an inhibin ␣-subunit and ␤A-subunit
ceptors with their respective liheterodimer is expected to be similar to those of other related TGF␤ ligands, with some unique
gands. T␤RII binds to the fingertips
elements. The predicted inhibin ␣-subunit is shown in the foreground, dimerized with a single
of
the TGF␤ ligands, whereas Act␤A-subunit in the background (light green). The proline-rich region of the inhibin ␣-subunit
RII
binds to the outer convex sur(A) is shown as a disrupted helix compared to the homologous ␣-helix wrist region in the
␤A-subunit. B, The N-terminal extension of the inhibin ␣-subunit is unique to inhibins and
face or knuckle regions of the aclacks structural homology to other TGF␤ ligands. [Reprinted from Y. Makanji et al:
tivin ligands. The significance of
Suppression of inhibin A biological activity by alterations in the binding site for betaglycan.
this apparent shift in binding reJ Biol Chem. 2008;283: 16743–16751 (229), with permission. © American Society for
Biochemistry and Molecular Biology.]
gions among members of the TGF␤
superfamily is not known.
Despite the lack of a crystal structure for the activin
An important aspect of the activin A-receptor interactype I receptor, mutagenesis studies identified a binding tion is the flexibility of the ligand dimer when “locked”
epitope for ALK4 on activin A. Harrison et al (149) iden- into place after binding a pair of type II receptors; this
allows for interaction of the activin
dimer with two type I receptors
Figure 6.
(ALK4; Figure 6, C and D). Greenwald et al (131) showed that binding
of the activin A dimer to the first type
II receptor led to an increase in activin concentration, decreased its rotational freedom, and resulted in a
decrease in entropy, allowing the
second type II receptor to bind.
These studies also showed that when
Figure 6. Ligand flexibility for receptor assembly in the membrane. A and B, Two views of a single
activin A was bound to a single type
activin dimer (light and dark gray subunits) bound to two ActRIIB extracellular domains (ECDs;
II receptor, the binding affinity (Kd)
green). C, BMP7 (light and dark gray subunits) bound to two ActRII-ECDs (green). D, Overlay of
was 6.9 nM, which increased 30-fold
panel C with the model of BMP2/BMPRIA-ECD to generate the six-chain BMP7/ActRII-ECD/
(to 0.24 nM) when bound to a second
BMPRIA-ECD model. The two subunits of BMP7 are light and dark gray, the type II receptor ECDs
are green, and the type I receptor is blue. The red surface on the ligands denotes the residues at
type II receptor. This suggests that
the type I receptor interface. Arrows indicate the axes of the 2-fold symmetry of the complexes,
bidentate binding to ActRIIB is reand the right sides of the complexes are aligned in the same orientation, with a black outline for
quired for subsequent binding to
clarity. The C termini of receptor ECDs (open circles) are connected via a colored line to the
putative membrane surface (horizontal line). Note that the left-side receptor ECDs in panels A
ALK4 and downstream signaling.
and B are faded and disconnected from the membrane surface to highlight the right-side
On the other hand, bidentate bindreceptor as the first binder. [Adapted from J. Greenwald et al: A flexible activin explains the
ing to ActRIIB did not further enmembrane-dependent cooperative assembly of TGF-␤ family receptors. Mol Cell. 2004;15:485–
489 (131), with permission. © Elsevier.]
hance affinity of inhibin A.
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Endocrine Reviews, October 2014, 35(5):747–794
Figure 7.
Figure 7. Binding interfaces of the ActRIIB:activin A complex. A, Positively charged (blue), negatively charged (red), hydrophobic (pink), and
hydrophilic (cyan) interfaces are shown on the ActRIIB and activin A monomers. B and C, Close-up of the residues involved in the hydrophobic (B)
and hydrophilic (C) interactions at the ActRIIB:activin A binding interface. [Adapted from T. B. Thompson et al: Structures of an ActRIIB:activin A
complex reveal a novel binding mode for TGF-␤ ligand:receptor interactions. EMBO J. 2003;22:1555–1566 (130), with permission. © John Wiley
and Sons.]
2. Activin A vs activin B
Most of our knowledge of activin action is based on
analysis of activin A, with much less information available
for activin B. In most systems, activin A is more potent
than activin B, perhaps due to the higher affinity of activin
A for its type II receptors compared with activin B (150,
151). Overall, activin B has a 6-fold lower affinity for
ActRII/IIB compared to activin A. Interestingly, of the 14
activin A residues at the activin A:ActRIIB binding interface, four residues are not conserved in activin B and account for the difference in affinity between activin A and
activin B for ActRIIB binding. Despite the lower affinity of
activin B for type II receptors, it has similar bioactivity
to activin A in some systems (eg, suppression of apoptosis
in human SH-SY5Y neuroblastoma cells [152]), and is
more potent than activin A in other systems (eg, stimulation of insulin release by mouse MIN6 pancreatic cells
[153]). To explain these differences, researchers speculated that activin A and B bound to type II receptors may
have different affinities for the type I receptor (ALK4).
However, most of the residues at the ALK4 binding interface, with the exception of Thr61(Ala) and His71(Leu),
are conserved between activin A and activin B, suggesting
that the activin isoforms are expected to have similar affinities for ALK4.
Tsuchida et al (153) showed that in mouse pancreatic
MIN6 cells, ALK7 preferentially mediates activin Band activin AB-stimulated (but not activin A-stimulated) insulin release in a dose-dependent manner. To
further understand the role of ALK7 in activin B biology, the phenotypes of ALK7 and activin B null mice
were compared. Both mice developed hyperinsulinemia, but the double knockouts had no additive effects,
suggesting that ALK7 and activin B cooperatively regulate insulin secretion (154). The increase in activin B
potency in MIN6 cells is attributed to the greater binding affinity of the ␤B-subunit for its type I receptor,
ALK7. The activin B residues involved in the interaction
with ALK7 are not known.
The roles of activin A and activin B can be inferred from
␤A- and ␤B-subunit knockout mice (Table 1). ␤A-subunit
null mice are neonatal lethal due to craniofacial defects
(155, 156), whereas the ␤B-subunit null mice are fertile
and viable but show delayed parturition and eyelid closure
and nursing defects (157, 158). In the double ␤A- and
␤B-subunit null mice, no additional defects apart from
those seen in the single knockouts were observed (159).
Initially, it was anticipated that mice null for any activin
receptor (ALK4, ActRII, or ActRIIB) would phenocopy
the ␤A/␤B-subunit double-knockout mice. However,
ActRIIB null mice die postnatally due to axial patterning
defects and disturbances of anterior-posterior patterning
and left-right asymmetry (160). Embryos of ALK4 null
mice have impaired primitive streak formation and fail to
form a normal egg cylinder during embryogenesis (161),
whereas ALK7 null mice are viable and fertile with no
observable defects (162). These studies highlight the pleiotropic nature of the activin receptors and indicate that
during embryogenesis, ALK4 and ActRIIB likely mediate
the actions of additional TGF␤ superfamily ligands. The
striking lack of overlap between the phenotypes of ActRIIdeficient and activin-deficient mice suggests that the ligands that signal through ActRII during embryonic development are not activins (159).
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Table 1. Phenotype Summary of Inhibin/Activin Subunit, Receptor, and Binding Protein Transgenic and Knockout
Mouse Models
Knockout/Transgenic Mouse
Line
␤A-subunit knockout
␤B-subunit knockout
␤C-subunit knockout, ␤E-subunit
knockout, and ␤C/␤E-double
knockout
␤B-subunit knock-in to the
␤A-subunit locus
␣-subunit knockout
␣-subunit transgenic
Activin receptor type II knockout
Betaglycan knockout
Follistatin knockout
␣-subunit and activin receptor
type II knockout
Follistatin transgene in ␣-inhibin
knockout
Phenotype
Refs.
Defects in eyelid development; lack of whiskers and incisors; cleft palates
Developmental failure of incisor and mandibular molars beyond bud stage; maxillary
molars unaffected
Failure of eyelid fusion during late embryonic development; in females, impaired
reproduction associated with perinatal lethality of offspring and increased gestational
time
Open eyes at birth in 40% of offspring, resulting in corneal opacity; completely viable
and fertile males and females
Normal
159
156
Rescue of craniofacial phenotype from ␤A-knockout; somatic, testicular, genital, and
hair growth grossly affected
Infertility in males and females due to development of gonadal tumors
Elevated levels of activin A and B; development of gonadal tumors followed by cachexia
wasting syndrome: hepatocellular necrosis around the central vein, parietal cell
depletion and mucosal atrophy in the glandular stomach; anemia; and severe weight
loss. If gonadectomized at an early age, wasting syndrome does not develop, but
adrenocortical sex steroidogenic tumors develop
␤A- and ␤B-subunit level reduction in ovaries; reduction in FSH levels; increase in LH
levels; 52% reduction in litter size for females. In males, litter size unaffected but
50% reduction in sperm count
Development of ovarian cysts, polyovular follicles, fewer mature antral follicles and
corpora lutea; increased serum T levels; reduced serum estradiol levels; 20 – 40%
reduction in testis size
Variable hypoplasia of the mandible (micrognathia); cleft palate; eyelid closure defects;
absence of incisors; defects in Meckel’s cartilage; neonatal fatality; in males, delayed
fertility and smaller gonads; infertility in females
Embryonic lethal at E13.5; proliferative defects in heart and apoptosis in liver
Growth retardation; decreased mass of diaphragm and intercostal muscles; shiny taut
skin; skeletal defects of hard plate; 13th pair of ribs; abnormal whisker and tooth
development; breathing failure; neonatal fatal
Normal in terms of weight loss and stomach and liver histology
Development of activin-secreting gonadal tumors without abnormalities in gastric
epithelium; ActRII-dependent signaling pathways in inhibin-deficient mice affects
gastric epithelial stem cell proliferation
Reduction of FSH␤ mRNA levels similar in mice with ActRII deficiency, ␣-subunit
deficiency, and combined deficiencies; significant reduction of pituitary FSH levels in
ActRII-deficient mice and slight increase in ␣-deficient mice; reduction of pituitary
FSH levels in mice with combined deficiencies compared to those with ␣-deficiency
alone; ActRII-deficiency does not affect GnRH biosynthesis or GnRH-receptor
expression
Less severe wasting syndrome; lower serum activin levels; prolonged survival
163
In order to understand the functional relationship between the activin A and B isoforms and to rescue the neonatal null phenotype of the ␤A-subunit mice, mice were
generated in which the mature region of the Inhbb gene
was knocked into the Inhba gene locus (163). The expression pattern of the chimeric allele was similar to that of the
endogenous ␤A-subunit, resulting in the correct processing of the mature ␤B-subunit. Unlike ␤A-subunit null
mice, the ␤B-subunit knock-in mice survived, although
not all defects were rescued. The mice showed hypogonadism and decreased body mass, female subfertility, decreased life expectancy, and reduced hair growth, indicat-
157
158
76
239
240
248
249
159
510
192
241
511
512
242
ing differences in biological activity between ␤A and ␤B
isoforms (163). The nonoverlapping functions of activin A
and activin B may be related to differences in affinities for
their receptors and antagonists, and in their spatial and
temporal expression patterns.
Rescue of neonatal lethality in ␤A-subunit null mice
with ␤B-subunit knock-in also permitted analysis of the
reproductive functions of activin A. Although male ␤Bsubunit null mice are fertile, mice homo- or hemizygous
for the ␤B-subunit knock-in allele have smaller testicular
volumes. Heterozygous male mice have delayed seminiferous tubule differentiation, whereas homozygous male
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Inhibin at 90, a Historical Review
mice display a delay in fertility. These phenotypes underscore the importance of activin A in the development of the
testes. Adult male mice, as in other species (with the exception of rams [164]), do not express the ␤A-subunit in
the testes. However, the developing testes (prior to postnatal day 23) of immature male mice express both ␤A- and
␤B-subunit, and the knock-in experiments demonstrate
that activin ␤B cannot substitute for ␤A during this developmental window.
A comprehensive characterization of mRNA and protein levels in mice testes from birth to adulthood found
that the inhibin/activin subunits, FSH, and follistatin are
tightly regulated during mouse testis development. The
observed changes in mRNA and protein levels reflect Sertoli cell and germ cell maturation. During the first wave of
spermatogenesis, rapid changes in activin A levels are
counteracted by synthesis of inhibin ␣-subunit, follistatin,
and ␤C-subunit. Together, these studies highlight the importance of activin subunits and their antagonists in the
developing testes (165).
3. Activin binding proteins
Activin A and B are pleiotropic factors that affect proliferation, differentiation, and apoptosis in a variety of cell
types. They control a number of important processes,
from embryogenesis through adulthood. The critical actions of activins are modulated by a group of binding proteins/antagonists that limit access of the ligands to their
signaling receptors (166 –169). Follistatin is a functional
antagonist of activin and other TGF␤ superfamily members, including myostatin; BMP2, -4, -6, and -7; and
GDF11 (170 –172). Of all the superfamily members, follistatin binds activin with the greatest affinity, followed by
myostatin, GDF11, and to a lesser extent BMPs. Follistatin was initially isolated from porcine (173) and bovine
(174) follicular fluid, binds activin with high affinity, has
an inhibitory effect on pituitary FSH release (143, 171),
and binds irreversibly to its protein target. In addition to
gonadal tissue and pituitary gonadotropes, follistatin expression has been reported in skeletal muscles (175),
pancreatic ␤-cells (176), placenta (177), bone (178), cerebrospinal fluid (179), intestinal epithelium (180), and
mammary tissue (181), suggesting a paracrine and/or autocrine role of activin in these organ systems.
Follistatin is a cysteine-rich, glycosylated, monomeric
protein (83) that is structurally homologous to the Kazal
serine protease enzyme inhibitor family. It is a gene product of chromosome 13 in mice and chromosome 5 in humans. Two alternative mRNA splicing events occur from
the single follistatin gene, and the primary mRNA transcript is post-translationally processed at its C terminal to
produce a total of three protein isoforms (182, 183). Dif-
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ferential processing of exon 6 at the C terminal results in
three follistatin glycoproteins of 288, 303, or 315 amino
acids (182, 184). The predominant isoform in the circulation is follistatin 315, and follistatin 288 is restricted to
tissues (184, 185). The three major follistatin isoforms
differ in their C-terminal sequences, which results in different affinities for cell surface heparin sulfate proteoglycans (183, 186). Follistatin 288 is able to bind to cell surface heparin sulfate proteoglycans with high affinity
(183), whereas follistatin 303 has comparatively less binding activity, and follistatin 315 has none (184). Follistatin
303 was originally discovered in porcine follicular fluid
(183), and it remains unclear why a follistatin isoform
with intermediate activity exists in ovarian follicular fluid.
Originally, it was postulated that follistatin 303 was a
cleavage product of follistatin 315 and that the circulating
315 was of gonadal origin; however, using a follistatin
315-specific assay, it was shown that follistatin 315 was
not present in ovarian follicular fluid. This suggests that
circulating follistatin has a nongonadal origin (184). Follistatin consists of four domains, a unique N-terminal domain and three follistatin domains: Fs1, 2, and 3 (187). Fs1
and Fs2 are the biologically relevant activin binding domains (188); the crystal structure of these binding domains
within the follistatin and activin A complex provides insights into the mechanism of activin antagonism (142).
Through its Fs domains, follistatin binds to the outer convex surface and fingertips of the activin subunit, thereby
blocking interactions with ActRII/IIB (142). Mutagenesis
(189) and crystallography (142) studies of activin A show
that follistatin and ActRII binding sites overlap. Asp27,
Leu92, Tyr94, Ile100, and Lys102 are key activin residues
that interact with residues in the Fs1 and Fs2 domains of
follistatin (142, 189). In particular, Arg192 in the Fs2
domain is crucial for follistatin binding to activin. Ser201
in the Fs2 domain is also important for binding stability
(142). Interestingly, the N-terminal domain, Fs1, and Fs3
are important for binding to myostatin. Although residues
in the Fs1 domain are crucial for specific binding to myostatin, the N-terminal and Fs3 domains are required for
complex stability (139, 172). Follistatin mutants with two
Fs1 domains and an absent Fs2 domain had greater affinity for myostatin and reduced affinity for activin (172).
As an antagonist of activin action, follistatin modulates
activin-mediated FSH secretion from the anterior pituitary. FSH stimulates the gonadotropes and folliculostellate cells of the pituitary to secrete follistatin 288 to bind
and neutralize activins locally (190). Interestingly, activin
isoforms have different affinities for follistatin—activin B
binds with a 10-fold lower affinity compared to activin A
(191)—and it is thought that the differential binding affinities of activin A and B for follistatin contribute to their
doi: 10.1210/er.2014-1003
nonoverlapping functions in vivo. Mice lacking the follistatin gene die within hours of birth due to their inability
to breathe, and they display several abnormalities including decreased mass of the diaphragm and intercostal muscles, growth retardation, shiny taut skin, skeletal defects of
the hard palate, a 13th pair of ribs, and abnormal whisker
and tooth development (192). To understand the isoformspecific effects of follistatin, Lin et al (193) developed
transgenic mice that expressed either human follistatin
288 or 315 in a mouse follistatin null background. Human
follistatin 315, but not follistatin 288, was able to rescue
the lethal phenotype of the follistatin null mice. Additional
defects were observed in human follistatin 315 transgenic
mice, such as growth retardation, abnormal tail formation, failure to form corpora lutea, and increased inflammatory activity in the uterus. Kimura et al (194) developed
a follistatin 288-only mouse; these mice survived to adulthood with significant defects to female fertility. Follistatin
288-only mice experienced rapid depletion of their ovarian reserve and were infertile by day 250, a phenotype
similar to that seen in premature ovarian failure (POF) in
women (194). Follistatin 288-only females also had reduced apoptosis during germ cell nest breakdown (postnatal day 0.5– 8.5), and nest breakdown lasted longer. As
a result, follistatin 288-only females had a larger primordial follicle pool on postnatal day 8.5 compared to wildtype littermates (195). Previously, Jorgez et al (196) had
conditionally knocked out follistatin in the granulosa cells
of the ovary of mice and reported a significant loss in
fertility and litter numbers.
Another activin binding protein, follistatin-related
gene (FLRG), also known as follistatin-related protein
(197) or follistatin-like 3 (191), shares structural and functional homology with follistatin (198). Although FLRG is
able to antagonize the actions of activin in vivo (199), it is
slightly less potent than follistatin (200). Interestingly,
FLRG cannot bind cell surface proteoglycans because it
lacks a heparin-binding domain (201). FLRG also binds
other TGF␤ superfamily members, including myostatin
(202), BMP2, BMP4 (203), and BMP15 (204), and regulates their actions. FLRG is highly expressed in placenta
(205), testis (199), skin (206), adrenal glands (199), endometrium (207), skeletal muscle (208), and cardiovascular tissue (166), whereas follistatin is highly expressed in
the pituitary (209, 210) and ovary (211).
To understand the role of FLRG in reproduction, Xia
et al (199) developed transgenic mice that overexpress human FLRG. Male FLRG transgenic mice have lower gonadal weights, sperm counts, and fertility, and females
have reduced litter size, suggesting a role for FLRG in the
regulation of activin-mediated gonadal development and
gametogenesis (199). Mukherjee et al (212) developed
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mice lacking the FLRG gene and observed that the mice
developed a distinct group of metabolic phenotypes, including increased pancreatic islet number and size, pancreatic ␤-cell hyperplasia, decreased visceral fat mass, improved glucose tolerance, enhanced insulin sensitivity,
hepatic steatosis, and mild hypertension, but exhibited no
alteration of muscle or body weight. The observed knockout mouse phenotype is attributed to increased levels of
activin- and myostatin-mediated processes (212).
E. Mechanisms of inhibin action and antagonism
Although the activin signal transduction pathway has
been well studied, the mechanisms of inhibin signaling and
the molecular mechanisms of activin antagonism are still
an area of active investigation. There are several working
models that have been proposed. The simplest mechanism
suggests that the ability of inhibin to antagonize activin is
based on the proportion of available ␣-subunits and the
preferential assembly of ␣␤ heterodimers over ␤␤ dimers.
It is now clear that the antagonism of activin action by
inhibin is more complex and likely involves interaction of
the inhibin ␤-subunit with the activin receptors. In this
model, functional antagonism of activin signaling by inhibin is achieved through the competitive binding to the
cell surface ActRII and ActRIIB, which subsequently prevents recruitment of ALK4 and initiation of the intracellular activin signaling cascade (146, 213, 214) (Figure 8).
Cook et al (215) found that when two residues within the
␤-subunit that are necessary for activin binding to and
activation of ActRIIB are substituted with corresponding
residues from the ␣-subunit, ActRIIB binding capacity and
activation are lost. Thus, whereas activin binds to two
ActRIIB molecules in a 2:1 ratio with its ␤-subunits, inhibin binds a single ActRIIB through its single ␤-subunit.
Interestingly, the N-terminal region of the inhibin ␣-subunit also interacts with ALK4 and may be an important
component of the inhibin antagonism complex (216) (Figure 8). Because inhibin binds the activin receptors with a
much lower binding affinity than activin (217), it was further proposed that ancillary proteins or coreceptors with
high-affinity inhibin binding capacity may be necessary
for effective inhibin signaling/activin antagonism. One
such ancillary protein, betaglycan, was found to play such
a role as an inhibin coreceptor in pituitary cell lines (217,
218).
Betaglycan, also known as the type III TGF␤ receptor,
is a large, single transmembrane proteoglycan that acts as
a TGF␤ (219) and bone morphogenetic protein (220) coreceptor. Several groups have demonstrated that the competitive antagonistic activity of inhibin toward activinmediated FSH release is potentiated by betaglycan.
Inhibin binds to betaglycan with high affinity [Ki ⫽ 0.6
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Figure 8.
Inhibin at 90, a Historical Review
Endocrine Reviews, October 2014, 35(5):747–794
ActRII and inhibin in binding experiments (151). Farnworth et al (223)
identified several inhibin A-specific
binding proteins ranging from ⬍20
to ⬎170 kDa. Another binding protein, p120/InhBP (224), was initially
thought to bind inhibin B, but further work found that it does not bind
inhibin A, inhibin B, or activin A
(218), and null mice were viable and
fertile with no effect on FSH synthesis or secretion (225). Although most
of the binding proteins identified by
Farnworth et al (223) were betaglycan-related, 40 kDa (p40) and 125
kDa (p125), binding candidates remain unidentified. The 125-kDa
protein is unrelated to p120/InhBP,
raising the question of a unique signaling mechanism (223). Betaglycan
(217), p125, and p40 (223, 226)
bind inhibin A with high affinity.
Specific inhibin B binding proteins
have not been identified; elucidation
of proteins that bind inhibin B with
high affinity and high specificity will
enable a greater understanding of the
discordant pattern of inhibin A and B
secretion in males and females. The
identification of high-affinity binding sites for inhibin A in ovine pituitary (227), murine adrenal, and rat
Figure 8. Mechanism of inhibin action. Inhibin antagonism of activin action is mediated by
primary adrenal cells (223, 226) and
binding to activin receptors and betaglycan. Inhibin binds with high affinity to the
the identification of binding proteins
ActRII/betaglycan complex and blocks activin from mediating downstream Smad signaling.
betaglycan (217), p125, and p40
(223) suggests that inhibin B binding
(0.5– 0.9) nM] (217), and whereas inhibin has low binding proteins may behave in a similar manner as those for inaffinity to ActRII receptors, cotransfection of betaglycan hibin A.
with ActRII or ActRIIB potentiates inhibin binding to the
The structure of the betaglycan ZP-C domain has been
activin receptors (217). Affinities of inhibins and activins solved and suggests that TGF␤-related ligands bind conbinding to their receptors are summarized in Table 2. vex surface pockets of the AB loop (228). To understand
Lewis et al (217) demonstrated betaglycan-dependent the structural basis for how betaglycan facilitates the anblockade of activin signaling by inhibin in corticotropes, tagonistic actions of inhibin, ␣-subunit mutants of inhibin
ovarian cells, and erythroleukemic cells in a dose-depen- A and B were generated and assessed for their ability to
dent manner. Disruption of betaglycan expression by bind betaglycan (78, 229). Mutagenesis of residues in the
RNA interference-mediated knockdown or immunoneu- “fingers” of the ␣-subunit, in particular, residues Val340
tralization in gonadotrope cells eliminated functional in- and Tyr352 and, to a lesser extent, Tyr282, Arg341,
hibin antagonism, further demonstrating the central role Thr342, Thr343, Ser344, Ser349, Phe350, and Lys351,
of betaglycan as a coreceptor in inhibin-mediated suppres- were found to be critical for high-affinity interactions of
sion of FSH release (221, 222).
inhibin with betaglycan. These residues form a contiguous
A soluble form of betaglycan was generated with a Fc epitope on the outer convex surface of the fingers, or
fusion tag, which formed a high-affinity complex with knuckle region, of the inhibin ␣-subunit. The betaglycan-
doi: 10.1210/er.2014-1003
Table 2.
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761
Reported Affinities of Inhibin and Activin Isoforms for Activin Receptors and Betaglycan
Competing Ligands
Receptor
ActRII Ki (HEK293 cells)
ActRII Kd - displacing I125 activin A or B
ActRII ED50 - displacing I125 activin A
ActRII ED50 - displacing I125 activin B
ActRIIB-ECD Kd - BIAcore chip analysis
ActRIIB-ECD (single) Kd -BIAcore chip analysis
ActRIIB2 (IC50) - displacing I125 inhibin A
ActRIIB2 (IC50) - displacing I125 inhibin B
BG Ki (HEK293 cells)
BG (IC50) - displacing I125 inhibin B
ActRII ⫹ BG Ki (HEK293 cells)
ActRII ⫹ BG Kd - displacing I125 inhibin A
ActRIIB ⫹ BG Kd - displacing I125 inhibin A
ActRIIB2 ⫹ BG (IC50) - displacing I125 inhibin B
TM4 cells ED50 - displacing I125 inhibin A
TM4 cells ED50 - displacing I125 inhibin B
BG (IC50) - displacing I125 inhibin A and
inhibin B, respectively
ActRII ⫹ BG (IC50) - displacing 31 kDa I125
inhibin A and inhibin B, respectively
ActRIIB⫹ BG (IC50) - displacing 31 kDa I125
inhibin A and inhibin B, respectively
Activin A
Activin B
Inhibin A
Inhibin B
6.3 nM
49.3 ⫾ 25 pM
0.73 ⫾ 0.09 ng
0.50 ⫾ 0.25 ng
0.24 nM
6.9 nM
2.2 nM
3240 ⫾ 224 pM
3.26 ⫾ 1.29 ng
3.75 ⫾ 1.02 ng
15.8 ⫾ 0.39 ng
19.3 ⫾ 6.02 ng
13.1 nM
3.7 nM
75.5 nM
5.9 nM
0.6 nM
329 pM
0.2 nM
1100 ⫾ 169 pM
433 ⫾ 27 pM
2.7 nM
75 pM
50 pM
4.5 nM (34 kDa)
0.2 nM (31 kDa)
1.1 nM (34 kDa)
0.08 nM (31 kDa)
1.8 nM (34 kDa)
0.3 nM (31 kDa)
23.7 ⫾ 2.59 ng
12.9 ⫾ 2.62 ng
20.7 nM
4.5 nM
533 pM
1270 ⫾ 37 pM
704 ⫾ 59 pM
4.0 nM
6000 pM
400 pM
5.0 nM (34 kDa)
2.2 nM (31 kDa)
3.5 nM (34 kDa)
0.4 nM (31 kDa)
3.8 nM (34 kDa)
0.8 nM (31 kDa)
Refs.
217
151
151
151
131
131
218
218
217
218
217
151
151
218
226
226
78
78
78
Abbreviations: ECD, extracellular domain; BG, betaglycan.
binding site on the ␣-subunit of inhibin A (229) and inhibin B (78) was disrupted by the simultaneous substitution to alanine of Thr43, Ser344, and Tyr352. The
resultant TSY-inhibin A and B variants were 70- and 50fold less active, respectively, compared to wild-type inhibin A and B at suppressing activin-induced FSH release
by a mouse pituitary gonadotrope cell line (L␤T2) and
primary rat pituitary cells in culture. Therefore, binding to
betaglycan is essential for the biological activity of both
inhibin A and inhibin B.
Glycosylation of Asn302 adversely affects inhibin A
and B in vitro biological activities, whereas glycosylation
at Asn268 appears to have no effect on bioactivity and
may even facilitate inhibin action. The effect of differential
Asn302 glycosylation on inhibin A and B binding affinities
to betaglycan is not understood; however, the lower biological activity of 34-kDa inhibin A and B (glycosylated at
both Asn268 and Asn302) is attributed to reduced binding
to the betaglycan⫹ActRII/IIB complex. Binding of the 34kDa forms of inhibin A and B to this receptor complex is
lower than for the 31-kDa forms of inhibin A and B (glycosylated only on Asn268). Asn302 is located in the proline-rich region of the ␣-subunit; however, betaglycan
binds to residues in the fingers of the inhibin ␣-subunit (78,
229). Interestingly, absence of the inhibin ␣-subunit’s proline-rich region in nonmammalian species removes its
functional dependence on betaglycan (144). Therefore,
reduced betaglycan binding due to glycosylation of
Asn302 in the proline-rich region is likely due to a con-
formational change. Crystal structures of glycosylated inhibins would enable us to define the binding sites involved.
Immunohistochemical analyses have localized betaglycan in the granulosa and theca cells of ovarian follicles and in testicular Leydig cells (217), as well as in
pituitary cells during the reproductive cycle (230). The
immunoprecipitation of a betaglycan/ActRII/inhibin
complex from ovarian KK-1 cells (217) further supports
the involvement of betaglycan in inhibin-mediated actions in the ovary (217).
The absence of signaling motifs in the intracellular domain of betaglycan makes it unlikely that it acts as an independent inhibin receptor. It is still possible that inhibin may
signal through its own receptor to mediate biological effects.
In situ radioligand studies have demonstrated inhibin-specific binding sites on granulosa cells in the ovaries (231) and
ovine pituitary cells (227); however, an inhibin-specific binding molecule that supports activin receptor-independent inhibin action has not been identified to date.
F. Soluble inhibin binding proteins
In addition to membrane-bound ActRII and betaglycan, other soluble proteins that demonstrate inhibin-binding capacity have been reported in the literature. ␣2-Macroglobulin binds to both inhibin and activin (232), but the
physiological role of this serum glycoprotein in activin/
inhibin biology is yet to be determined. Binding of ␣2macroglobulin to either inhibin or activin does not affect
bioactivity or immunoreactivity (232). ␣2-Macroglobulin
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Makanji et al
Inhibin at 90, a Historical Review
exists in circulation in both the native and transformed
forms (233). Inhibin, activin, and follistatin bind preferentially to the transformed species of ␣2-macroglobulin,
although activin also shows binding affinity to the native
form (233). Because proteins that bind to the transformed
species are rapidly cleared from circulation through the
␣2-macroglobulin receptor, it is proposed that ␣2-macroglobulin could play a role in inhibin, activin, and follistatin clearance (233).
III. Physiological Roles of Inhibin
Within the reproductive axis, inhibins have been studied
extensively as endocrine negative regulators of FSH release from the anterior pituitary. With the development of
inhibin immunoassays and better molecular tools, inhibin
transcripts and protein expression have been found in organs other than the gonads and pituitary. In addition, we
are learning more about the processing of inhibin forms
that are found in circulation, drawing on insights from the
TGF␤ superfamily. Discoveries suggesting expanded biological activities and bioavailabilities of inhibin precursors
and mature forms are discussed in several recent reviews
(20, 99, 234 –238). The following sections focus on the
physiological expression and roles of the mature forms of
inhibin.
A. Dissecting the roles of inhibins using knockout and
transgenic mouse models
Knockout of the inhibin ␣-subunit, shared by both inhibin A and B, in mice led to the development of gonadal
stromal tumors as early as 4 weeks of age that progressively worsen and are accompanied by cachexia and thoracic kyphoscoliosis, eventually resulting in death (239).
The lethal cachectic-wasting syndrome is characterized by
severe hepatocellular necrosis, parietal cell depletion and
mucosal atrophy in the stomach, severe weight loss, and
anemia. Secretion of activins by the gonadal tumors leads
to a ⬎10-fold increase in circulating activins, which is
most likely culpable for the wasting syndrome (240). Bilateral gonadectomy slightly increases the survival of these
mice; however, they succumb to adrenal cortical sex steroidogenic tumors by 21 weeks of age, developing the
same lethal cachectic-wasting syndrome as observed in
intact null animals by 4 weeks of age (240).
To understand the apparent tumor suppressor function
of inhibin, several additional knockout and transgenic
mouse models have been created (Table 3). Coerver et al
(241) crossed the inhibin ␣-subunit null mouse with the
ActRII null mouse to determine the functional significance
of the elevated activin levels observed in the inhibin ␣-sub-
Endocrine Reviews, October 2014, 35(5):747–794
unit null mice. These double-knockout mice still develop
gonadal sex-cord stromal tumors with elevated serum activins; however, they do not develop the cachectic wasting
syndrome. Similarly, when follistatin was overexpressed
in the inhibin ␣-subunit-deficient mice, development of
the wasting syndrome was delayed and reduced in severity
(242). Li et al (243) crossed the inhibin ␣-subunit null
mouse with Smad3 null mouse to further clarify the role of
activins in gonadal tumorigenesis and development of the
cachectic-wasting syndrome. Interestingly, 90% of the
␣-subunit-Smad3 double-knockout males survived until
26 weeks of age and developed only unilateral small tumors with occasional contralateral cyst formation, and no
cachexia (243). These double-mutant male mice also had
reduced levels of serum activins compared to the inhibin
␣-subunit null mice (243). The double-knockout female
mice demonstrate abnormal ovarian histology and oocyte
degeneration by 6 weeks of age and develop multifocal
hemorrhagic ovarian cysts by 16 weeks of age. Unlike
males, females have elevated levels of serum activins and
develop cachexia, albeit delayed compared with inhibin
␣-subunit null mice (243).
Adult inhibin-deficient mice are infertile due to severe
disruption of the normal architecture of the ovaries and
the testes; however, the gonadal development of the ovaries and testis is normal, with all the hallmarks of secondary sexual development before tumor formation. Because
inhibin negatively regulates FSH, both male and female
knockout mice have a 2- to 3-fold increase in FSH levels
(239). To examine the role of elevated FSH in gonadal
tumor development, Kumar et al (244) crossed the hypogonadal (hpg) mouse (contains a naturally occurring
mutation in the GnRH gene, resulting in reduced levels of
FSH and LH) with the inhibin ␣-subunit null mouse. Interestingly, these compound mutant mice do not develop
gonadal or adrenal tumors or a wasting syndrome and
survive for more than a year (244). Surprisingly, when the
inhibin ␣-subunit null mouse was crossed with the FSH
␤-subunit null mouse, these double-knockout mice developed gonadal tumors after 12 weeks of age, later than seen
in the inhibin ␣-subunit null mice (245). Parallel efforts by
Nagaraja et al (246) led to the creation of inhibin ␣-subunit and LH ␤-subunit double-mutant mice. The doubleknockout mice developed gonadal tumors; however, the
tumors were slow growing and less aggressive, with mice
surviving for more than a year. These mice also had lower
levels of serum FSH and estradiol compared with the
inhibin ␣-subunit null mice. All mice developed the cachectic-wasting syndrome. Together, these results suggest that although LH is not involved in tumor formation in the absence of inhibins, it may aid tumor
progression (246).
doi: 10.1210/er.2014-1003
Table 3.
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763
Inhibin ␣-Subunit Knockout Mouse Models Created to Investigate the Tumor Suppressor Role of Inhibin
Mutant Mouse Model
Male Phenotype
Inhibin ␣-subunit knockout
(Inha⫺/⫺)
Sertoli cell tumors evident by 4 wk of age; mice
succumb to a wasting syndrome with 95% death
by 12 wk of age
Female Phenotype
Granulosa cell tumors evident by 4 wk
of age; mice succumb to a wasting
syndrome with 95% death by 17 wk
of age
Inha⫺/⫺; Gnrh mutant (hpg/hpg)
Loss of GnRH protects from tumorigenesis and the
Protection from tumorigenesis and the
associated wasting syndrome; double-mutant mice
associated wasting syndrome; doublesurvive ⬎1 y
mutant mice survive ⬎1 y. Disrupted
folliculogenesis; halted at primary
follicle stage
Inha⫺/⫺; FSH knockout (Fshb⫺/⫺)
Males develop testicular tumors with a delayed onset Females develop slow growing and less
and less aggressive course
hemorrhagic tumors, and 70% live
beyond 17 wk of age
Inha⫺/⫺; anti-Müllerian hormone
Sertoli cell tumors earlier, and tumors grow faster
Granulosa cell tumors develop similar to
(Amh) knockout (Amh⫺/⫺)
than in inhibin-deficient mice; tumors are less
the inhibin-deficient mouse
hemorrhagic; additional Leydig cell tumors are
observed as early as 1 wk of age
Inha⫺/⫺; anti-Müllerian hormone
Sertoli cell tumors earlier, and tumors grow faster
Phenocopies Inha⫺/⫺; Amh⫺/⫺ double
receptor (Amhr) knockout
than those of inhibin deficient mice; tumors are
mutants
(Amhr⫺/⫺)
less hemorrhagic; additional Leydig cell tumors are
observed at 1 wk of age
Testicular tumors progress less rapidly and are less
Ovarian tumors progress less rapidly and
Inha⫺/⫺; androgen receptor (Ar)
hemorrhagic; mice have a prolonged survival
are less hemorrhagic
mutant mice (Xtfm,Y)
compared to inhibin knockouts (50% at 17 wk)
Inha⫺/⫺; activin receptor II (ActRII)
Loss of ACVR2 protects from the wasting syndrome; Loss of ACVR2 protects from the
knockout (Acvr2⫺/⫺)
stomach and liver histology are normal in the
wasting syndrome; stomach and liver
absence of inhibins despite tumor progression
histology are normal in the absence
of inhibins despite tumor progression
Inha⫺/⫺; treatment with ActRII
Development of late-onset, reduced size testicular
Development of ovarian tumor and cysts
fused to Fc region of murine IgG2a
tumors and testicular cysts. Mice do not develop
and disrupted ovarian architecture.
cachexia
Mice do not develop cachexia
Inha⫺/⫺; inhibin A (␣:␤A) transgenic Induction of a bi-cistronic transgene allows
Rescues the tumor phenotype in Inha⫺/⫺
overexpresser
production of mouse inhibin A from the liver and
females and displays disrupted
rescues the tumor phenotype in Inha⫺/⫺ males
folliculogenesis; halted at early antral
stage and lack corpora lutea
Inha⫺/⫺; follistatin transgenic
Despite tumor formation, mice exhibit a less severe
Despite tumor formation, mice exhibit a
overexpresser
wasting syndrome, lower serum activin levels, and
less severe wasting syndrome, lower
prolonged survival compared with mice deficient
serum activin levels, and prolonged
in inhibins alone
survival as compared with mice
deficient in inhibins alone
Inha⫺/⫺; p27 knockout
Sertoli cell tumors evident by 2 wk of age; mice
Granulosa cell tumors evident by 4 wk
(Cdkn1b⫺/⫺)
succumb to a wasting syndrome with 100% death
of age; mice succumb to a wasting
by 10 wk of age
syndrome with 100% death by 18 wk
of age
Inha⫺/⫺; cyclin D2 knockout (cyclin Males do not develop tumors; 50% survive for up to Ovarian tumors with late onset (29 wk)
D2⫺/⫺)
41 wk and 29% survive for more than 1 y and are
and less aggressive; development of
fertile
cachexia and death by 39 wk of age
Inha⫺/⫺; Smad3 knockout
Mice develop less aggressive, slower growing
Ovaries display abnormal histology with
testicular tumors with late onset. Mice do not
oocyte degeneration by 6 wk of age.
develop cachectic-wasting syndrome and survive
Hemorrhagic tumors develop by 16
beyond 26 wk
wk, which manifests the lethal
cachectic-wasting syndrome
Inha⫺/⫺; activin ␤C-subunit
Prolonged survival of male mice with reduced
Mice develop less aggressive, slower
transgenic
incidence of tumors
growing tumors with no significant
loss in body weight
Refs.
239, 240
244
245
514
515
516
241
517
251
242
518
519
243
520
Modified and reprinted from W. Yan et al: Genetic engineering to study testicular tumorigenesis. APMIS. 2003;111:174 –181 (513), with permission. © John
Wiley and Sons.
The reproductive phenotype of most of the inhibindeficient mutant mouse strains described above is complicated by the development of gonadal tumors; however,
those models with delayed onset of tumorigenesis permit
us to examine the roles of inhibins in male and female
reproduction. Both male and female hpg mutant mice that
are also null for the inhibin ␣-subunit display disrupted
reproductive phenotypes (245), clearly indicating the im-
764
Makanji et al
Inhibin at 90, a Historical Review
portance of both inhibins and FSH for normal reproductive function. ␣-Subunit knockout mice are infertile secondary to the development of gonadal tumors and have
elevated activin A and activin B levels (239, 240, 247).
Conversely, a marked reduction in FSH levels is observed
in both female and male mice that overexpress the inhibin
␣-subunit (248).
In female mice, overexpression of the ␣-subunit results
in a dramatic reduction in litter size that is associated with
reduced ovulating oocyte number (248). Ovaries from
these mice contain fluid-filled cysts and fewer mature antral follicles and corpora lutea (249). These mice also have
a lower uterine weight and develop abnormal steroid hormone production, with higher serum T and lower serum
estradiol levels compared with wild-type mice (249). FSH
␤-subunit null mice also display abnormal reproductive
phenotypes: females are infertile with small ovaries and
folliculogenesis arrested at the preantral stage, and males
are fertile but have small testes and reduced sperm number
and motility (250). Overexpression of human FSH in
FSH␤ transgenic mice results in infertility; males have enlarged seminal vesicles due to elevated serum T, and the
females have enlarged hemorrhagic cystic ovaries with
edematous uteri and disrupted folliculogenesis (245).
In male mice, overexpression of the ␣-subunit results in
decreased sperm number (248), smaller testes (249), and
reduced seminiferous tubule volume (249). Mice lacking
the inhibin ␣-subunit gene develop testicular tumors very
early, by 4 weeks of age (239, 240), making it difficult to
study the effects of a loss of inhibins on spermatogenesis.
Nonetheless, the homozygous male inhibin-deficient mice
have enlarged testes, and spermatogenesis is active between weeks 5 and 7 but declines rapidly with tumor progression. The inhibin null animals also display a loss of
Leydig cells compared to wild-type animals. Interestingly,
the secondary sexual characteristics in these mice are normally developed before tumor formation, suggesting a
negligible role of inhibins in embryonic gonadal development. It is important to note that some inhibin null male
mice have testes devoid of tumors but still show halted
germ cell maturation, perhaps due to the large quantities
of activins the tumors secrete into circulation (239). Because the tumors hinder the assessment of the roles of
inhibins in adult spermatogenesis, several double-mutant
mice have been developed to prevent gonadal tumorigenesis (Table 3). Using gene manipulation technology, inhibin A expressed in the livers of 3-week-old inhibin
␣-subunit null male mice rescued the gonadal tumor phenotype, resulting in fertile mice with normal testes (251).
A similar induction of inhibin A in 3-week-old wild-type
mice resulted in the reduction of testis weight and seminiferous tubule volume and diameter (251). The hpg/
Endocrine Reviews, October 2014, 35(5):747–794
inha⫺/⫺ double-mutant mice do not develop tumors; the
male mice are fertile despite small testes, reduced sperm
number, and reduced sperm motility (250). Overall, these
studies fail to dissect the roles of inhibins from those of
activins, because manipulating the ␣-subunit also affects
dimerization of the ␤-subunits, and therefore activin levels. Nonetheless, whether inhibins have a direct role or an
indirect role via antagonism of the activins, they are critical for successful spermatogenesis.
Together, these studies highlight the importance of inhibin’s dual role in the reproductive axis: the endocrine
regulation of FSH from the anterior pituitary, and the
paracrine regulation of gametogenesis in the gonads.
B. Sites of inhibin expression
Levels of circulating inhibin are undetectable after gonadectomy in both male and female rats, providing strong
evidence that the gonads are the primary site of inhibin
production (252, 253). Inhibin ␣- and ␤B-subunits are
expressed in the Sertoli and Leydig cells of the testes (254),
and in the ovaries, inhibin subunit expression is detected
in granulosa and luteal cells in certain species (255). Inhibin subunits are detectable as early as 51 days of gestation in the embryonic gonads in humans (256). In addition
to the reproductive organs, immunoreactive inhibin is
present in the adrenal glands (257), eye (258), lung (259),
kidney (259), pituitary (259), and spleen (259). Inhibin
subunit (␣, ␤A, and ␤B) mRNA expression is also observed in the placenta, pituitary, adrenal glands, bone
marrow, kidney, spinal cord, and brain (260). During
pregnancy, inhibin is expressed in the placenta (261, 262);
specifically, ␣- and ␤B-subunits are present in the placental syncytiotrophoblast (263).
Protein and mRNA expression of inhibin ␣- and ␤Bsubunits have also been extensively characterized from
Carnegie stages 7 through to 23 in the human embryo by
Harkness and Baird (256). The developing liver, digestive
system (esophagus, stomach, gut), cardiovascular system
(pericardium), urogenital system, adrenal glands, and respiratory system are immunoreactive for both ␣- and ␤Asubunits (256). Inhibin is also present in the developing
eye (264), embryonic nervous system, skin, and mesenchyme (256). mRNA expression of the ␤A subunit is detected in the embryonic heart, digestive tract, urogenital
tract, respiratory tract, and skin (265). ActRII and ActRIIB mRNA expression is also present during early embryonic development (266).
The widespread expression of inhibin subunit mRNA
and protein, as well as the activin receptors and betaglycan
(Table 4), suggests a possible biological role of inhibin in
various systems during development and embryogenesis.
A number of studies have reported various physiological
doi: 10.1210/er.2014-1003
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765
Table 4. Tissue Distribution of Activin and Inhibin Subunits, Betaglycan, Activin Receptors, and Follistatin mRNA
Transcripts in Adult Human Tissues
Tissue
␤A
␤B
␣
BG
ActRIIA
ActRIIB
ALK4
ALK7
Follistatin
Ovary
Placenta
Uterus/decidua
Oocytes
Small antral follicles
Granulosa cells
Theca cells
Large dominant follicles
Granulosa cells
Theca cells
Testis
Sertoli cells
Leydig cells
Prostate
Brain
Anterior pituitary
Gonadotropes
Adrenal
Bone marrow
Breast
Spleen
Heart
Lung
Thymus
Skeletal muscle
Kidney
Pancreas
Liver/hepatocytes
⫹
⫹
⫹
⫹
⫹
⫹
?
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
?
?
⫹
⫺
⫺
⫺
?
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
?
?
⫹
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?
?
⫹
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⫹
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?
⫺
⫺
⫹
⫹
⫹
⫹
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⫹
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⫹
⫹
⫺
⫺
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⫹
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⫹
⫹
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⫹
⫹
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⫹
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⫹
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⫹
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⫹
⫹
?
?
⫹
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?
?
?
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⫺?
⫹
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⫺
⫹
⫹
?
⫹
⫹
?
⫹
?
?
⫹
⫹
⫹
⫹
⫹
⫹
⫹
?
⫹
⫹
⫺
⫺
?
?
?
⫹
⫺
?
⫺
⫺
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫺
?
⫹
⫹
?
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
Abbreviations: BG, betaglycan; ?, insufficient evidence. Adapted from Refs 150, 278, 299, 300, 336, 391, 400, 486, 521–545 and expression profiles from
http://www.ncbi.nlm.nih.gov/sites/entrez?db⫽unigene of Hs.583348-INHBA, Hs.1735-INHBB, Hs.470174-ACVR2A Hs.174273-ACVR2B, Hs.4389818-ACVR1B,
Hs.562901-ACVR1C and Hs.9914-FST.
roles of inhibin and activin, including a series of studies on
inhibin/activin subunit and receptor knockout mouse
models (Table 1). Here, we discuss some of the organ systems in which inhibin appears to have significant physiological functions (Figure 9).
C. Pituitary-gonadal axis
Inhibin acts as an endocrine hormone as part of a negative feedback loop within the pituitary-gonadal axis to
regulate synthesis of FSH by the anterior pituitary gonadotropes (267). In adults, serum inhibin inversely correlates with serum FSH concentration (253). Loss of inhibins at the time of menopause in women leads to a rise
in FSH production, confirming the central role of inhibins
in restraining FSH via negative feedback (268). In vivo
studies have demonstrated that inhibin is vital to normal
reproductive function in nonhuman primates (247, 269).
1. Regulation of FSH and LH in the pituitary
Specialized cells in the anterior pituitary called gonadotropes produce and secrete gonadotropins, FSH and LH.
FSH and LH are heterodimeric proteins made up of a
common ␣-subunit and their respective ␤-subunits.
GnRH from the hypothalamus, T from the testis, estradiol
and progesterone from the ovaries, gonadal inhibins, local
activins, and follistatin regulate the synthesis and secretion
of gonadotropins from the pituitary. In males, T synthesized by Leydig cells in the testes provides feedback to the
hypothalamus (to modulate GnRH secretion) and the anterior pituitary. In females, estradiol produced by developing follicles and progesterone by the corpus luteum provide feedback to the hypothalamus and the pituitary.
Because the ␣-subunit is common to both FSH and LH,
regulation of the individual gonadotropins is achieved by
modulation of the FSH␤ or LH␤ gene. Modulation of the
GnRH pulsatile frequency, which regulates GnRH receptor (GnRHR) expression, determines the biosynthesis of
FSH and LH by gonadotropes (270, 271). High-frequency
GnRH pulses result in the maximal expression of GnRHR
and correlate with elevated levels of LH/FSH ␣-subunit
and LH␤. On the other hand, low GnRH pulse frequencies
lower the expression of GnRHR and result in the elevation
of FSH␤ mRNA (272). In this manner, GnRH is able to
modulate the synthesis and expression of both LH and
FSH. In addition, in in vitro rodent models, activin alone
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Figure 9.
Inhibin at 90, a Historical Review
Endocrine Reviews, October 2014, 35(5):747–794
shown to express the inhibin coreceptor betaglycan (230, 278). Regulatable expression of inhibin A from
the liver of inhibin ␣-subunit null
mice resulted in a reduction in serum
FSH levels and development of reproductive defects as a consequence
(251). Inhibins modulate gonadotrope function (ie, FSH synthesis) by
antagonizing the actions of activins.
Gonadal inhibins and locally produced activin B (279) directly regulate the biosynthesis of FSH␤; in
vitro studies using rat (280) and
mouse (281, 282) pituitary cell cultures show that activin stimulates
and inhibin (283) decreases the levels
of FSH␤ mRNA and FSH protein
(284). In addition, inhibin (285) reduces and activin (286) increases the
half-life of FSH␤ mRNA.
Follistatin, produced by folliculostellate cells and gonadotropes in the
pituitary, suppresses FSH secretion
by sequestering activins (190). Activins may also play a role in the regulation of the LH␤-subunit. Activin
A was able to increase LH␤ mRNA
and LH levels in the mouse gonadotrope cell line, L␤T2 (287). In combination with GnRH, activin A is a
potent activator of the LH␤ promoter in L␤T2 cells (287, 288). Interestingly, treatment with follistatin
does not diminish LH␤ levels in
Figure 9. Physiological roles of inhibins. Inhibin’s diverse role in the gonadal and nongonadal
L␤T2 cells, although there is a 50%
tissues is shown.
reduction of FSH␤ levels (289). Coss
et al (288) investigated the roles of
and in combination with GnRH can increase GnRHR activins and their related Smads in the regulation of LH␤.
transcriptional activity (273). In contrast, inhibin (274, They utilized Smad3-deficient mice to show that the re275) and follistatin (276) down-regulate activin- stimu- duction in LH␤ and FSH␤ levels can be attributed to imlated GnRHR transcriptional activity. GnRH pulse fre- paired activin signaling. In addition, overexpression of
quency can also regulate activin B production by modu- Smad7 (inhibitor of activin signaling) in L␤T2 cells relating the activin ␤B-subunit mRNA levels in the pituitary sulted in abolishment of activin-induced LH␤ expression
(277).
(288). The role of inhibins in the modulation of LH is not
FSH drives the Sertoli cells of the testis and granulosa understood.
cells of the ovary into secreting inhibins. Despite the expression of the inhibin ␣-subunit by cells of the anterior 2. Inhibins and the female
pituitary, gonadal inhibin is the main peptide hormone reproductive axis
In females, the granulosa cells of the ovary produce
that regulates FSH synthesis and secretion during folliculogenesis and spermatogenesis. Gonadotropes are the pri- inhibin, and inhibin production by each follicle increases
mary site of action for inhibin action; these cells have been as the granulosa cell population expands during normal
doi: 10.1210/er.2014-1003
follicle growth and maturation (290, 291). During germ
cell nest breakdown, activin is in part responsible for the
formation of the primordial follicle pool. Treatment of
neonatal mice with activin A results in a greater number of
primordial follicles entering the initial follicle pool (292),
the establishment of which determines future fertility
(293). Inhibin and activin also act as intraovarian paracrine signaling molecules that regulate follicular dominance during the preovulatory phase of the menstrual cycle (290). Activin is an important paracrine factor in the
early stages of follicle growth, before follicles begin to
express the FSH receptor and acquire FSH responsiveness
(294). Once follicles express FSH receptor (FSHR), further development is FSH-dependent. Lu et al (295) reported that inhibin A modulates FSH action at the
intrafollicular level by suppressing FSH-induced FSHR
promoter activity and mRNA expression in cultured rat
granulosa cells. Inhibin A also inhibits FSH-induced steroidogenesis in vitro via a mechanism involving transcription factors SF-1, AR, and DAX-1. Activin A treatment of
granulosa cells increases estrogen receptor gene expression (296), and neonatal mice exposed to estrogen have
reduced levels of inhibin ␤A-subunit and reduced serum
inhibin A (297). Inhibin is involved in the paracrine modulation of ovarian androgen (androstenedione and dehydroepiandrosterone) production. Hillier et al (298) demonstrated that treatment of human theca cell cultures with
inhibin results in a 2-fold increase in androgen synthesis.
The elevation in androgen synthesis was additive when
theca cell cultures were cotreated with LH and inhibin,
and inhibin opposed activin-induced inhibition of LHstimulated androgen synthesis (298).
In humans, the inhibin ␣-subunit is expressed by the
granulosa cells of follicles of all sizes, unlike the ␤A- and
␤B-subunit mRNA, which are expressed in a discordant
pattern by different sized follicles (299, 300). The ␤Asubunit mRNA is expressed in the granulosa cells of large
follicles and the corpus luteum, whereas the ␤B-subunit
mRNA is detected in the granulosa cells of small antral
follicles (300). Granulosa cells, theca cells, and oocytes of
all follicle classes express follistatin and activin type I and
II receptors. Thus, all the components for activin action
are present in the ovary (Table 4). Granulosa cells of developing follicles have the ability to produce activins, inhibins, and follistatin from an early stage. The pattern of
inhibin A and B secretion during folliculogenesis is discordant; small antral follicles produce mainly inhibin B,
whereas the dominant follicles and corpus luteum secrete
inhibin A (301, 302). Betaglycan and ActRII mRNA are
expressed by theca and granulosa cells and oocytes during
all stages of folliculogenesis (303). During the primary
follicle to antral stage, inhibins may modulate activin-me-
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diated granulosa cell proliferation and differentiation;
however, its precise role in unknown. However, during the
antral follicle development stage, inhibin A slows down
oocyte maturation and development (304), promotes LHdependent androgen production by theca cells (305, 306),
and promotes FSH-induced estradiol secretion by granulosa cells (305). The formation of the corpus luteum marks
the end of the follicular cycle, and if fertilization and subsequent implantation occurs, it serves as an endocrine organ in early pregnancy. Along with estrogen and progesterone, the corpus luteum is a major source of inhibin A
(299). As previously reported, activin A attenuates LHinduced progesterone secretion in vitro (307, 308). The
local role of inhibin A in the corpus luteum is unclear;
however, some reports suggest that it may promote LHinduced progesterone secretion (309, 310). Perhaps the
elevated levels of inhibin A secreted by the corpus luteum
have a role in pregnancy. In addition, inhibin also promotes LH-induced androgen secretion from theca cells in
a paracrine manner (311, 312). Together, these studies
highlight the important roles of inhibins and activins during follicle growth and development.
In in vitro culture of mouse ovarian follicles encapsulated in alginate beads, the inhibin ␤A-subunit gene is upregulated during follicle growth (313). Transcripts of inhibin ␣- and ␤B-subunit are also up-regulated in
encapsulated follicles and cumulus oocyte complexes
(314). In vitro treatment of rat granulosa cells isolated
from immature follicles with FSH results in increased inhibin and estradiol production (290), and FSH up-regulates the transcription of the ␣-, ␤A-, and ␤B-subunits
(315, 316, 317). By comparison, the oviducts of alligators
express inhibin ␤A- and ␤B-subunit transcripts, but minimal inhibin ␣-subunit, in response to FSH (318). Interestingly, endocrine-disrupting contaminants in polluted
lakes alter FSH responsiveness and gene expression of inhibin subunits and related ligands. Ovaries of alligators
from polluted lakes respond poorly to FSH stimuli and
have increased serum estradiol and T and decreased expression of inhibin ␣-, ␤A-, and ␤B-subunit transcripts
compared to animals from unpolluted sites (319). In addition, offspring of alligators from polluted lakes have
altered expression of gonadal inhibin ␣-, ␤A-, and ␤Bsubunit, GDF9, and follistatin transcripts (320). These
studies highlight the disruptive nature of environmental
contaminants on the sensitive balance that inhibin and
related ligands maintain in reproduction.
In humans and primates, levels of circulating inhibin A
and B oscillate throughout the menstrual cycle: inhibin A
remains at low levels during the follicular phase, then rises
rapidly through ovulation and peaks at the midluteal
phase, whereas maximum inhibin B levels occur during the
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Inhibin at 90, a Historical Review
early-follicular and early-luteal phases (63, 253, 291).
Normal-cycling rhesus monkeys injected with inhibin A
daily during the early follicular phase (269) or luteal phase
(247) over several days had a reduction in serum FSH.
Serum LH levels were unaffected in animals injected with
inhibin during the luteal phase (247). However, animals
injected during the follicular phase had decreased serum
estradiol and increased serum LH 2–3 days after treatment. This was attributed to insufficient aromatase activity in granulosa cells because of the low FSH and subsequent low estrogen feedback to the hypothalamus/
pituitary (269).
Due to the inherent problems associated with disrupting the homeostatic balance that inhibins provide to activins, FSH, and LH, it is difficult to isolate the roles of
inhibins in most knockout/knock-in mouse models. Nonetheless, these models do provide some insight into the important roles of inhibins in folliculogenesis. The inhibin
␣-subunit null mice develop gonadal tumors; thus, the
primary effects of inhibin loss cannot be determined in this
model. However, in the hpg:inh⫺/⫺ double-knockout
mouse, the absence of gonadal tumors gives some insights
into the reproductive phenotype (Table 3). The female
double-knockout mice display disrupted folliculogenesis,
with the process being halted at the primary antral stage
(244). In addition, overexpression of inhibin ␣-subunit
results in disrupted folliculogenesis due to the formation
of ovarian cysts and elevated theca cell androgen production in female transgenic mice (249). Furthermore, the loss
of the activin A or inhibin A in the ␤B-subunit knock-in
mouse model resulted in fewer preovulatory follicles and
a reduction in fertility (163). These mouse models highlight the importance of inhibins in folliculogenesis, with
the latter study suggesting differential roles of inhibin A
and B during the process.
3. Inhibins and the male reproductive axis
In contrast to the ovary, the Sertoli cells of the adult
human testes produce only inhibin B (321, 322). Inhibin B
is also the only detectable inhibin form in male rodents
(253) and nonhuman primates (323). Inhibin A levels are
undetectable in human males, although inhibin A is found
in the serum of bulls (324), boars (325), and rams (164).
In male rats, inhibin B levels begin rising between 3 and 6
months of age and remain elevated up to 12 months of age
(326). Inhibin levels then remain low until puberty, at
which point serum inhibin gradually rises to a steady level
throughout adulthood (326, 327). The inhibin ␣-subunit
is expressed by Sertoli cells and to a lesser extent Leydig
cells (328, 329), with maximal mRNA levels between
stages XIII–I and minimal between stages VII–VIII of the
rat seminiferous cycle (330, 331). As a result, inhibin B
Endocrine Reviews, October 2014, 35(5):747–794
protein secretion is highest from stage IX–I and lowest at
stage VII (332). The ␤B-subunit is expressed by Sertoli
cells and germ cells; spermatogonia, primary spermatocytes, and round spermatids (333). Leydig cells also express mRNA for the ␤B-subunit (334). Betaglycan is expressed in the male pituitary gonadotropes and Leydig
cells and tubule-specific germ cells of the testis (278).
In adult males, serum inhibin B levels are negatively
correlated with serum FSH and positively correlated with
sperm count (335), sperm concentration (336), and testicular volume (335). Inhibin B levels in human males (16,
337, 338), rats (253), and monkeys (339) are significantly
inversely correlated with FSH levels. As in females, inhibin
B in males is a primary negative regulator of FSH in humans (338), rodents (340), and nonhuman primates (341,
342). In turn, FSH promotes the production of inhibin B
and free inhibin ␣-subunit in cultured Sertoli cells, forming
a classic feedback loop within the male pituitary-gonadal
axis (50, 51). Interestingly, the proinflammatory cytokine
IL-1 also modulates inhibin B secretion; addition of IL-1
to Sertoli cell cultures results in the elevation of activin A
and reduction in inhibin B levels (343). Serum inhibin B
levels positively correlate with Sertoli cell numbers (340,
344). Some studies have reported a strong positive correlation between inhibin B and testicular volume and sperm
concentration in the ejaculate, suggesting a paracrine role
of inhibin in regulating spermatogenesis (337, 345–349).
Others have disputed this correlation, suggesting that FSH
is by far a superior marker for sperm concentration
(350 –352).
The paracrine roles of inhibins in the developing testis
and later in the adult testis are unclear. Inhibin ␣-subunit
mRNA is present in the developing fetal testis, and the
levels increase up to the time of birth (353, 354). Men with
impaired spermatogenesis have lower levels of circulating
inhibin B compared to normozoospermic men (335, 355,
356). In adult animals, activin A induces Sertoli cell proliferation, inhibits the proliferation of differentiating type
A spermatogonia (357), promotes DNA synthesis in early
germ cells, and inhibits T production by Leydig cells (31).
Mather et al (358) showed that inhibin failed to block or
reduce the effects of activin on DNA synthesis in the spermatogonia of Chinese hamsters in culture, in contrast to
the effects of inhibin suppression on activin-mediated FSH
release in the pituitary. In addition, inhibin reduces the
number of spermatogonia in the testes of mice and Chinese
hamsters (359). There is mounting evidence to suggest that
germ cells modulate inhibin B secretion via interactions
with the Sertoli cell. In Sertoli cell cocultures with germ
cells, pachytene spermatocytes suppressed FSH-induced
␤B-subunit mRNA and inhibin B levels without affecting
inhibin ␣-subunit mRNA (360). Therefore, inhibin B is
doi: 10.1210/er.2014-1003
vital to the interplay between germ cells and their supporting Sertoli and Leydig cells in order to regulate spermatogenesis and indirectly modulate FSH synthesis.
D. Bone metabolism
Bone mass is regulated through a balance of bone formation (osteoblastogenesis) and bone resorption (osteoclastogenesis). It was believed for some time that hypogonadism associated with declining estrogen levels and
increasing FSH concentrations during the menopausal
transition were primarily responsible for postmenopausal
bone loss and osteoporosis. There is accumulating evidence that now points to a role of inhibin in modulating
changes in bone turnover during this transition.
Studies in primary murine bone marrow cultures have
demonstrated an inhibitory effect of inhibin and a stimulatory effect of activin on obsteoblast and osteoclast formation (361). Perrien et al (362) reported increased bone
mass and strength in an inducible transgenic mouse model
that overexpresses liver-derived human inhibin A. In this
model, inhibin also prevented gonadectomy-induced bone
loss in male mice, suggesting that inhibin may play a larger
role than sex steroids in regulating bone turnover.
Another line of evidence that supports an estrogen-independent mechanism of bone homeostasis during the
perimenopausal period comes from studies performed by
Sun et al (363). They showed that FSHR-deficient mice
had normal bone mass despite hypogonadism, with a concomitant elevation in circulating FSH levels. In FSHR
heterozygous mice, increased bone volume due to decreased bone-resorbing osteoclasts was observed in the
presence of normal ovarian function (363). These findings
suggest that FSH directly modulates bone metabolism independent of estrogen in vivo. Sun et al (363) also showed
that FSH-stimulated osteoclastogenesis is mediated
through FSHR, MEK/ERK, NF-␬B, and Akt signaling
pathways. Based on these findings, it is plausible that inhibin has an indirect effect on bone homeostasis, via regulation of FSH levels during the perimenopausal period.
Indeed, clinical studies investigating perimenopausal
changes in bone metabolism have demonstrated a correlation between inhibin B levels and bone mass (364). Increasing gonadotropin (FSH and LH) levels and decreasing inhibin B levels correlated with increasing bone
resorption. By contrast, estradiol levels were not significantly correlated with markers for either bone formation
or resorption (364). Multivariate analyses from a crosssectional, age-stratified study showed that serum inhibin
is a better predictor of bone turnover than either FSH or
estradiol during premenopause (365). In this study, Perrien et al (365) found that decreased serum inhibin levels
correlated with elevated markers of bone formation and
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769
resorption in pre- and perimenopausal women. Thus, it is
becoming clear that estrogen deficiency alone cannot fully
explain postmenopausal bone loss. The regulation of bone
metabolism during the perimenopause phase appears to be
a complex process, and FSH and inhibin might play a more
significant role than previously thought.
E. Adrenal gland growth and function
The physiological roles of both activin and inhibin in
adrenal gland development and function have been intensely investigated over the past two decades. The adrenal cortex and gonads share a common embryonic origin;
these are specific urogenital cells called adrenogonadal
primordium that characteristically stain for SF-1 (366 –
368). The presence of activin and inhibin (257, 369) along
with functional components of their signaling pathway
(ActRII, ActRIIB, ALK4, Smad2/3/4, and betaglycan) in
both the fetal and adult adrenal glands (370, 371) (Table
4) suggests a possible paracrine and/or autocrine role of
activin/inhibin in the regulation of adrenal function. The
role of inhibin in adrenal steroidogenesis is unclear, although inhibin A was unable to affect steroidogenesis in
human fetal adrenal cells (257). On the other hand, activins either stimulate (human fetal adrenal cells) (257) or
inhibit (bovine adrenal cells) (372) steroidogenesis. Activin A induces apoptosis in cultured adrenal cells whereas
inhibin A had no effect (370, 373). Adrenal glucocorticoids exert a clear negative feedback on ACTH secretion,
corticosterone also enhances pituitary FSH synthesis and
secretion in vivo or in vitro. Follistatin suppresses these
steroid-induced changes in FSH, indicating that activin is
involved in the positive feedback on FSH (374).
ACTH regulates the production of adrenal inhibin and
activin, and it has been postulated that these ligands, in
turn, may modulate ACTH-mediated adrenal steroidogenesis in an autocrine and/or paracrine manner (369,
370). Up-regulation of steroidogenic enzymes was observed after activin A treatment of the NCI-H295R adrenocortical carcinoma cell line, and this effect was attenuated by follistatin (371). Inhibin treatment also led to
up-regulation of the steroidogenic enzyme P450c17 (371).
In cultured fetal adrenal cells, activin A, but not inhibin,
treatment increased ACTH-stimulated cortisol production (257). Vanttinen et al (370) showed that activin A
inhibits steroidogenesis and stimulates apoptosis in NCIH295R cells, but inhibin does not affect either process.
The inconsistency of these study findings warrants further
investigation into the roles of activin and inhibin in adrenal steroidogenesis.
It was initially suggested that inhibin acts as a tumor
suppressor in the adrenal glands. In humans, inhibin immunoreactivity has been detected in adrenal cortical car-
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Makanji et al
Inhibin at 90, a Historical Review
cinomas and adenomas (375–377). In mice, the absence of
inhibin results in the development of malignant adrenal
tumors (240). Adrenal tumorigenesis in gonadectomized
inhibin-deficient mice is driven by LH-induced differentiation and proliferation of subcapsular adrenocortical
progenitor cells (378, 379). Gonadectomized inhibin
␣-subunit null mice develop adrenal cortical tumors by 40
weeks (females) or 70 weeks (males) of age. The fate of
these animals is similar to the intact null mice; mice succumb to cachectic wasting syndrome due to excess of activins secreted by the tumors (240). Disruption of the inhibin ␣-subunit gene results in a switch from Gata6 to
Gata4 expression in the adrenal gland due to high levels of
FSH (380). The inhibin ␣-subunit gene promoter contains
specific Gata4 binding sites, and ␣-subunit expression can
be directly driven by Gata4 (108). Gata4 expression is
gonad specific; expression in the adrenal gland results in
the expression of gonad-restricted genes and subsequent
tumor formation. In addition, the adrenal cortical tumors
express elevated levels of activin ␤A- and ␤B-subunit
(380). Furthermore, inhibin functionally antagonizes
TGF␤2 signaling in vitro (381). Both inhibin and TGF␤2
have high affinity for betaglycan, and each ligand induces
betaglycan internalization by a distinct mechanism (381).
Up-regulated TGF␤2 activity in adrenocortical tumors
may be due to betaglycan binding that is unopposed by
inhibin, suggesting an important role for inhibin in adrenal growth (381). Together, these studies suggest a tumor
suppressor role of the inhibin ␣-subunit in the adrenal
cortex.
F. Retinal development and vision
Little is known about the physiological role of retinal
inhibin, but limited evidence suggests that inhibin may be
involved in retinal development. The signaling mechanisms and progenitor cell differentiation processes that
give rise to the ordered cell type-specific laminar layers of
the retina are not well understood. In the developing
mouse eye, inhibin immunoreactivity is present in the lens
and in migrating cells between the lens and optic disc as
early as gestation day 10 (E10) (264). Inhibin is also expressed in various cell type-specific layers of the retina
between E16 and postnatal day 12 (P12); inhibin immunolocalization is apparent in the neuroblastic layer at E16
and in the ganglion and amacrine cells at birth, migrates
toward the inner layers of the retina, and concentrates at
the interphotoreceptor matrix by P12 (264). The distribution pattern of inhibin in the developing mouse retina
by P12 is similar to that of the adult mouse (264). BeleckyAdams et al (382) demonstrated that the developing retina
also expresses follistatin, ActRII, and ActRIIB, which are
primarily localized in the amacrine and ganglion cells be-
Endocrine Reviews, October 2014, 35(5):747–794
tween E8 and E18, with some immunoreactivity in the
outer nuclear layer and retinal pigmented epithelium. The
expression of inhibin mRNA and protein and its signaling
pathway components in the retina implies a possible paracrine and/or autocrine modulation of eye development by
inhibin.
In the adult rat eye, the inhibin ␣-subunit has been detected in the inner and outer segments of photoreceptor
cells, but not in the photoreceptor nuclei or in other retinal
cell types (258). In situ hybridization revealed the presence
of inhibin ␣-subunit mRNA in the cell bodies of photoreceptor cells (258). The physiological role of photoreceptor inhibin is presently unknown, but the expression of
inhibin in these cells could suggest a plausible paracrine
role in phototransduction and vision.
Finally, mice deficient in the ␤B-subunit have impaired
eyelid development (157, 158) and are born with their eyes
open; these mice show development of corneal hyperkeratinization and squamous metaplasia associated with
acute inflammation of all corneal layers and eyelids in the
first day of life (157). Although this phenotype suggests
that activin B, activin AB, and/or inhibin B are involved in
prenatal eyelid development during embryogenesis, inhibin-deficient mice have normal eyelid development, implying that activin, and not inhibin, is likely involved
(157).
G. Hematopoiesis
Hematopoiesis is the formation of the cellular components of blood arising from the myeloid (erythrocytes,
megakaryocytes, granulocytes, monocytes) and lymphoid
(T cells, B cells, natural killer cells) cell lineages. Bone
marrow is the primary source of multipotent hematopoietic stem and progenitor cells that give rise to mature blood
cells. The granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units (CFU-GEMM) are progenitor cells derived from the common myeloid progenitor
cells, and through a series of differentiation processes,
CFU-GEMM progenitor cells can be committed to become erythrocytes or megakaryocytes.
Both activin and inhibin have been reported to affect
human erythropoiesis in vitro. Activin was initially purified and characterized as a “erythroid differentiation factor” before being identified as a homodimer of inhibin
␤A-subunits (383, 384). Erythroid CFUs are derived from
the erythroid burst-forming units (BFU-E) after erythropoietin-stimulated differentiation. Spontaneous erythroid
differentiation of K562 human myelogenous leukemia
cells is reduced after inhibin treatment in culture (385).
Inhibin also suppresses activin-induced differentiation of
K562 cells (385) and human bone marrow cultures. At low
doses of erythropoietin, inhibin suppresses erythropoie-
doi: 10.1210/er.2014-1003
tin-induced erythroid CFU formation (385). Collectively,
these observations demonstrate an antagonistic role of inhibin in modulating erythroid progenitor cell differentiation (385).
Broxmeyer et al (386) also demonstrated an indirect
effect of activin A and inhibin A on hematopoietic processes in vitro. They showed that inhibin suppresses activin-stimulated BFU-E and CFU-GEMM colony formation in the presence of erythropoietin in a dose-dependent
manner in human bone marrow cultures (386). Removal
of monocytes and T-lymphocytes from culture abolished
the effect of inhibin and activin in these cells. The authors
speculated that activin and inhibin may manifest their respective stimulatory and inhibitory effects on these progenitor cells indirectly, through the release of growth factors from accessory cells. Interestingly, suppression of
colony formation by inhibin in granulocyte-macrophage
progenitor cells (CFU-GM) cells was not observed by this
group (386). This implies that the modulatory effect of
activin/inhibin might be restricted to the megakaryocyte
and erythrocyte developmental pathways. Contrary to
this observation, a myelosuppressive effect of inhibin in
vivo was reported by Hangoc et al (387). Decreased turnover of bone marrow and splenic CFU-GM, CFU-GEMM,
and BFU-E cells was noted after iv inhibin administration
in mice (387). Reduction in the number of bone marrow
CFU-GEMM cells, as well as splenic CFU-GM, CFUGEMM, and BFU-E cells, was reported after inhibin treatment, thus implying that the inhibitory effect of inhibin is
not confined to the erythropoietic developmental pathway
in vivo (387).
H. Placenta
1. Inhibins in the placenta and early pregnancy
From early pregnancy through to the third trimester,
inhibin ␣-subunit, ␤A- and ␤B-subunit mRNA levels increase to reach maximal levels in the third trimester (388).
At the end of the menstrual cycle, during decidualization,
and in early pregnancy, the inhibin ␣-subunit mRNA expression shifts from epithelial to stromal cells (389). Decidualized human endometrial cells in culture respond to
activin A treatment by elevating matrix metalloproteinase
2 secretion; inhibin A blocks this activin-mediated response (390). In addition, betaglycan mRNA is also upregulated in decidua of early pregnancy, correlating with
the increase in inhibin ␣-subunit mRNA secretion by stromal cells (391). Placentation is a complex process modulated by various factors. Inhibin A plays some important
roles in placentation and in pregnancy. In contrast to low
levels of inhibin A produced by the nonpregnant uterus,
the syncytiotrophoblast cells of the placenta abundantly
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produce inhibin A (26) along with betaglycan (391, 392).
In the placenta, inhibin is a potent antagonist of activinmediated steroidogenesis and hCG production by syncytiotrophoblasts (390, 393). Throughout gestation, levels
of activin A and inhibin A continue to rise until parturition
(394). The precise roles of inhibins in the placenta are not
understood; however, in several placental diseases inhibin
A is implicated. Wallace et al (395) measured the levels of
inhibin A and activin A in normal and failed pregnancy;
they found that women who had miscarried had approximately 2-fold lower levels of serum inhibin A compared
to controls, whereas activin A levels were not affected.
Further investigation revealed that the inhibin ␣-subunit
mRNA expression was not altered; however, there was a
reduction in placental mass (396). Elevated levels of serum
inhibin A in the second trimester of pregnancy are indicative of fetal Down syndrome and are utilized as a marker
for this disease in combination with other factors, eg hCG
and ␣-fetoprotein (AFP) (397, 398). Similarly, increased
secretion of serum inhibin A and activin A has also been
reported for women with pre-eclampsia (399) due to elevated expression of inhibin ␣-subunit and activin ␤A-subunit mRNA in the placenta (400). Studies have shown that
inclusion of the measurement of maternal serum inhibin A
and activin A along with other tests in the second trimester
of pregnancy may improve the predictive efficacy of earlyonset pre-eclampsia screening (401– 404).
I. Branching morphogenesis
During development, epithelial tissues of glandular organs undergo complex transformation to form branched
tubular networks. Initially, these processes are tightly controlled by the genetic blueprint, and later they become
more stochastic, taking cues from their surroundings. Salivary glands, mammary glands, lungs, pancreas, kidneys,
and prostate among others undergo branching morphogenesis during organogenesis (for reviews, see Refs. 405
and 406). The role of activin during organogenesis has
been described in these tissues and organs (see review in
Ref. 407). However, the role of inhibins on the actions of
activins during these early development processes has not
been explored.
IV. Clinical Applications of Inhibin
The role of inhibin in modulating pituitary FSH secretion
in reproduction is well established in both males and females. Consequently, inhibin has been evaluated and applied in the clinical setting for the assessment of fertility,
pregnancy-related conditions, and reproductive function.
Research efforts are assessing the potential clinical use of
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serum inhibin levels as a biochemical marker in the diagnosis and/or monitoring of nonviable pregnancies, ectopic
pregnancies, and pre-eclampsia (Table 5). Currently, inhibin is being used as a diagnostic marker as part of the
quadruple test in antenatal screening for Down syndrome.
In this section, we will highlight the various clinical applications and potential diagnostic or prognostic applications for which inhibin is currently being evaluated.
A. Early pregnancy viability
Spontaneous abortion is the most common complication of early pregnancy. The incidence of spontaneous
abortions is highest during the first 12 weeks of gestation
but decreases with increasing fetal gestational age. Loss of
subclinical pregnancies and clinical pregnancies accounts
for 22 and 31% of all pregnancies, respectively (408).
Increased risk of early pregnancy loss is associated with
advanced maternal age, high consumption of alcohol and
caffeine, heavy smoking, prior history of pregnancy loss,
and low plasma folate, among others.
Maternal serum inhibin A progressively increases over
the course of pregnancy, remaining relatively low during
the first and second trimesters, then rising dramatically
during the third trimester (394, 409). Similar patterns are
also observed in pregnant rats (410). Maternal serum activin levels also progressively increase during pregnancy,
almost 10-fold by term (409), and are even higher in
women with pre-eclampsia (411), preterm labor, and gestational diabetes (412). In addition, activin levels are regulated in a pulsatile manner, and the pulse amplitude is
higher in gestational diabetes and preterm labor (413).
Chorionic and amniotic ActRIIB mRNA levels were also
Table 5. Summary of Changes in Serum Inhibin and
Activin in Various Diseases and Conditions
Clinical Condition
Pregnancy
Early spontaneous abortion/
pregnancy loss
Ectopic pregnancy
Prenatal diagnosis of fetal
Down syndrome
Pre-eclampsia
Normal/preterm labor
Infertility
Declining ovarian reserve
Impaired spermatogenesis
Cancer
Cancer cachexia
Bone loss
Anemia
Chemotherapy
Amenorrhea in early-stage
breast cancer
Serum Inhibin
A/Inhibin B
Serum Activin
A/Activin B
Decreased
Decreased
Increased
Increased
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elevated in preterm labor (414). Serum follistatin, either
free or bound to activin/inhibin, is also elevated in late
pregnancy with a high activin:follistatin ratio, implying
an important biological role for activin at term (415).
Radiolabeled activin A was localized to the endometrium/myometrium in pregnant rats (416). Together, these
data suggest that inhibins and activins are dynamically
controlled throughout pregnancy and are biologically
relevant.
The primary source of inhibin during early stages of
pregnancy is not clear. The placenta (25, 417), fetal membranes (418), fetoplacental unit (419), and the corpus luteum (420) have all been suggested as possible candidates.
During the later stages of pregnancy, the rapidly expanding fetoplacental mass appears to be the major source of
inhibin. The physiological functions of inhibin during
pregnancy and the mechanisms underlying changes in inhibin levels in pregnancy-associated conditions are not
well understood. In a pilot study, Muttukrishna et al (421)
found a significant decrease in maternal serum inhibin A
levels in cases of spontaneous abortion. Serum inhibin A
was also considerably lower in women with previous obstetric history of pregnancy loss (421). The authors proposed that inhibin A, in addition to hCG levels, may be
useful in predicting spontaneous abortion as early as week
6 of gestation in patients with a history of recurring early
pregnancy loss.
Inhibin A levels have also been investigated as a predictor of pregnancy viability in women undergoing IVF
(422). Increased probability of pregnancy loss has been
associated with lower serum inhibin A after IVF (419,
422). Another group showed a trend toward lower levels
of inhibin A in early IVF pregnancies that resulted in spontaneous abortions compared with ongoing pregnancies,
although differences in serum inhibin levels in this study
did not reach statistical significance (420). Inconsistencies
in the results of studies of inhibin in IVF outcomes could
be attributed to small sample size, differences in ovarian
stimulation protocols, and serum sampling times (420).
Nevertheless, the authors of these studies proposed that
serum inhibin A levels might be a useful biochemical
marker of subclinical pregnancies (420) and spontaneous
abortions (422).
Increased
B. Ectopic pregnancy
Decreased
Decreased
Increased
Increased
Increased
Decreased
Ectopic pregnancy occurs when the developing gestational sac is implanted at extrauterine sites, such as the
fallopian tubes. Ectopic implantation occurs in approximately 2% of pregnancies in the United States (423) and,
if left undetected, can lead to potentially life-threatening
tubal rupture. Ectopic pregnancies account for approximately 14% of pregnancy-related mortality in the first
doi: 10.1210/er.2014-1003
trimester (423). Women with a previous history of ectopic
pregnancy or who have disrupted tubal structure due to
surgery, infection, tumors, or congenital abnormalities are
at higher risk of having an ectopic pregnancy. Presently,
transvaginal ultrasounds with serial serum hCG monitoring are used to diagnose ectopic pregnancy.
The mechanism of embryo transport along the fallopian tubes to successful implantation onto the uterine endometrial wall is not well understood. Decreased maternal
serum inhibin has been reported in ectopic pregnancies
when compared to normal intrauterine pregnancies (424).
Microarray analysis of the decidualized endometrium
demonstrated decreased expression of inhibin ␤B-subunit
in tubal ectopic pregnancies compared to normal intrauterine pregnancies (425). Less advanced endometrial
stromal decidualization in ectopic pregnancies is associated with decreased prolactin and IGF binding protein
(IGFBP)-1 expression, as well as lower serum activin B
concentrations (425). A prospective case-control study
also showed that patients with ectopic pregnancies have
significantly lower serum inhibin A levels compared to
women with normal pregnancies (426). Immunohistochemistry of fallopian tube samples from ectopic pregnancies revealed elevated expression of the ␤A- and
␤B-subunits, ActRII, ActRIIB, and follistatin (427). Collectively, these findings imply a possible role of the inhibins and activins in embryonic transport and/or attachment
to the endometrial wall; however, further research is
needed to gain a better understanding of the roles of activin and inhibin in early pregnancy outcomes.
C. Down syndrome
Down syndrome is caused by trisomy of chromosome
21 and is characterized in part by short stature, characteristic facial features, intellectual disability, and hearing
and vision impairment. The risk of fetal Down syndrome
is highest among pregnant women over 35 years of age,
affecting about 52% of pregnancies between 2000 and
2004 (428). Between 1985 and 2004, Down syndrome
affected 1.72 in 1000 total births (428).
Prenatal screening for Down syndrome in the first trimester includes maternal serum analysis of pregnancyassociated plasma protein A and hCG, which are interpreted along with risk related to maternal age (429).
Ultrasound is also performed to determine fetal age and
nuchal translucency measurements, the most widely used
and reliable marker for detection of trisomy 21 (429). To
confirm a positive screen, amniocentesis and/or chorionic
villus sampling are performed for fetal karyotyping (430).
Both the serum and ultrasound analyses carry a high likelihood of false positives, leading to invasive sampling procedures that carry an elevated risk of pregnancy loss of a
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potentially normal fetus. Improving specificity and reliability of existing noninvasive screening procedures to reduce the false-positive rate will be beneficial in lowering
costs and maternal morbidity associated with invasive
procedures.
The association between elevated maternal serum inhibin and Down syndrome pregnancies was initially reported in 1992 (431) and was subsequently confirmed by
other investigators (432– 435). Inhibin A has since become
a useful biochemical marker in predicting the risk of fetal
chromosomal abnormalities and has been incorporated as
a fourth marker in the quadruple screening test performed
at 15–18 weeks of gestation (436). Test specificity increased when inhibin A testing was added to the conventional triple test—for hCG, AFP, and unconjugated estriol
(uE3)— used to predict the risk of Down syndrome. Wald
et al (437) reported that when these four serum markers
were evaluated in combination with maternal age, 70% of
fetal Down syndrome cases were accurately detected,
compared to 59% using the conventional triple test in the
same sample population. On average, serum AFP (438)
and uE3 (439) are lower and serum hCG and inhibin A are
elevated in the second trimester of Down syndrome pregnancies (436). Term placentas from mothers with Down
syndrome pregnancies had significantly reduced levels of
inhibin ␣-subunit repressors, NR5A2 and WT1 (440).
This may be one of the mechanisms that explains elevated
levels of maternal serum inhibin A.
D. Pre-eclampsia
A multiple-biomarker diagnostic approach that includes inhibin is also being considered for screening of
pre-eclampsia, a condition characterized by the onset
of high blood pressure and proteinuria after 20 weeks of
pregnancy in previously normotensive women. Pre-eclampsia affects approximately 5–10% of nulliparous
pregnancies (441). Although pre-eclampsia can be reversed with the induction of labor, the decision to induce
premature labor requires careful evaluation of fetal maturity and maternal morbidity. Currently, there are numerous biochemical markers that can be employed for the
screening of pre-eclampsia, but none of these have accurately predicted disease onset with a high degree of specificity and sensitivity (442).
Serum inhibin A is elevated in women with pre-eclampsia compared to women with a normal pregnancy, when
matched for duration of gestation, parity, and maternal
age (443). Elevated maternal serum inhibin A correlates
with both onset and severity of pre-eclampsia (444). The
cellular mechanisms underlying increased maternal inhibin A levels in pre-eclampsia are unclear, but it has been
suggested that elevated serum inhibin reflects trophoblast
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viability. In addition to established pre-eclampsia, Muttukrishna et al (445) found that serum inhibin A was
higher in asymptomatic patients at risk of developing preeclampsia. Increased serum inhibin A levels were observed
in women who went on to develop early-onset pre-eclampsia, term pre-eclampsia, gestational hypertension, or
pre-eclampsia delivery at 34 –36 weeks. It has been noted
that maternal serum inhibin alone may not be a sufficient
prognostic marker in screening for pre-eclampsia risk;
however, serum inhibin A in conjunction with endoglin
and placental growth factor during the second trimester
are highly predictive for early-onset pre-eclampsia
(446). Placental growth factor is a member of the vascular endothelial growth factor family and promotes
angiogenesis during embryogenesis, and endoglin is an
antiangiogenic factor. Marked elevations in circulating
endoglin are seen before and at the onset of pre-eclampsia (447). Although uE3 and AFP are not known to be
predictive for pre-eclampsia, mothers who are screened
for fetal Down syndrome using the quadruple test could
also be assessed for pre-eclampsia risk with little to no
increase in cost.
E. Ovarian reserve
Assisted reproductive technology (ART) is defined by
the Centers for Disease Control and Prevention as technology used to treat infertility in which the ovum and
sperm are manipulated ex vivo (448). This includes technologies such as IVF, intracytoplasmic sperm injection,
and other related techniques. In 2005, approximately 1%
of infants born in the United States were conceived
through ART (448). The pregnancy rate after IVF was
approximately 42% in 2005 (448) and is, in part, determined by the ovarian reserve, a measure of oocyte quality
and quantity. Ovarian reserve determines the ability of the
ovaries to produce ova that are capable of fertilization and
result in a successful pregnancy.
A repertoire of tests is currently available for the assessment of ovarian reserve in women undergoing ART.
Numerous hormone markers (FSH, estradiol, progesterone, inhibin, anti-Müllerian hormone [AMH]), ultrasound parameters (antral follicle count, ovarian volume),
and dynamic testing protocols (clomiphene citrate challenge, exogenous FSH ovarian reserve test [Effort], GnRH
agonist stimulation test) are employed, often in combination, to accurately estimate ovarian reserve (449). Reduced ovarian function has been associated with increasing age, missed menstrual cycles, increased serum FSH,
low baseline estradiol, decreased inhibins, elevated
progesterone, decreased AMH, decreased antral follicle
count, and low ovarian volume (449).
Endocrine Reviews, October 2014, 35(5):747–794
How does inhibin level relate to ovarian reserve and
predict a successful pregnancy after ART? The granulosa
cells of growing ovarian follicles release inhibins; as follicles progress to the antral stage, the population of inhibin-producing granulosa cells increases. Thus, inhibin
levels provide a good surrogate measure of follicle health
and viability. In women with declining ovarian reserve,
reduced serum inhibin B levels at day 3 of the cycle are
observed in conjunction with a low FSH concentration
(450). Conversely, high levels of follicular fluid inhibin A
and inhibin B levels correlate with increased pregnancy
rate and better ovarian response in women undergoing
hormonal stimulation for oocyte retrieval in IVF (451).
Consistent with this observation, Seifer et al (452) noted
that women with low serum inhibin B at day 3 of the cycle
had a poorer response to ovulation stimulation and, consequently, were less likely to become pregnant after ART
when compared with women who had high day 3 serum
inhibin concentrations.
Others have shown that maternal age and basal FSH are
better predictors of pregnancy success than inhibin B levels
after the first IVF/intracytoplasmic sperm injection treatment cycle (453). Information is still emerging about the
possible clinical applications of serum inhibin as an ancillary marker of ovarian reserve and predictor of ART
outcomes. There is strong evidence that supports an association between low inhibin levels and decreased fertility. It may be useful to consider using a panel of serum
AMH, FSH, and inhibin measurements during the follicular phase of the menstrual cycle to gauge ovarian reserve
(454, 455). Serum AMH is more widely used to measure
ovarian reserve (for review, see Ref. 456).
F. Polycystic ovarian syndrome (PCOS)
PCOS is characterized by irregular menses, hyperandrogenism, polycystic ovarian morphology, and arrested
folliculogenesis manifested as an elevated number of small
antral follicles on ultrasound. Initial studies reported elevated total serum inhibins in women with PCOS (457,
458); however, studies utilizing specific inhibin A (459,
460) and inhibin B (460, 461) ELISAs have shown that this
is not the case. In fact, Welt et al (462) showed that levels
of inhibin A and B are significantly reduced in the follicular
fluid of women with PCOS, and inhibin ␣-subunit and
␤A-subunit mRNA were lower in arrested follicles from
women with PCOS (463). Nevertheless, a handful of studies continue to report elevated serum inhibin A (464) and
inhibin B (464, 465), as well as slightly lower activin A
levels and higher follistatin levels in women with PCOS
compared to controls (466). Tsigkou et al (467) reported
that whereas total serum inhibins are elevated, inhibin A
is only slightly elevated, and inhibin B levels are unaffected
doi: 10.1210/er.2014-1003
in women with PCOS. Overall, these studies highlight the
need for further investigation into the regulation of inhibin
and related ligands in women with PCOS to better understand disease etiology.
G. Premature ovarian failure
POF is a condition associated with amenorrhea, infertility, hypoestrogenism, and elevated gonadotropin concentrations in women under the age of 40 years, and it
occurs in 1% of all women (468). Women with POF have
elevated levels of serum FSH and reduced levels of serum
estradiol. In these women, serum inhibin A and B levels are
also reduced, which explains the observed elevation in
FSH (469).
Several studies have reported a significant association
of the INHA 769G⬎A (Ala257Thr) missense mutation in
the inhibin ␣-subunit gene in women with POF (468, 470,
471). The most recent report by Dixit et al (472) demonstrated the presence of this variant in 10.5% of cases of
POF, 10% of cases of primary amenorrhea, and 0.5% of
controls. To investigate the effect of this mutation on inhibin biological activity, L␤T2 gonadotrope cells were
cotransfected with an activin-sensitive luciferase reporter
construct (p3xGRAS) and increasing amounts of either
wild-type or mutant ␣-subunit. Transfection efficiency
was assessed by the measurement of secreted inhibin B
(L␤T2 cells express inhibin ␤B but not ␤A). The transfection of wild-type inhibin ␣-subunit resulted in a dose-dependent decrease in activin-induced p3xGRAS-Luc activity. In contrast, activin-induced p3xGRAS-Luc activity
was unaffected by transfection of increasing doses of inhibin ␣-subunit mutant DNA, indicating that inhibin B
(Ala257Thr) has compromised biological activity (473).
Subsequent transfection studies in the ovarian tumor cell
line COV434 suggest that the A257T inhibin ␣-subunit
mutation significantly blunts the effect of inhibin A on
activin-induced reporter activity (473). These preliminary
results indicate that Ala257 and/or juxtaposed residues
are important for inhibin B activity and further highlight
the mechanistic differences between the inhibin isoforms.
H. Ovarian cancer
Ovarian cancer is the most common fatal malignancy in
women and is usually not detected until the end stage of the
disease. Ovarian cancers are classified as epithelial, stromal sex cord, or germ cell cancer. Epithelial cancers make
up 90% of all ovarian cancers and are further characterized as serous (70%), mucinous (10 –15%), endometrioid
(10 –15%), or other subtypes. Granulosa cell tumors present in only 5% of cases (474). Inhibins have been implicated in the pathogenesis of ovarian cancer (55, 475). Inhibin B is the major form of inhibin secreted by granulosa
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cell tumors (14, 476, 477). Lappöhn et al (478) have
shown that total inhibin levels are elevated in postmenopausal women with granulosa cell tumors. Furthermore,
serum inhibins are elevated in women with mucinous epithelial cancers (479). Preoperative serum inhibin A levels
can be prognostic for postmenopausal women with epithelial ovarian cancers, independent of age or stage (480).
Women with serous cancers have elevated serum, cystic,
and peritoneal fluid activin A and may be involved in tumor progression. Analysis of downstream signaling genes
from cancer tissues from these patients suggests that activins may promote Akt and suppress glycogen synthase
kinase signaling to promote tumor proliferation (481).
Despite the rapid onset of ovarian tumors in the inhibin
␣-subunit null female mouse, the molecular mechanisms
underlying disease etiology are not understood (239).
Hempel et al (482) reported the loss of betaglycan mRNA
and protein in epithelial-derived ovarian cancer cell lines,
suggesting that a consequent loss of inhibin activity may
promote tumorigenesis. Reintroduction of betaglycan
into these cells reduced cancer cell motility and invasiveness by specifically enhancing inhibin-mediated suppression of matrix metalloproteinases (482).
Advances in assay methodology have resulted in the
development of assays capable of detecting various types
of ovarian cancer in postmenopausal women. Due to the
very low levels of inhibins in postmenopausal women,
inhibin-producing tumors can be accurately identified;
however, inhibin is less effective as a marker of ovarian
cancer in premenopausal women due the cyclic nature and
higher levels of inhibin secretion (474). A dual assay system that detects the inhibin ␣-subunit (inhibin ␣C-subunit
ELISA) (69) and CA125, an ovarian tumor marker (483),
provides a specific and sensitive detection system for most
ovarian cancers (14, 474).
I. Breast cancer
Activins and inhibins have been implicated in the progression and diagnosis of breast cancer. Activins maintain
cell cycle arrest in breast cancer cell lines via Smad2/3
mediated expression of cyclin-dependent kinase inhibitors, p21 and p27, and increase expression of p15 and
reduced phosphorylation of retinoblastoma protein
(484). Activin and estrogen cross talk in breast cancer cells
is important for cell cycle control in these cells. Activins
reduce the expression of estrogen-induced trefoil factor 1
mRNA expression, and estrogen-blocked activin induced
Smad3 signaling and reduced activin B mRNA and protein
levels (484). In low-grade breast cancer, activin signaling
components (␤-subunit, type II and I receptors, and
Smads) are maintained, whereas in high-grade breast cancer, these signaling components are diminished (484).
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Therefore, a change in activin signaling is tied with the
progress of the disease.
Serum activin A is elevated in women with breast cancer
(485), whereas serum inhibin is not significantly elevated
(69). However, women with breast cancer that underwent
surgical treatment had a decrease in serum inhibin levels,
suggesting that these tumors may be secreting inhibins
(486). A subset of postmenopausal women with breast
cancer are at risk of developing chemotherapy-related
amenorrhea—reduced serum inhibin B and AMH can be
predictive in these women (487). In postmenopausal
women, where inhibin levels are already very low, changes
in serum inhibins along with other markers can be used to
diagnose breast cancer.
J. Menopause
Menopause is defined as the complete absence of menstruation due to loss of ovarian follicular activity. The
period approaching menopause is referred to as the menopause transition, perimenopause, or climacteric period.
The menopause transition is a dynamic process involving
several factors that ultimately culminates in menopause
(488 – 490). To standardize the reproductive staging process, researchers and clinicians adopted the Stages of Reproductive Aging Workshop (STRAW) system (491).
During menopause transition, the pool of follicles is
depleted, inhibin levels are drastically reduced, FSH levels
are elevated, estrogen and androgen levels are reduced,
and the menstrual cycle becomes irregular with variable
cycle lengths (488 – 490). The most characteristic feature
of reproductive aging, evident throughout the reproductive life of women, is increasing FSH levels. As women age,
ovarian follicle numbers decline, resulting in a drop in
inhibin B levels, loss of negative feedback to the pituitary,
and elevation of FSH levels (490, 492, 493). In older cycling women, elevated FSH was correlated to diminished
serum inhibin B and not inhibin A, activin A, or estradiol
(494, 495). Estradiol also negatively regulates FSH; the
decrease in estradiol levels with depletion of the follicle
pool may also contribute to elevated FSH in the menopause transition (490, 493). However, estradiol is poorly
correlated to FSH levels and more likely sets an overall
inhibitory tone across the cycle (495, 496). Follicular fluid
from perimenopausal women with normal cycles had normal levels of steroids, inhibin A and B, IGF-II, IGFBP-2,
and IGFBP-3; increased concentrations of follistatin, activin A, and vascular endothelial growth factor; and decreased concentrations of IGF-I (497). Others have suggested that activins also contribute to the rising levels of
FSH in perimenopause; in aging women, serum activin A
levels are elevated on day 6 whereas follistatin levels are
unaffected (498). Interestingly, the luteal phase is also af-
Endocrine Reviews, October 2014, 35(5):747–794
fected by reproductive aging, marked by stable progesterone levels but declining inhibin A levels, suggesting that
separate mechanisms regulate the corpus luteum during
the aging process (454, 490, 499).
Burger and colleagues (360, 500, 501) reclassified the
menopause transition based on the STRAW staging system: group 1, women with normal cycles; group 2, women
with irregular bleeding (bleeding once in the last 3
months); group 3, women with highly irregular bleeding
(bleeding at least once in the last year but not once in the
last 3 months); and group 4, absence of bleeding for 12
months (after menopause). FSH levels increase across the
four groups, estradiol and inhibin A levels only decrease in
groups 3 and 4, and inhibin B levels decrease between
groups 1 and 2 (490, 500). Together, these studies show
that the rise in serum FSH in aging women is primarily a
result of the declining serum inhibin B secondary to the
loss of growing follicles; there is a subsequent reduction in
inhibin A and estradiol levels as folliculogenesis ceases.
K. Male reproductive function
Inhibin B is the biologically active form of inhibin in
men, and circulating levels of inhibin B and FSH are inversely correlated in healthy and subfertile individuals
(322). Both inhibin B subunits (␣ and ␤B) are expressed in
Sertoli cells and Leydig cells (502), and although inhibin B
dimers are produced by Sertoli cells (503), germ cells
(502), and Leydig cells, there is general agreement that the
Sertoli cells generate most circulating inhibin B levels in
men.
Serum inhibin B has been investigated as a potential
biochemical marker of male fertility. Serum inhibin B
levels may be a direct marker of Sertoli cell function and
an indirect marker of spermatogenesis (504). Studies
over the last decade have demonstrated a strong correlation between spermatogenesis and serum inhibin B
concentrations in men (335, 336, 506). In contrast to
men with normospermia, significantly lower levels of
serum inhibin B are observed in patients with conditions
associated with impaired spermatogenesis, such as
severe oligozoospermia, idiopathic azoospermia, Klinefelter syndrome, and cryptorchidism (335). Serum inhibin B also correlates with sperm concentration in men
with normal (336) and impaired (507) spermatogenesis.
Serum inhibin B concentration positively correlates
with sperm count (336, 507) and testicular volume
(335). In a large, multicenter, cross-sectional study of
nearly 1800 fertile men, a positive correlation was
found between sperm count, sperm concentration, and
serum inhibin B levels, particularly at times during the
day when inhibin levels are relatively low (508). As in
females, serum inhibin B and serum FSH are inversely
doi: 10.1210/er.2014-1003
correlated (335, 336, 507). Collectively, these observations strongly support the potential clinical application
of serum inhibin B as a marker of spermatogenic function. Of note, in patients with nonobstructive azoospermia who underwent testicular sperm extraction, serum
inhibin B levels demonstrated a diagnostic sensitivity of
0.65 and a specificity of 0.85, suggesting that inhibin B
on its own is not a reliable marker for the presence of
sperm (504). Consistent with this finding, Mitchell et al
(509) also showed that seminal AMH and inhibin B
levels were not predictive for successful sperm recovery.
More research is needed to more accurately assess the
predictive value of inhibin B as a marker of fertility in
men.
V. Conclusion
Inhibin is a major hormone in reproductive biology, secreted primarily by testicular Sertoli cells and ovarian
granulosa cells. Its role in negative feedback regulation of
pituitary FSH production is well established, as is its mechanism of action as an antagonist of activin signaling via
activin receptors and betaglycan. In the 90 years since its
discovery, it is becoming increasingly clear that inhibin has
actions outside the reproductive axis. Inhibin subunits and
dimers are expressed in many organ systems, and researchers will continue to investigate the physiological functions
of extragonadal inhibins. As one of the major hormones
that regulate folliculogenesis, inhibin has great potential
to be exploited and used as a diagnostic marker in the
assessment and management of infertility- and pregnancyrelated conditions. Ongoing research is rapidly filling the
gaps in our understanding and knowledge of inhibin
physiology.
Acknowledgments
The authors dedicate this review to the memory of Prof. Wylie Vale, a
long-time friend, mentor, and champion of the field of inhibin biology.
The authors thank Mr. Andrew Russell for collating the information
in Table 1. The authors also thank the National Institutes of Health and
Australian National Health and Medical Research Council for funding
support.
Address requests for reprints to: Teresa K. Woodruff, PhD, Department of Obstetrics and Gynecology, Feinberg School of Medicine,
Northwestern University, 303 East Superior Street, Lurie Building 10121, Chicago, IL 60611. E-mail: [email protected].
The study was funded by National Institutes of Health Grants
R01HD037096 (to T.K.W.) and P01HD021921 (to K.E.M.) from the
Eunice Kennedy Shriver National Institute of Child Health and Human
Development and the Australian National Health and Medical Research
Council Grant GNT1016460 (to Y.M.).
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Editorial assistance was provided by Dr. Stacey C. Tobin. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
Disclosure Summary: The authors have nothing to disclose.
References
1. McCullagh DR. Dual endocrine activity of the testes. Science. 1932;76:19 –20.
2. Mottram JC, Cramer W. On the general effects of exposure
to radium on metabolism and tumour growth in the rat and
the special effects on testis and pituitary. J Exp Physiol.
1923;13:209 –229.
3. Schwartz NB, Channing CP. Evidence for ovarian “inhibin”: suppression of the secondary rise in serum follicle
stimulating hormone levels in proestrous rats by injection
of porcine follicular fluid. Proc Natl Acad Sci USA. 1977;
74:5721–5724.
4. Keogh EJ, Lee VW, Rennie GC, Burger HG, Hudson B, De
Kretser DM. Selective suppression of FSH by testicular extracts. Endocrinology. 1976;98:997–1004.
5. Robertson DM, Foulds LM, Leversha L, et al. Isolation of
inhibin from bovine follicular fluid. Biochem Biophys Res
Commun. 1985;126:220 –226.
6. Rivier J, Spiess J, McClintock R, Vaughan J, Vale W. Purification and partial characterization of inhibin from porcine follicular fluid. Biochem Biophys Res Commun. 1985;
133:120 –127.
7. Ling N, Ying SY, Ueno N, Esch F, Denoroy L, Guillemin R.
Isolation and partial characterization of a Mr 32,000 protein with inhibin activity from porcine follicular fluid. Proc
Natl Acad Sci USA. 1985;82:7217–7221.
8. Miyamoto K, Hasegawa Y, Fukuda M, et al. Isolation of
porcine follicular fluid inhibin of 32K daltons. Biochem
Biophys Res Commun. 1985;129:396 – 403.
9. Esch FS, Shimasaki S, Cooksey K, et al. Complementary
deoxyribonucleic acid (cDNA) cloning and DNA sequence
analysis of rat ovarian inhibins. Mol Endocrinol. 1987;1:
388 –396.
10. Forage RG, Ring JM, Brown RW, et al. Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc Natl
Acad Sci USA. 1986;83:3091–3095.
11. Mason AJ, Hayflick JS, Ling N, et al. Complementary
DNA sequences of ovarian follicular fluid inhibin show
precursor structure and homology with transforming
growth factor-␤. Nature. 1985;318:659 – 663.
12. Mayo KE, Cerelli GM, Spiess J, et al. Inhibin A-subunit
cDNAs from porcine ovary and human placenta. Proc Natl
Acad Sci USA. 1986;83:5849 –5853.
13. Woodruff TK, Meunier H, Jones PB, Hsueh AJ, Mayo KE.
Rat inhibin: molecular cloning of ␣- and ␤-subunit complementary deoxyribonucleic acids and expression in the
ovary. Mol Endocrinol. 1987;1:561–568.
14. Burger HG, Fuller PJ, Chu S, et al. The inhibins and ovarian
cancer. Mol Cell Endocrinol. 2001;180:145–148.
15. de Kretser DM, Hedger MP, Loveland KL, Phillips DJ.
Inhibins, activins and follistatin in reproduction. Hum Reprod Update. 2002;8:529 –541.
16. de Kretser DM, Loveland KL, Meehan T, O’Bryan MK,
778
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Makanji et al
Inhibin at 90, a Historical Review
Phillips DJ, Wreford NG. Inhibins, activins and follistatin:
actions on the testis. Mol Cell Endocrinol. 2001;180:
87–92.
Hillier SG, Miró F. Inhibin, activin, and follistatin. Potential roles in ovarian physiology. Ann N Y Acad Sci. 1993;
687:29 –38.
Jaffe RB, Spencer SJ, Rabinovici J. Activins and inhibins:
gonadal peptides during prenatal development and adult
life. Ann N Y Acad Sci. 1993;687:1–9.
Lambert-Messerlian GM, Pinar H, Laprade E, Tantravahi
U, Schneyer A, Canick JA. Inhibins and activins in human
fetal abnormalities. Mol Cell Endocrinol. 2004;225:101–
108.
Makanji Y, Harrison CA, Robertson DM. Feedback regulation by inhibins A and B of the pituitary secretion of
follicle-stimulating hormone. Vitam Horm. 2011;85:299 –
321.
Mather JP, Woodruff TK, Krummen LA. Paracrine regulation of reproductive function by inhibin and activin. Proc
Soc Exp Biol Med. 1992;201:1–15.
Matzuk MM, Kumar TR, Shou W, et al. Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development. Recent Prog Horm
Res. 1996;51:123–154; discussion 155–127.
Mayo KE. Inhibin and activin molecular aspects of regulation and function. Trends Endocrinol Metab. 1994;5:
407– 415.
McNeilly AS. Diagnostic applications for inhibin and activins. Mol Cell Endocrinol. 2012;359:121–125.
Petraglia F, Garuti GC, Calza L, et al. Inhibin subunits in
human placenta: localization and messenger ribonucleic
acid levels during pregnancy. Am J Obstet Gynecol. 1991;
165:750 –758.
Qu J, Thomas K. Inhibin and activin production in human
placenta. Endocr Rev. 1995;16:485–507.
Robertson DM. Inhibins and activins in blood: predictors
of female reproductive health? Mol Cell Endocrinol. 2012;
359:78 – 84.
De Jong FH, Sharpe RM. Evidence for inhibin-like activity
in bovine follicular fluid. Nature. 1976;263:71–72.
Setchell BP, Jacks F. Inhibin-like activity in rete testis fluid.
J Endocrinol. 1974;62:675– 676.
Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP.
Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology. 1990;127:3196 –3205.
Hsueh AJ, Dahl KD, Vaughan J, et al. Heterodimers and
homodimers of inhibin subunits have different paracrine
action in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Proc Natl Acad Sci USA.
1987;84:5082–5086.
Baly DL, Allison DE, Krummen LA, et al. Development of
a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.
Endocrinology. 1993;132:2099 –2108.
Betteridge A, Craven RP. A two-site enzyme-linked immunosorbent assay for inhibin. Biol Reprod. 1991;45:748 –
754.
Wong WL, Garg SJ, Woodruff T, Bald L, Fendly B,
Lofgren JA. Monoclonal antibody based ELISAs for measurement of activins in biological fluids. J Immunol Methods. 1993;165:1–10.
Endocrine Reviews, October 2014, 35(5):747–794
35. Woodruff T, Krummen L, Baly D, et al. Quantitative twosite enzyme-linked immunosorbent assays for inhibin A,
activin A and activin B. Hum Reprod. 1993;8(suppl 2):
133–137.
36. Chari S, Duraiswami S, Franchimont P. A convenient and
rapid bioassay for inhibin. Horm Res. 1976;7:129 –137.
37. Nandini SG, Lipner H, Moudgal NR. A model system for
studying inhibin. Endocrinology. 1976;98:1460 –1465.
38. Ramasharma K, Murthy HM, Moudgal NR. A rapid bioassay for measuring inhibin activity. Biol Reprod. 1979;
20:831– 835.
39. de Jong FH, Smith SD, van der Molen HJ. Bioassay of
inhibin-like activity using pituitary cells in vitro. J Endocrinol. 1979;80:91–102.
40. Eddie LW, Baker HW, Higginson RE, Hudson B. A bioassay for inhibin using pituitary cell cultures. J Endocrinol.
1979;81:49 – 60.
41. Hudson B, Baker HW, Eddie LW, et al. Bioassays for inhibin: a critical review. J Reprod Fertil Suppl. 1979;26:
17–29.
42. Scott RS, Burger HG, Quigg H. A simple and rapid in vitro
bioassay for inhibin. Endocrinology. 1980;107:1536 –
1542.
43. Leversha LJ, Robertson DM, de Vos FL, et al. Isolation of
inhibin from ovine follicular fluid. J Endocrinol. 1987;113:
213–221.
44. Good TE, Weber PS, Ireland JL, et al. Isolation of nine
different biologically and immunologically active molecular variants of bovine follicular inhibin. Biol Reprod. 1995;
53:1478 –1488.
45. Ireland JL, Good TE, Knight PG, Ireland JJ. Alterations in
amounts of different forms of inhibin during follicular atresia. Biol Reprod. 1994;50:1265–1276.
46. Knight PG, Beard AJ, Wrathall JH, Castillo RJ. Evidence
that the bovine ovary secretes large amounts of monomeric
inhibin ␣ subunit and its isolation from bovine follicular
fluid. J Mol Endocrinol. 1989;2:189 –200.
47. Miyamoto K, Hasegawa Y, Fukuda M, Igarashi M. Demonstration of high molecular weight forms of inhibin in
bovine follicular fluid (bFF) by using monoclonal antibodies to bFF 32K inhibin. Biochem Biophys Res Commun.
1986;136:1103–1109.
48. Sugino K, Nakamura T, Takio K, et al. Purification and
characterization of high molecular weight forms of inhibin
from bovine follicular fluid. Endocrinology. 1992;130:
789 –796.
49. Moore KH, Dunbar BS, Bousfield GR, Ward DN. Initial
characterization of equine inhibin. Biol Reprod. 1994;51:
63–71.
50. Grootenhuis AJ, Timmerman MA, Hordijk PL, de Jong
FH. Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa ␣ ␤-B dimer. Mol Cell Endocrinol. 1990;
70:109 –116.
51. Hancock AD, Robertson DM, de Kretser DM. Inhibin and
inhibin ␣-chain precursors are produced by immature rat
Sertoli cells in culture. Biol Reprod. 1992;46:155–161.
52. Noguchi J, Hikono H, Sato S, et al. Ontogeny of inhibin
secretion in the rat testis: secretion of inhibin-related proteins from fetal Leydig cells and of bioactive inhibin from
Sertoli cells. J Endocrinol. 1997;155:27–34.
53. Majumdar SS, Winters SJ, Plant TM. A study of the relative
doi: 10.1210/er.2014-1003
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
roles of follicle-stimulating hormone and luteinizing hormone in the regulation of testicular inhibin secretion in the
rhesus monkey (Macaca mulatta). Endocrinology. 1997;
138:1363–1373.
Winters SJ, Plant TM. Partial characterization of circulating inhibin-B and pro-␣C during development in the male
rhesus monkey. Endocrinology. 1999;140:5497–5504.
Robertson D, Burger HG, Sullivan J, et al. Biological and
immunological characterization of inhibin forms in human
plasma. J Clin Endocrinol Metab. 1996;81:669 – 676.
Robertson DM, Cahir N, Findlay JK, Burger HG, Groome
N. The biological and immunological characterization of
inhibin A and B forms in human follicular fluid and plasma.
J Clin Endocrinol Metab. 1997;82:889 – 896.
Findlay JK, Tsonis CG, Doughton B, et al. Immunisation
against the amino-terminal peptide (␣ N) of the ␣ 43 subunit of inhibin impairs fertility in sheep. Endocrinology.
1989;124:3122–3124.
Robertson DM, de Vos FL, Foulds LM, et al. Isolation of
a 31 kDa form of inhibin from bovine follicular fluid. Mol
Cell Endocrinol. 1986;44:271–277.
McLachlan RI, Robertson DM, Burger HG, de Kretser
DM. The radioimmunoassay of bovine and human follicular fluid and serum inhibin. Mol Cell Endocrinol. 1986;
46:175–185.
McLachlan RI, Robertson DM, Healy DL, Burger HG, de
Kretser DM. Circulating immunoreactive inhibin levels
during the normal human menstrual cycle. J Clin Endocrinol Metab. 1987;65:954 –961.
Groome N, Hancock J, Betteridge A, Lawrence M, Craven
R. Monoclonal and polyclonal antibodies reactive with the
1–32 amino terminal sequence of the ␣ subunit of human
32K inhibin. Hybridoma. 1990;9:31– 42.
Groome NP, Illingworth PJ, O’Brien M, et al. Detection of
dimeric inhibin throughout the human menstrual cycle by
two-site enzyme immunoassay. Clin Endocrinol (Oxf).
1994;40:717–723.
Groome NP, Illingworth PJ, O’Brien M, et al. Measurement of dimeric inhibin B throughout the human menstrual
cycle. J Clin Endocrinol Metab. 1996;81:1401–1405.
Groome N, Lawrence M. Preparation of monoclonal antibodies to the ␤ A subunit of ovarian inhibin using a synthetic peptide immunogen. Hybridoma. 1991;10:309 –
316.
Knight PG, Muttukrishna S, Groome NP. Development
and application of a two-site enzyme immunoassay for the
determination of ‘total’ activin-A concentrations in serum
and follicular fluid. J Endocrinol. 1996;148:267–279.
Knight PG, Muttukrishna S. Measurement of dimeric inhibin using a modified two-site immunoradiometric assay
specific for oxidized (Met O) inhibin. J Endocrinol. 1994;
141:417– 425.
Ludlow H, Muttukrishna S, Hyvönen M, Groome NP. Development of a new antibody to the human inhibin/activin
␤B subunit and its application to improved inhibin B
ELISAs. J Immunol Methods. 2008;329:102–111.
Robertson DM, Stephenson T, Cahir N, et al. Development of an inhibin ␣ subunit ELISA with broad specificity.
Mol Cell Endocrinol. 2001;180:79 – 86.
Robertson DM, Stephenson T, Pruysers E, et al. Characterization of inhibin forms and their measurement by an
edrv.endojournals.org
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
779
inhibin ␣-subunit ELISA in serum from postmenopausal
women with ovarian cancer. J Clin Endocrinol Metab.
2002;87:816 – 824.
Ling N, Ying SY, Ueno N, et al. Pituitary FSH is released
by a heterodimer of the ␤-subunits from the two forms of
inhibin. Nature. 1986;321:779 –782.
Vale W, Rivier J, Vaughan J, et al. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature. 1986;321:776 –779.
Ling N, Ying SY, Ueno N, et al. A homodimer of the ␤-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone. Biochem Biophys Res Commun. 1986;138:1129 –1137.
Mason AJ, Berkemeier LM, Schmelzer CH, Schwall RH.
Activin B: precursor sequences, genomic structure and in
vitro activities. Mol Endocrinol. 1989;3:1352–1358.
H tten G, Neidhardt H, Schneider C, Pohl J. Cloning of a
new member of the TGF-␤ family: a putative new activin ␤
C chain. Biochem Biophys Res Commun. 1995;206:608 –
613.
Hashimoto O, Tsuchida K, Ushiro Y, et al. cDNA cloning
and expression of human activin ␤E subunit. Mol Cell Endocrinol. 2002;194:117–122.
Lau AL, Kumar TR, Nishimori K, Bonadio J, Matzuk
MM. Activin ␤C and ␤E genes are not essential for mouse
liver growth, differentiation, and regeneration. Mol Cell
Biol. 2000;20:6127– 6137.
Gold E, Jetly N, O’Bryan MK, et al. Activin C antagonizes
activin A in vitro and overexpression leads to pathologies
in vivo. Am J Pathol. 2009;174:184 –195.
Makanji Y, Temple-Smith PD, Walton KL, Harrison CA,
Robertson DM. Inhibin B is a more potent suppressor of rat
follicle-stimulating hormone release than inhibin A in vitro
and in vivo. Endocrinology. 2009;150:4784 – 4793.
Woodruff TK, Krummen LA, Chen S, et al. Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. I. Serum profile of rh-inhibin
A and rh-activin A in the immature female rat. Endocrinology. 1993;132:715–724.
Woodruff TK, Krummen L, Chen SA, et al. Pharmacokinetic profile of recombinant human (rh) inhibin A and activin A in the immature rat. II. Tissue distribution of
[125I]rh-inhibin A and [125I]rh-activin A in immature female and male rats. Endocrinology. 1993;132:725–734.
Mason AJ, Farnworth PG, Sullivan J. Characterization and
determination of the biological activities of noncleavable
high molecular weight forms of inhibin A and activin A.
Mol Endocrinol. 1996;10:1055–1065.
Woodruff TK, Mayo KE. Regulation of inhibin synthesis in
the rat ovary. Annu Rev Physiol. 1990;52:807– 821.
Esch FS, Shimasaki S, Mercado M, et al. Structural characterization of follistatin: a novel follicle-stimulating hormone release-inhibiting polypeptide from the gonad. Mol
Endocrinol. 1987;1:849 – 855.
Mason AJ, Niall HD, Seeburg PH. Structure of two human
ovarian inhibins. Biochem Biophys Res Commun. 1986;
135:957–964.
Gray AM, Mason AJ. Requirement for activin A and transforming growth factor–␤ 1 pro-regions in homodimer assembly. Science. 1990;247:1328 –1330.
Antenos M, Zhu J, Jetly NM, Woodruff TK. An activin/
780
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
Makanji et al
Inhibin at 90, a Historical Review
furin regulatory loop modulates the processing and secretion of inhibin ␣- and ␤B-subunit dimers in pituitary gonadotrope cells. J Biol Chem. 2008;283:33059 –33068.
Antenos M, Lei L, Xu M, Malipatil A, Kiesewetter S,
Woodruff TK. Role of PCSK5 expression in mouse ovarian
follicle development: identification of the inhibin ␣- and
␤-subunits as candidate substrates. PLoS One. 2011;6:
e17348.
Hauburger A, von Einem S, Schwaerzer GK, et al. The
pro-form of BMP-2 interferes with BMP-2 signalling by
competing with BMP-2 for IA receptor binding. FEBS J.
2009;276:6386 – 6398.
Jasuja R, Ge G, Voss NG, et al. Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling
by bone morphogenetic proteins 2 and 4. J Biol Chem.
2007;282:9053–9062.
Gregory KE, Ono RN, Charbonneau NL, et al. The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix. J Biol Chem. 2005;280:27970 –27980.
Thies RS, Chen T, Davies MV, et al. GDF-8 propeptide
binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding. Growth Factors. 2001;
18:251–259.
Makanji Y, Harrison CA, Stanton PG, Krishna R, Robertson DM. Inhibin A and B in vitro bioactivities are modified
by their degree of glycosylation and their affinities to betaglycan. Endocrinology. 2007;148:2309 –2316.
Antenos M, Stemler M, Boime I, Woodruff TK. N-linked
oligosaccharides direct the differential assembly and secretion of inhibin ␣- and ␤A-subunit dimers. Mol Endocrinol.
2007;21:1670 –1684.
Walton KL, Makanji Y, Wilce MC, Chan KL, Robertson
DM, Harrison CA. A common biosynthetic pathway governs the dimerization and secretion of inhibin and related
transforming growth factor ␤ (TGF␤) ligands. J Biol Chem.
2009;284:9311–9320.
Barton DE, Yang-Feng TL, Mason AJ, Seeburg PH,
Francke U. Mapping of genes for inhibin subunits ␣, ␤ A,
and ␤ B on human and mouse chromosomes and studies of
jsd mice. Genomics. 1989;5:91–99.
Stewart AG, Milborrow HM, Ring JM, Crowther CE, Forage RG. Human inhibin genes. Genomic characterisation
and sequencing. FEBS Lett. 1986;206:329 –334.
Debieve F, Thomas K. Control of the human inhibin ␣
chain promoter in cytotrophoblast cells differentiating into
syncytium. Mol Hum Reprod. 2002;8:262–270.
Su JG, Hsueh AJ. Characterization of mouse inhibin ␣ gene
and its promoter. Biochem Biophys Res Commun. 1992;
186:293–300.
Walton KL, Makanji Y, Robertson DM, Harrison CA. The
synthesis and secretion of inhibins. Vitam Horm. 2011;85:
149 –184.
Vidal SM, Epstein DJ, Malo D, Weith A, Vekemans M,
Gros P. Identification and mapping of six microdissected
genomic DNA probes to the proximal region of mouse
chromosome 1. Genomics. 1992;14:32–37.
Justice MJ, Silan CM, Ceci JD, Buchberg AM, Copeland
NG, Jenkins NA. A molecular genetic linkage map of
mouse chromosome 13 anchored by the beige (bg) and
satin (sa) loci. Genomics. 1990;6:341–351.
Tanimoto K, Yoshida E, Mita S, Nibu Y, Murakami K,
Endocrine Reviews, October 2014, 35(5):747–794
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
Fukamizu A. Human activin ␤A gene. Identification of
novel 5⬘ exon, functional promoter, and enhancers. J Biol
Chem. 1996;271:32760 –32769.
Tanimoto K, Handa S, Ueno N, Murakami K, Fukamizu
A. Structure and sequence analysis of the human activin ␤
A subunit gene. DNA Seq. 1991;2:103–110.
Erämaa M, Heikinheimo K, Voutilainen R. Developmental and cyclic adenosine 3⬘,5⬘monophosphate-dependent
regulation of inhibin subunit messenger ribonucleic acids
in human fetal testes. J Clin Endocrinol Metab. 1992;75:
806 – 811.
Dykema JC, Mayo KE. Two messenger ribonucleic acids
encoding the common ␤ B-chain of inhibin and activin
have distinct 5⬘-initiation sites and are differentially regulated in rat granulosa cells. Endocrinology. 1994;135:
702–711.
Feng ZM, Li YP, Chen CL. Analysis of the 5⬘-flanking
regions of rat inhibin ␣- and ␤-B-subunit genes suggests
two different regulatory mechanisms. Mol Endocrinol.
1989;3:1914 –1925.
Robert NM, Miyamoto Y, Taniguchi H, Viger RS. LRH1/NR5A2 cooperates with GATA factors to regulate inhibin ␣-subunit promoter activity. Mol Cell Endocrinol.
2006;257–258:65–74.
Tremblay JJ, Viger RS. GATA factors differentially activate multiple gonadal promoters through conserved
GATA regulatory elements. Endocrinology. 2001;142:
977–986.
Bicsak TA, Tucker EM, Cappel S, et al. Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology. 1986;119:2711–2719.
Tsonis CG, Hillier SG, Baird DT. Production of inhibin
bioactivity by human granulosa-lutein cells: stimulation by
LH and testosterone in vitro. J Endocrinol. 1987;112:
R11–R14.
Conti M. Specificity of the cyclic adenosine 3⬘,5⬘-monophosphate signal in granulosa cell function. Biol Reprod.
2002;67:1653–1661.
Richards JS. New signaling pathways for hormones and
cyclic adenosine 3⬘,5⬘-monophosphate action in endocrine
cells. Mol Endocrinol. 2001;15:209 –218.
Ito M, Park Y, Weck J, Mayo KE, Jameson JL. Synergistic
activation of the inhibin ␣-promoter by steroidogenic factor-1 and cyclic adenosine 3⬘,5⬘-monophosphate. Mol Endocrinol. 2000;14:66 – 81.
Mukherjee A, Park-Sarge OK, Mayo KE. Gonadotropins
induce rapid phosphorylation of the 3⬘,5⬘-cyclic adenosine
monophosphate response element binding protein in ovarian granulosa cells. Endocrinology. 1996;137:3234 –
3245.
Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE.
Regulation of the ␣ inhibin gene by cyclic adenosine 3⬘,5⬘monophosphate after transfection into rat granulosa cells.
Mol Endocrinol. 1991;5:521–534.
Ardekani AM, Romanelli JC, Mayo KE. Structure of the
rat inhibin and activin ␤A-subunit gene and regulation in
an ovarian granulosa cell line. Endocrinology. 1998;139:
3271–3279.
Matulis CK, Mayo KE. The LIM domain protein FHL2
interacts with the NR5A family of nuclear receptors and
doi: 10.1210/er.2014-1003
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
CREB to activate the inhibin-␣ subunit gene in ovarian
granulosa cells. Mol Endocrinol. 2012;26:1278 –1290.
Weck J, Mayo KE. Switching of NR5A proteins associated
with the inhibin ␣-subunit gene promoter after activation
of the gene in granulosa cells. Mol Endocrinol. 2006;20:
1090 –1103.
Woodruff TK, D’Agostino J, Schwartz NB, Mayo KE. Dynamic changes in inhibin messenger RNAs in rat ovarian
follicles during the reproductive cycle. Science. 1988;239:
1296 –1299.
Camp TA, Rahal JO, Mayo KE. Cellular localization and
hormonal regulation of follicle-stimulating hormone and
luteinizing hormone receptor messenger RNAs in the rat
ovary. Mol Endocrinol. 1991;5:1405–1417.
Hickey GJ, Chen SA, Besman MJ, et al. Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat
ovarian follicles and corpora lutea: relationship to estradiol biosynthesis. Endocrinology. 1988;122:1426 –1436.
Ronen-Fuhrmann T, Timberg R, King SR, et al. Spatiotemporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the
rat ovary. Endocrinology. 1998;139:303–315.
Meldi KM, Gaconnet GA, Mayo KE. DNA methylation
and histone modifications are associated with repression of
the inhibin ␣ promoter in the rat corpus luteum. Endocrinology. 2012;153:4905– 4917.
Lenton EA, de Kretser DM, Woodward AJ, Robertson
DM. Inhibin concentrations throughout the menstrual cycles of normal, infertile, and older women compared with
those during spontaneous conception cycles. J Clin Endocrinol Metab. 1991;73:1180 –1190.
Burkart AD, Mukherjee A, Mayo KE. Mechanism of repression of the inhibin ␣-subunit gene by inducible 3⬘,5⬘cyclic adenosine monophosphate early repressor. Mol Endocrinol. 2006;20:584 –597.
Burkart AD, Mukherjee A, Sterneck E, Johnson PF, Mayo
KE. Repression of the inhibin ␣-subunit gene by the transcription factor CCAAT/enhancer-binding protein-␤. Endocrinology. 2005;146:1909 –1921.
Daopin S, Piez KA, Ogawa Y, Davies DR. Crystal structure
of transforming growth factor-␤ 2: an unusual fold for the
superfamily. Science. 1992;257:369 –373.
Hart PJ, Deep S, Taylor AB, Shu Z, Hinck CS, Hinck AP.
Crystal structure of the human T␤R2 ectodomain–
TGF-␤3 complex. Nat Struct Biol. 2002;9:203–208.
Groppe J, Hinck CS, Samavarchi-Tehrani P, et al. Cooperative assembly of TGF-␤ superfamily signaling complexes is mediated by two disparate mechanisms and
distinct modes of receptor binding. Mol Cell. 2008;29:
157–168.
Thompson TB, Woodruff TK, Jardetzky TS. Structures of
an ActRIIB:activin A complex reveal a novel binding mode
for TGF-␤ ligand:receptor interactions. EMBO J. 2003;
22:1555–1566.
Greenwald J, Vega ME, Allendorph GP, Fischer WH, Vale
W, Choe S. A flexible activin explains the membrane-dependent cooperative assembly of TGF-␤ family receptors.
Mol Cell. 2004;15:485– 489.
Lin SJ, Lerch TF, Cook RW, Jardetzky TS, Woodruff TK.
The structural basis of TGF-␤, bone morphogenetic pro-
edrv.endojournals.org
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
781
tein, and activin ligand binding. Reproduction. 2006;132:
179 –190.
Kirsch T, Sebald W, Dreyer MK. Crystal structure of the
BMP-2-BRIA ectodomain complex. Nat Struct Biol. 2000;
7:492– 496.
Nickel J, Dreyer MK, Kirsch T, Sebald W. The crystal
structure of the BMP-2:BMPR-IA complex and the generation of BMP-2 antagonists. J Bone Joint Surg Am. 2001;
83-A(suppl 1):S7–S14.
Allendorph GP, Isaacs MJ, Kawakami Y, Izpisua Belmonte
JC, Choe S. BMP-3 and BMP-6 structures illuminate the
nature of binding specificity with receptors. Biochemistry.
2007;46:12238 –12247.
Saremba S, Nickel J, Seher A, Kotzsch A, Sebald W, Mueller TD. Type I receptor binding of bone morphogenetic
protein 6 is dependent on N-glycosylation of the ligand.
FEBS J. 2008;275:172–183.
Greenwald J, Groppe J, Gray P, et al. The BMP7/ActRII
extracellular domain complex provides new insights into
the cooperative nature of receptor assembly. Mol Cell.
2003;11:605– 617.
Brown MA, Zhao Q, Baker KA, et al. Crystal structure of
BMP-9 and functional interactions with pro-region and
receptors. J Biol Chem. 2005;280:25111–25118.
Cash JN, Rejon CA, McPherron AC, Bernard DJ, Thompson TB. The structure of myostatin:follistatin 288: insights
into receptor utilization and heparin binding. EMBO J.
2009;28:2662–2676.
Vitt UA, Hsu SY, Hsueh AJ. Evolution and classification of
cystine knot-containing hormones and related extracellular signaling molecules. Mol Endocrinol. 2001;15:681–
694.
Mason AJ. Functional analysis of the cysteine residues of
activin A. Mol Endocrinol. 1994;8:325–332.
Harrington AE, Morris-Triggs SA, Ruotolo BT, Robinson
CV, Ohnuma S, Hyvönen M. Structural basis for the inhibition of activin signalling by follistatin. EMBO J. 2006;
25:1035–1045.
Thompson TB, Lerch TF, Cook RW, Woodruff TK, Jardetzky TS. The structure of the follistatin:activin complex
reveals antagonism of both type I and type II receptor binding. Dev Cell. 2005;9:535–543.
Zhu J, Braun EL, Kohno S, et al. Phylogenomic analyses
reveal the evolutionary origin of the inhibin ␣-subunit, a
unique TGF␤ superfamily antagonist. PLoS One. 2010;5:
e9457.
Attisano L, Wrana JL. Signal transduction by the TGF-␤
superfamily. Science. 2002;296:1646 –1647.
Gray PC, Greenwald J, Blount AL, et al. Identification of
a binding site on the type II activin receptor for activin and
inhibin. J Biol Chem. 2000;275:3206 –3212.
Greenwald J, Fischer WH, Vale WW, Choe S. Three-finger
toxin fold for the extracellular ligand-binding domain of
the type II activin receptor serine kinase. Nat Struct Biol.
1999;6:18 –22.
Wuytens G, Verschueren K, de Winter JP, et al. Identification of two amino acids in activin A that are important
for biological activity and binding to the activin type II
receptors. J Biol Chem. 1999;274:9821–9827.
Harrison CA, Gray PC, Koerber SC, Fischer W, Vale W.
Identification of a functional binding site for activin on the
782
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
165.
Makanji et al
Inhibin at 90, a Historical Review
type I receptor ALK4. J Biol Chem. 2003;278:21129 –
21135.
Mathews LS, Vale WW. Expression cloning of an activin
receptor, a predicted transmembrane serine kinase. Cell.
1991;65:973–982.
del Re E, Sidis Y, Fabrizio DA, Lin HY, Schneyer A. Reconstitution and analysis of soluble inhibin and activin
receptor complexes in a cell-free system. J Biol Chem.
2004;279:53126 –53135.
Kupershmidt L, Amit T, Bar-Am O, Youdim MB, Blumenfeld Z. The neuroprotective effect of activin A and B:
implication for neurodegenerative diseases. J Neurochem.
2007;103:962–971.
Tsuchida K, Nakatani M, Yamakawa N, Hashimoto O,
Hasegawa Y, Sugino H. Activin isoforms signal through
type I receptor serine/threonine kinase ALK7. Mol Cell
Endocrinol. 2004;220:59 – 65.
Bertolino P, Holmberg R, Reissmann E, Andersson O,
Berggren PO, Ibáñez CF. Activin B receptor ALK7 is a
negative regulator of pancreatic ␤-cell function. Proc Natl
Acad Sci USA. 2008;105:7246 –7251.
Matzuk MM, Kumar TR, Vassalli A, et al. Functional
analysis of activins during mammalian development. Nature. 1995;374:354 –356.
Ferguson CA, Tucker AS, Christensen L, Lau AL, Matzuk
MM, Sharpe PT. Activin is an essential early mesenchymal
signal in tooth development that is required for patterning
of the murine dentition. Genes Dev. 1998;12:2636 –2649.
Vassalli A, Matzuk MM, Gardner HA, Lee KF, Jaenisch R.
Activin/inhibin ␤ B subunit gene disruption leads to defects
in eyelid development and female reproduction. Genes
Dev. 1994;8:414 – 427.
Schrewe H, Gendron-Maguire M, Harbison ML, Gridley
T. Mice homozygous for a null mutation of activin ␤ B are
viable and fertile. Mech Dev. 1994;47:43–51.
Matzuk MM, Kumar TR, Bradley A. Different phenotypes
for mice deficient in either activins or activin receptor type
II. Nature. 1995;374:356 –360.
Oh SP, Li E. The signaling pathway mediated by the type
IIB activin receptor controls axial patterning and lateral
asymmetry in the mouse. Genes Dev. 1997;11:1812–
1826.
Gu Z, Nomura M, Simpson BB, et al. The type I activin
receptor ActRIB is required for egg cylinder organization
and gastrulation in the mouse. Genes Dev. 1998;12:844 –
857.
Jörnvall H, Blokzijl A, ten Dijke P, Ibáñez CF. The orphan
receptor serine/threonine kinase ALK7 signals arrest of
proliferation and morphological differentiation in a neuronal cell line. J Biol Chem. 2001;276:5140 –5146.
Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues
the Inhba-null phenotype and reveals new activin functions. Nat Genet. 2000;25:453– 457.
McNeilly AS, Souza CJ, Baird DT, et al. Production of
inhibin A not B in rams: changes in plasma inhibin A during
testis growth, and expression of inhibin/activin subunit
mRNA and protein in adult testis. Reproduction. 2002;
123:827– 835.
Barakat B, O’Connor AE, Gold E, de Kretser DM, Loveland KL. Inhibin, activin, follistatin and FSH serum levels
Endocrine Reviews, October 2014, 35(5):747–794
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
and testicular production are highly modulated during the
first spermatogenic wave in mice. Reproduction. 2008;
136:345–359.
Takehara-Kasamatsu Y, Tsuchida K, Nakatani M, et al.
Characterization of follistatin-related gene as a negative
regulatory factor for activin family members during mouse
heart development. J Med Invest. 2007;54:276 –288.
Wang Y, Ge W. Developmental profiles of activin ␤A, ␤B,
and follistatin expression in the zebrafish ovary: evidence
for their differential roles during sexual maturation and
ovulatory cycle. Biol Reprod. 2004;71:2056 –2064.
Phillips DJ. New developments in the biology of inhibins,
activins and follistatins. Trends Endocrinol Metab. 2001;
12:94 –96.
Kocamis H, Kirkpatrick-Keller DC, Richter J, Killefer J.
The ontogeny of myostatin, follistatin and activin-B
mRNA expression during chicken embryonic development. Growth Dev Aging. 1999;63:143–150.
Glister C, Kemp CF, Knight PG. Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle
cells: actions of BMP-4, -6 and -7 on granulosa cells and
differential modulation of Smad-1 phosphorylation by follistatin. Reproduction. 2004;127:239 –254.
Sidis Y, Mukherjee A, Keutmann H, Delbaere A, Sadatsuki
M, Schneyer A. Biological activity of follistatin isoforms
and follistatin-like-3 is dependent on differential cell surface binding and specificity for activin, myostatin, and
bone morphogenetic proteins. Endocrinology. 2006;147:
3586 –3597.
Schneyer AL, Sidis Y, Gulati A, Sun JL, Keutmann H, Krasney PA. Differential antagonism of activin, myostatin and
growth and differentiation factor 11 by wild-type and mutant follistatin. Endocrinology. 2008;149:4589 – 4595.
Ueno N, Ling N, Ying SY, Esch F, Shimasaki S, Guillemin
R. Isolation and partial characterization of follistatin: a
single-chain Mr 35,000 monomeric protein that inhibits
the release of follicle-stimulating hormone. Proc Natl Acad
Sci USA. 1987;84:8282– 8286.
Robertson DM, Klein R, de Vos FL, et al. The isolation of
polypeptides with FSH suppressing activity from bovine
follicular fluid which are structurally different to inhibin.
Biochem Biophys Res Commun. 1987;149:744 –749.
Lima AR, Martinez PF, Okoshi K, et al. Myostatin and
follistatin expression in skeletal muscles of rats with
chronic heart failure. Int J Exp Pathol. 2010;91:54 – 62.
Ogawa K, Abe K, Kurosawa N, et al. Expression of ␣, ␤ A
and ␤ B subunits of inhibin or activin and follistatin in rat
pancreatic islets. FEBS Lett. 1993;319:217–220.
Petraglia F, Gallinelli A, Grande A, et al. Local production
and action of follistatin in human placenta. J Clin Endocrinol Metab. 1994;78:205–210.
Inoue S, Nomura S, Hosoi T, Ouchi Y, Orimo H, Muramatsu M. Localization of follistatin, an activin-binding
protein, in bone tissues. Calcif Tissue Int. 1994;55:395–
397.
Michel U, Ebert S, Schneider O, et al. Follistatin (FS) in
human cerebrospinal fluid and regulation of FS expression
in a mouse model of meningitis. Eur J Endocrinol. 2000;
143:809 – 816.
Sonoyama K, Rutatip S, Kasai T. Gene expression of activin, activin receptors, and follistatin in intestinal epithe-
doi: 10.1210/er.2014-1003
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
lial cells. Am J Physiol Gastrointest Liver Physiol. 2000;
278:G89 –G97.
Bloise E, Couto HL, Massai L, et al. Differential expression
of follistatin and FLRG in human breast proliferative disorders. BMC Cancer. 2009;9:320.
Shimasaki S, Koga M, Esch F, et al. Porcine follistatin gene
structure supports two forms of mature follistatin produced by alternative splicing. Biochem Biophys Res Commun. 1988;152:717–723.
Sugino K, Kurosawa N, Nakamura T, et al. Molecular
heterogeneity of follistatin, an activin-binding protein.
Higher affinity of the carboxyl-terminal truncated forms
for heparan sulfate proteoglycans on the ovarian granulosa
cell. J Biol Chem. 1993;268:15579 –15587.
Schneyer AL, Wang Q, Sidis Y, Sluss PM. Differential distribution of follistatin isoforms: application of a new
FS315-specific immunoassay. J Clin Endocrinol Metab.
2004;89:5067–5075.
Hashimoto O, Nakamura T, Shoji H, Shimasaki S,
Hayashi Y, Sugino H. A novel role of follistatin, an activinbinding protein, in the inhibition of activin action in rat
pituitary cells. Endocytotic degradation of activin and its
acceleration by follistatin associated with cell-surface
heparan sulfate. J Biol Chem. 1997;272:13835–13842.
Lerch TF, Shimasaki S, Woodruff TK, Jardetzky TS. Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions. J Biol Chem. 2007;
282:15930 –15939.
Harrison CA, Gray PC, Vale WW, Robertson DM. Antagonists of activin signaling: mechanisms and potential
biological applications. Trends Endocrinol Metab. 2005;
16:73–78.
Keutmann HT, Schneyer AL, Sidis Y. The role of follistatin
domains in follistatin biological action. Mol Endocrinol.
2004;18:228 –240.
Harrison CA, Chan KL, Robertson DM. Activin-A binds
follistatin and type II receptors through overlapping binding sites: generation of mutants with isolated binding activities. Endocrinology. 2006;147:2744 –2753.
Kaiser UB, Lee BL, Carroll RS, Unabia G, Chin WW,
Childs GV. Follistatin gene expression in the pituitary: localization in gonadotropes and folliculostellate cells in diestrous rats. Endocrinology. 1992;130:3048 –3056.
Schneyer A, Schoen A, Quigg A, Sidis Y. Differential binding and neutralization of activins A and B by follistatin and
follistatin like-3 (FSTL-3/FSRP/FLRG). Endocrinology.
2003;144:1671–1674.
Matzuk MM, Lu N, Vogel H, Sellheyer K, Roop DR, Bradley A. Multiple defects and perinatal death in mice deficient
in follistatin. Nature. 1995;374:360 –363.
Lin SY, Craythorn RG, O’Connor AE, et al. Female infertility and disrupted angiogenesis are actions of specific follistatin isoforms. Mol Endocrinol. 2008;22:415– 429.
Kimura F, Sidis Y, Bonomi L, Xia Y, Schneyer A. The
follistatin-288 isoform alone is sufficient for survival but
not for normal fertility in mice. Endocrinology. 2010;151:
1310 –1319.
Kimura F, Bonomi LM, Schneyer AL. Follistatin regulates
germ cell nest breakdown and primordial follicle formation. Endocrinology. 2011;152:697–706.
Jorgez CJ, Klysik M, Jamin SP, Behringer RR, Matzuk
edrv.endojournals.org
197.
198.
199.
200.
201.
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
783
MM. Granulosa cell-specific inactivation of follistatin
causes female fertility defects. Mol Endocrinol. 2004;18:
953–967.
Schneyer A, Tortoriello D, Sidis Y, Keutmann H, Matsuzaki T, Holmes W. Follistatin-related protein (FSRP): a
new member of the follistatin gene family. Mol Cell Endocrinol. 2001;180:33–38.
Tortoriello DV, Sidis Y, Holtzman DA, Holmes WE, Schneyer AL. Human follistatin-related protein: a structural
homologue of follistatin with nuclear localization. Endocrinology. 2001;142:3426 –3434.
Xia Y, Sidis Y, Schneyer A. Overexpression of follistatinlike 3 in gonads causes defects in gonadal development and
function in transgenic mice. Mol Endocrinol. 2004;18:
979 –994.
Tsuchida K, Arai KY, Kuramoto Y, Yamakawa N, Hasegawa Y, Sugino H. Identification and characterization of a
novel follistatin-like protein as a binding protein for the
TGF-␤ family. J Biol Chem. 2000;275:40788 – 40796.
Sidis Y, Schneyer AL, Keutmann HT. Heparin and activinbinding determinants in follistatin and FSTL3. Endocrinology. 2005;146:130 –136.
Hill JJ, Qiu Y, Hewick RM, Wolfman NM. Regulation of
myostatin in vivo by growth and differentiation factorassociated serum protein-1: a novel protein with protease
inhibitor and follistatin domains. Mol Endocrinol. 2003;
17:1144 –1154.
Maguer-Satta V, Bartholin L, Jeanpierre S, et al. Regulation of human erythropoiesis by activin A, BMP2, and
BMP4, members of the TGF␤ family. Exp Cell Res. 2003;
282:110 –120.
Otsuka F, Moore RK, Iemura S, Ueno N, Shimasaki S.
Follistatin inhibits the function of the oocyte-derived factor BMP-15. Biochem Biophys Res Commun. 2001;289:
961–966.
Ciarmela P, Florio P, Toti P, et al. Human placenta and
fetal membranes express follistatin-related gene mRNA
and protein. J Endocrinol Invest. 2003;26:641– 645.
Wankell M, Kaesler S, Zhang YQ, Florence C, Werner S,
Duan R. The activin binding proteins follistatin and follistatin-related protein are differentially regulated in vitro
and during cutaneous wound repair. J Endocrinol. 2001;
171:385–395.
Florio P, Ciarmela P, Toti P, et al. Human endometrium
and decidua express follistatin-related gene (FLRG)
mRNA and peptide. Mol Cell Endocrinol. 2004;218:129 –
135.
Allen DL, Cleary AS, Speaker KJ, et al. Myostatin, activin
receptor IIb, and follistatin-like-3 gene expression are altered in adipose tissue and skeletal muscle of obese mice.
Am J Physiol Endocrinol Metab. 2008;294:E918 –E927.
Bilezikjian LM, Blount AL, Leal AM, Donaldson CJ, Fischer WH, Vale WW. Autocrine/paracrine regulation of
pituitary function by activin, inhibin and follistatin. Mol
Cell Endocrinol. 2004;225:29 –36.
Besecke LM, Guendner MJ, Sluss PA, et al. Pituitary follistatin regulates activin-mediated production of folliclestimulating hormone during the rat estrous cycle. Endocrinology. 1997;138:2841–2848.
Arai KY, Ohshima K, Watanabe G, Arai K, Uehara K,
Taya K. Dynamics of messenger RNAs encoding inhibin/
784
212.
213.
214.
215.
216.
217.
218.
219.
220.
221.
222.
223.
224.
225.
226.
227.
Makanji et al
Inhibin at 90, a Historical Review
activin subunits and follistatin in the ovary during the rat
estrous cycle. Biol Reprod. 2002;66:1119 –1126.
Mukherjee A, Sidis Y, Mahan A, et al. FSTL3 deletion
reveals roles for TGF-␤ family ligands in glucose and fat
homeostasis in adults. Proc Natl Acad Sci USA. 2007;104:
1348 –1353.
Lebrun JJ, Vale WW. Activin and inhibin have antagonistic
effects on ligand-dependent heteromerization of the type I
and type II activin receptors and human erythroid differentiation. Mol Cell Biol. 1997;17:1682–1691.
Donaldson CJ, Vaughan JM, Corrigan AZ, Fischer WH,
Vale WW. Activin and inhibin binding to the soluble extracellular domain of activin receptor II. Endocrinology.
1999;140:1760 –1766.
Cook RW, Thompson TB, Kurup SP, Jardetzky TS,
Woodruff TK. Structural basis for a functional antagonist
in the transforming growth factor ␤ superfamily. J Biol
Chem. 2005;280:40177– 40186.
Zhu J, Lin SJ, Zou C, Makanji Y, Jardetzky TS, Woodruff
TK. Inhibin ␣-subunit N terminus interacts with activin
type IB receptor to disrupt activin signaling. J Biol Chem.
2012;287:8060 – 8070.
Lewis KA, Gray PC, Blount AL, et al. Betaglycan binds
inhibin and can mediate functional antagonism of activin
signalling. Nature. 2000;404:411– 414.
Chapman SC, Bernard DJ, Jelen J, Woodruff TK. Properties of inhibin binding to betaglycan, InhBP/p120 and the
activin type II receptors. Mol Cell Endocrinol. 2002;196:
79 –93.
López-Casillas F, Cheifetz S, Doody J, Andres JL, Lane WS,
Massagué J. Structure and expression of the membrane
proteoglycan betaglycan, a component of the TGF-␤ receptor system. Cell. 1991;67:785–795.
Kirkbride KC, Townsend TA, Bruinsma MW, Barnett JV,
Blobe GC. Bone morphogenetic proteins signal through
the transforming growth factor-␤ type III receptor. J Biol
Chem. 2008;283:7628 –7637.
Wiater E, Lewis KA, Donaldson C, Vaughan J, Bilezikjian
L, Vale W. Endogenous betaglycan is essential for highpotency inhibin antagonism in gonadotropes. Mol Endocrinol. 2009;23:1033–1042.
Escalona RM, Stenvers KL, Farnworth PG, Findlay JK,
Ooi GT. Reducing betaglycan expression by RNA interference (RNAi) attenuates inhibin bioactivity in L␤T2 gonadotropes. Mol Cell Endocrinol. 2009;307:149 –156.
Farnworth PG, Harrison CA, Leembruggen P, et al. Inhibin
binding sites and proteins in pituitary, gonadal, adrenal
and bone cells. Mol Cell Endocrinol. 2001;180:63–71.
Chapman SC, Woodruff TK. Modulation of activin signal
transduction by inhibin B and inhibin-binding protein
(INhBP). Mol Endocrinol. 2001;15:668 – 679.
Bernard DJ, Burns KH, Haupt B, Matzuk MM, Woodruff
TK. Normal reproductive function in InhBP/p120-deficient mice. Mol Cell Biol. 2003;23:4882– 4891.
Harrison CA, Farnworth PG, Chan KL, et al. Identification
of specific inhibin A-binding proteins on mouse Leydig
(TM3) and Sertoli (TM4) cell lines. Endocrinology. 2001;
142:1393–1402.
Hertan R, Farnworth PG, Fitzsimmons KL, Robertson
DM. Identification of high affinity binding sites for inhibin
Endocrine Reviews, October 2014, 35(5):747–794
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
239.
240.
241.
242.
243.
244.
on ovine pituitary cells in culture. Endocrinology. 1999;
140:6 –12.
Lin SJ, Hu Y, Zhu J, Woodruff TK, Jardetzky TS. Structure
of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and
TGF-␤ binding. Proc Natl Acad Sci USA. 2011;108:5232–
5236.
Makanji Y, Walton KL, Wilce MC, Chan KL, Robertson
DM, Harrison CA. Suppression of inhibin A biological
activity by alterations in the binding site for betaglycan.
J Biol Chem. 2008;283:16743–16751.
Chapman SC, Woodruff TK. Betaglycan localization in the
female rat pituitary: implications for the regulation of follicle-stimulating hormone by inhibin. Endocrinology.
2003;144:5640 –5649.
Woodruff TK, Krummen L, McCray G, Mather JP. In situ
ligand binding of recombinant human [125I] activin-A and
recombinant human [125I]inhibin-A to the adult rat
ovary. Endocrinology. 1993;133:2998 –3006.
Krummen LA, Woodruff TK, DeGuzman G, et al. Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids.
Endocrinology. 1993;132:431– 443.
Phillips DJ, McFarlane JR, Hearn MT, de Kretser DM.
Inhibin, activin and follistatin bind preferentially to the
transformed species of ␣ 2-macroglobulin. J Endocrinol.
1997;155:65–71.
Cook RW, Thompson TB, Jardetzky TS, Woodruff TK.
Molecular biology of inhibin action. Semin Reprod Med.
2004;22:269 –276.
Lerch TF, Xu M, Jardetzky TS, et al. The structures that
underlie normal reproductive function. Mol Cell Endocrinol. 2007;267:1–5.
Phillips DJ, Woodruff TK. Inhibin: actions and signalling.
Growth Factors. 2004;22:13–18.
Thompson TB, Cook RW, Chapman SC, Jardetzky TS,
Woodruff TK. ␤A versus ␤B: is it merely a matter of expression? Mol Cell Endocrinol. 2004;225:9 –17.
Walton KL, Makanji Y, Harrison CA. New insights into
the mechanisms of activin action and inhibition. Mol Cell
Endocrinol. 2012;359:2–12.
Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A.
␣-Inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature. 1992;360:313–319.
Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H,
Bradley A. Development of cancer cachexia-like syndrome
and adrenal tumors in inhibin-deficient mice. Proc Natl
Acad Sci USA. 1994;91:8817– 8821.
Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, Matzuk MM. Activin signaling through activin receptor type II causes the cachexia-like symptoms in inhibin-deficient mice. Mol Endocrinol. 1996;10:534 –543.
Cipriano SC, Chen L, Kumar TR, Matzuk MM. Follistatin
is a modulator of gonadal tumor progression and the activin-induced wasting syndrome in inhibin-deficient mice.
Endocrinology. 2000;141:2319 –2327.
Li Q, Graff JM, O’Connor AE, Loveland KL, Matzuk
MM. SMAD3 regulates gonadal tumorigenesis. Mol Endocrinol. 2007;21:2472–2486.
Kumar TR, Wang Y, Matzuk MM. Gonadotropins are
essential modifier factors for gonadal tumor development
doi: 10.1210/er.2014-1003
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
258.
259.
260.
in inhibin-deficient mice. Endocrinology. 1996;137:
4210 – 4216.
Kumar TR, Palapattu G, Wang P, et al. Transgenic models
to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol
Endocrinol. 1999;13:851– 865.
Nagaraja AK, Agno JE, Kumar TR, Matzuk MM. Luteinizing hormone promotes gonadal tumorigenesis in inhibindeficient mice. Mol Cell Endocrinol. 2008;294:19 –28.
Stouffer RL, Dahl KD, Hess DL, Woodruff TK, Mather JP,
Molskness TA. Systemic and intraluteal infusion of inhibin
A or activin A in rhesus monkeys during the luteal phase of
the menstrual cycle. Biol Reprod. 1994;50:888 – 895.
Cho BN, McMullen ML, Pei L, Yates CJ, Mayo KE. Reproductive deficiencies in transgenic mice expressing the
rat inhibin ␣-subunit gene. Endocrinology. 2001;142:
4994 –5004.
McMullen ML, Cho BN, Yates CJ, Mayo KE. Gonadal
pathologies in transgenic mice expressing the rat inhibin
␣-subunit. Endocrinology. 2001;142:5005–5014.
Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hormone is required for ovarian follicle maturation
but not male fertility. Nat Genet. 1997;15:201–204.
Pierson TM, Wang Y, DeMayo FJ, Matzuk MM, Tsai SY,
Omalley BW. Regulable expression of inhibin A in wildtype and inhibin ␣ null mice. Mol Endocrinol. 2000;14:
1075–1085.
Robertson DM, Hayward S, Irby D, et al. Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation
and gonadectomy. Mol Cell Endocrinol. 1988;58:1– 8.
Woodruff TK, Besecke LM, Groome N, Draper LB,
Schwartz NB, Weiss J. Inhibin A and inhibin B are inversely
correlated to follicle-stimulating hormone, yet are discordant during the follicular phase of the rat estrous cycle, and
inhibin A is expressed in a sexually dimorphic manner.
Endocrinology. 1996;137:5463–5467.
Anderson RA, Irvine DS, Balfour C, Groome NP, Riley SC.
Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod. 1998;13:920 –926.
Cuevas P, Ying SY, Ling N, et al. Immunohistochemical
detection of inhibin in the gonad. Biochem Biophys Res
Commun. 1987;142:23–30.
Harkness LM, Baird DT. Morphological and molecular
characteristics of living human fetuses between Carnegie
stages 7 and 23: immunolocalization of inhibin ␣ and ␤ a
subunits. Hum Reprod Update. 1997;3:35–57.
Spencer SJ, Rabinovici J, Mesiano S, Goldsmith PC, Jaffe
RB. Activin and inhibin in the human adrenal gland. Regulation and differential effects in fetal and adult cells. J Clin
Invest. 1992;90:142–149.
Ying SY, Li SQ, Zhang Z. Expression and localization of
inhibin ␣-subunit in rat retinal photoreceptor cells. Life
Sci. 1995;57:45–52.
Peeters R, Vanmontfort D, Van Isterdael J, Verhoeven G,
Rombauts L, Decuypere E. Evidence for the presence of
immunoreactive inhibin in extragonadal tissues of ovariectomized ewes. Anim Reprod Sci. 1997;48:257–268.
Meunier H, Rivier C, Evans RM, Vale W. Gonadal and
extragonadal expression of inhibin ␣, ␤ A, and ␤ B subunits
in various tissues predicts diverse functions. Proc Natl
Acad Sci USA. 1988;85:247–251.
edrv.endojournals.org
785
261. McLachlan RI, Healy DL, Robertson DM, Burger HG, de
Kretser DM. The human placenta: a novel source of inhibin. Biochem Biophys Res Commun. 1986;140:485–
490.
262. Mylonas I, Schiessl B, Jeschke U, et al. Expression of inhibin/activin subunits ␣ (-␣), ␤ A (-␤ (A)) and ␤ B (-␤ (B))
in placental tissue of normal and intrauterine growth restricted (IUGR) pregnancies. J Mol Histol. 2006;37:43–52.
263. Kandiel MM, Watanabe G, Sosa GA, et al. Profiles of circulating steroid hormones, gonadotropins, immunoreactive inhibin and prolactin during pregnancy in goats and
immunolocalization of inhibin subunits, steroidogenic enzymes and prolactin in the corpus luteum and placenta.
J Reprod Dev. 2010;56:243–250.
264. Ying SY, Zhan Y, Zhang Z. Changes in the immunoreactivity for inhibin in mouse retina during development of
photoreceptor cells. Dev Neurosci. 1997;19:184 –188.
265. Harkness LM, Baird DT. Morphological and molecular
characteristics of living human fetuses between Carnegie
stages 7 and 23: developmental stages in the post-implantation embryo. Hum Reprod Update. 1997;3:3–23.
266. Feijen A, Goumans MJ, van den Eijnden-van Raaij AJ.
Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development. 1994;120:3621–3637.
267. Bilezikjian LM, Blount AL, Donaldson CJ, Vale WW. Pituitary actions of ligands of the TGF-␤ family: activins and
inhibins. Reproduction. 2006;132:207–215.
268. Burger HG. The endocrinology of the menopause. J Steroid
Biochem Mol Biol. 1999;69:31–35.
269. Molskness TA, Woodruff TK, Hess DL, Dahl KD, Stouffer
RL. Recombinant human inhibin-A administered early in
the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys.
J Clin Endocrinol Metab. 1996;81:4002– 4006.
270. Sealfon SC, Weinstein H, Millar RP. Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev. 1997;18:180 –205.
271. Shacham S, Harris D, Ben-Shlomo H, et al. Mechanism of
GnRH receptor signaling on gonadotropin release and
gene expression in pituitary gonadotrophs. Vitam Horm.
2001;63:63–90.
272. Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM,
Chin WW. A mechanism for the differential regulation of
gonadotropin subunit gene expression by gonadotropinreleasing hormone. Proc Natl Acad Sci USA. 1995;92:
12280 –12284.
273. Norwitz ER, Xu S, Jeong KH, et al. Activin A augments
GnRH-mediated transcriptional activation of the mouse
GnRH receptor gene. Endocrinology. 2002;143:985–997.
274. Wang QF, Farnworth PG, Findlay JK, Burger HG. Effect
of purified 31K bovine inhibin on the specific binding of
gonadotropin-releasing hormone to rat anterior pituitary
cells in culture. Endocrinology. 1988;123:2161–2166.
275. Winters SJ, Pohl CR, Adedoyin A, Marshall GR. Effects of
continuous inhibin administration on gonadotropin secretion and subunit gene expression in immature and adult
male rats. Biol Reprod. 1996;55:1377–1382.
276. Fernández-Vázquez G, Kaiser UB, Albarracin CT, Chin
WW. Transcriptional activation of the gonadotropin-re-
786
277.
278.
279.
280.
281.
282.
283.
284.
285.
286.
287.
288.
289.
290.
291.
Makanji et al
Inhibin at 90, a Historical Review
leasing hormone receptor gene by activin A. Mol Endocrinol. 1996;10:356 –366.
Burger LL, Dalkin AC, Aylor KW, Haisenleder DJ, Marshall JC. GnRH pulse frequency modulation of gonadotropin subunit gene transcription in normal gonadotropesassessment by primary transcript assay provides evidence
for roles of GnRH and follistatin. Endocrinology. 2002;
143:3243–3249.
MacConell LA, Leal AM, Vale WW. The distribution of
betaglycan protein and mRNA in rat brain, pituitary, and
gonads: implications for a role for betaglycan in inhibinmediated reproductive functions. Endocrinology. 2002;
143:1066 –1075.
Corrigan AZ, Bilezikjian LM, Carroll RS, et al. Evidence
for an autocrine role of activin B within rat anterior pituitary cultures. Endocrinology. 1991;128:1682–1684.
Attardi B, Miklos J. Rapid stimulatory effect of activin-A
on messenger RNA encoding the follicle-stimulating hormone ␤-subunit in rat pituitary cell cultures. Mol Endocrinol. 1990;4:721–726.
Suszko MI, Balkin DM, Chen Y, Woodruff TK. Smad3
mediates activin-induced transcription of follicle-stimulating hormone ␤-subunit gene. Mol Endocrinol. 2005;19:
1849 –1858.
Suszko MI, Lo DJ, Suh H, Camper SA, Woodruff TK.
Regulation of the rat follicle-stimulating hormone ␤-subunit promoter by activin. Mol Endocrinol. 2003;17:318 –
332.
Attardi B, Vaughan J, Vale W. Regulation of FSH ␤ messenger ribonucleic acid levels in the rat by endogenous inhibin. Endocrinology. 1992;130:557–559.
Carroll RS, Corrigan AZ, Gharib SD, Vale W, Chin WW.
Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Mol
Endocrinol. 1989;3:1969 –1976.
Attardi B, Winters SJ. Decay of follicle-stimulating hormone-␤ messenger RNA in the presence of transcriptional
inhibitors and/or inhibin, activin, or follistatin. Mol Endocrinol. 1993;7:668 – 680.
Carroll RS, Corrigan AZ, Vale W, Chin WW. Activin stabilizes follicle-stimulating hormone-␤ messenger ribonucleic acid levels. Endocrinology. 1991;129:1721–1726.
Yamada Y, Yamamoto H, Yonehara T, et al. Differential
activation of the luteinizing hormone ␤-subunit promoter
by activin and gonadotropin-releasing hormone: a role for
the mitogen-activated protein kinase signaling pathway in
L␤T2 gonadotrophs. Biol Reprod. 2004;70:236 –243.
Coss D, Thackray VG, Deng CX, Mellon PL. Activin regulates luteinizing hormone ␤-subunit gene expression
through Smad-binding and homeobox elements. Mol Endocrinol. 2005;19:2610 –2623.
Pernasetti F, Vasilyev VV, Rosenberg SB, et al. Cell-specific
transcriptional regulation of follicle-stimulating hormone-␤ by activin and gonadotropin-releasing hormone in
the L␤T2 pituitary gonadotrope cell model. Endocrinology. 2001;142:2284 –2295.
Hillier SG, Wickings EJ, Illingworth PI, et al. Control of
immunoactive inhibin production by human granulosa
cells. Clin Endocrinol (Oxf). 1991;35:71–78.
Welt CK. Regulation and function of inhibins in the nor-
Endocrine Reviews, October 2014, 35(5):747–794
292.
293.
294.
295.
296.
297.
298.
299.
300.
301.
302.
303.
304.
305.
306.
307.
mal menstrual cycle. Semin Reprod Med. 2004;22:187–
193.
Bristol-Gould SK, Kreeger PK, Selkirk CG, et al. Postnatal
regulation of germ cells by activin: the establishment of the
initial follicle pool. Dev Biol. 2006;298:132–148.
Bristol-Gould SK, Kreeger PK, Selkirk CG, et al. Fate of the
initial follicle pool: empirical and mathematical evidence
supporting its sufficiency for adult fertility. Dev Biol.
2006;298:149 –154.
Trombly DJ, Woodruff TK, Mayo KE. Roles for transforming growth factor ␤ superfamily proteins in early folliculogenesis. Semin Reprod Med. 2009;27:14 –23.
Lu C, Yang W, Chen M, et al. Inhibin A inhibits folliclestimulating hormone (FSH) action by suppressing its receptor expression in cultured rat granulosa cells. Mol Cell
Endocrinol. 2009;298:48 –56.
Kipp JL, Kilen SM, Woodruff TK, Mayo KE. Activin regulates estrogen receptor gene expression in the mouse
ovary. J Biol Chem. 2007;282:36755–36765.
Kipp JL, Kilen SM, Bristol-Gould S, Woodruff TK, Mayo
KE. Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary. Endocrinology.
2007;148:1968 –1976.
Hillier SG, Yong EL, Illingworth PJ, Baird DT, Schwall
RH, Mason AJ. Effect of recombinant inhibin on androgen
synthesis in cultured human thecal cells. Mol Cell Endocrinol. 1991;75:R1–R6.
Roberts VJ, Barth S, el-Roeiy A, Yen SS. Expression of
inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus
luteum during the human menstrual cycle. J Clin Endocrinol Metab. 1993;77:1402–1410.
Knight PG, Glister C. TGF-␤ superfamily members and
ovarian follicle development. Reproduction. 2006;132:
191–206.
Welt CK, Schneyer AL. Differential regulation of inhibin B
and inhibin A by follicle-stimulating hormone and local
growth factors in human granulosa cells from small antral
follicles. J Clin Endocrinol Metab. 2001;86:330 –336.
Welt CK, Smith ZA, Pauler DK, Hall JE. Differential regulation of inhibin A and inhibin B by luteinizing hormone,
follicle-stimulating hormone, and stage of follicle development. J Clin Endocrinol Metab. 2001;86:2531–2537.
Drummond AE, Le MT, Ethier JF, Dyson M, Findlay JK.
Expression and localization of activin receptors, Smads,
and beta glycan to the postnatal rat ovary. Endocrinology.
2002;143:1423–1433.
Silva CC, Groome NP, Knight PG. Demonstration of a
suppressive effect of inhibin ␣-subunit on the developmental competence of in vitro matured bovine oocytes. J Reprod Fertil. 1999;115:381–388.
Campbell BK, Baird DT. Inhibin A is a follicle stimulating
hormone-responsive marker of granulosa cell differentiation, which has both autocrine and paracrine actions in
sheep. J Endocrinol. 2001;169:333–345.
Wrathall JH, Knight PG. Effects of inhibin-related peptides
and oestradiol on androstenedione and progesterone secretion by bovine theca cells in vitro. J Endocrinol. 1995;
145:491–500.
Di Simone N, Lanzone A, Petraglia F, Ronsisvalle E,
Caruso A, Mancuso S. Effect of activin-A on progesterone
doi: 10.1210/er.2014-1003
308.
309.
310.
311.
312.
313.
314.
315.
316.
317.
318.
319.
320.
321.
322.
synthesis in human luteal cells. Fertil Steril. 1994;62:1157–
1161.
Rabinovici J, Spencer SJ, Jaffe RB. Recombinant human
activin-A promotes proliferation of human luteinized preovulatory granulosa cells in vitro. J Clin Endocrinol
Metab. 1990;71:1396 –1398.
Webley GE, Marsden PL, Knight PG. Differential control
of immunoreactive ␣-inhibin and progesterone production
by marmoset luteal cells in vitro: evidence for a paracrine
action of ␣-inhibin on basal and gonadotropin-stimulated
progesterone production. Biol Reprod. 1994;50:1394 –
1402.
Smith KB, Fraser HM. Control of progesterone and inhibin
secretion during the luteal phase in the macaque. J Endocrinol. 1991;128:107–113.
Knight PG, Glister C. Potential local regulatory functions
of inhibins, activins and follistatin in the ovary. Reproduction. 2001;121:503–512.
Findlay JK. An update on the roles of inhibin, activin, and
follistatin as local regulators of folliculogenesis. Biol Reprod. 1993;48:15–23.
Skory RM, Bernabé BP, Galdones E, Broadbelt LJ, Shea
LD, Woodruff TK. Microarray analysis identifies COMP
as the most differentially regulated transcript throughout
in vitro follicle growth. Mol Reprod Dev. 2013;80:132–
144.
Parrish EM, Siletz A, Xu M, Woodruff TK, Shea LD. Gene
expression in mouse ovarian follicle development in vivo
versus an ex vivo alginate culture system. Reproduction.
2011;142:309 –318.
D’Agostino J, Woodruff TK, Mayo KE, Schwartz NB. Unilateral ovariectomy increases inhibin messenger ribonucleic acid levels in newly recruited follicles. Endocrinology.
1989;124:310 –317.
Woodruff TK, D’Agostino J, Schwartz NB, Mayo KE. Decreased inhibin gene expression in preovulatory follicles
requires primary gonadotropin surges. Endocrinology.
1989;124:2193–2199.
Turner IM, Saunders PT, Shimasaki S, Hillier SG. Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells. Endocrinology.
1989;125:2790 –2792.
Moore BC, Forouhar S, Kohno S, Botteri NL, Hamlin HJ,
Guillette LJ Jr. Gonadotropin-induced changes in oviducal
mRNA expression levels of sex steroid hormone receptors
and activin-related signaling factors in the alligator. Gen
Comp Endocrinol. 2012;175:251–258.
Moore BC, Kohno S, Cook RW, et al. Altered sex hormone
concentrations and gonadal mRNA expression levels of
activin signaling factors in hatchling alligators from a contaminated Florida lake. J Exp Zool A Ecol Genet Physiol.
2010;313:218 –230.
Moore BC, Milnes MR, Kohno S, et al. Altered gonadal
expression of TGF-␤ superfamily signaling factors in environmental contaminant-exposed juvenile alligators.
J Steroid Biochem Mol Biol. 2011;127:58 – 63.
Marchetti C, Hamdane M, Mitchell V, et al. Immunolocalization of inhibin and activin ␣ and ␤B subunits and
expression of corresponding messenger RNAs in the human adult testis. Biol Reprod. 2003;68:230 –235.
Illingworth PJ, Groome NP, Byrd W, et al. Inhibin-B: a
edrv.endojournals.org
323.
324.
325.
326.
327.
328.
329.
330.
331.
332.
333.
334.
335.
336.
787
likely candidate for the physiologically important form of
inhibin in men. J Clin Endocrinol Metab. 1996;81:1321–
1325.
Plant TM, Padmanabhan V, Ramaswamy S, et al. Circulating concentrations of dimeric inhibin A and B in the male
rhesus monkey (Macaca mulatta). J Clin Endocrinol
Metab. 1997;82:2617–2621.
Kaneko H, Noguchi J, Kikuchi K, Hasegawa Y. Molecular
weight forms of inhibin A and inhibin B in the bovine testis
change with age. Biol Reprod. 2003;68:1918 –1925.
Ohnuma K, Kaneko H, Noguchi J, Kikuchi K, Ozawa M,
Hasegawa Y. Production of inhibin A and inhibin B in
boars: changes in testicular and circulating levels of dimeric
inhibins and characterization of inhibin forms during testis
growth. Domest Anim Endocrinol. 2007;33:410 – 421.
Andersson AM, Toppari J, Haavisto AM, et al. Longitudinal reproductive hormone profiles in infants: peak of
inhibin B levels in infant boys exceeds levels in adult men.
J Clin Endocrinol Metab. 1998;83:675– 681.
Byrd W, Bennett MJ, Carr BR, Dong Y, Wians F, Rainey
W. Regulation of biologically active dimeric inhibin A and
B from infancy to adulthood in the male. J Clin Endocrinol
Metab. 1998;83:2849 –2854.
Riccardi E, Grieco V, Greco V, Verganti S, Finazzi M.
Immunohistochemical diagnosis of canine ovarian epithelial and granulosa cell tumors. J Vet Diagn Invest. 2007;
19:431– 435.
Risbridger GP, Clements J, Robertson DM, et al. Immunoand bioactive inhibin and inhibin ␣-subunit expression in
rat Leydig cell cultures. Mol Cell Endocrinol. 1989;66:
119 –122.
Bhasin S, Krummen LA, Swerdloff RS, et al. Stage dependent expression of inhibin ␣ and ␤-B subunits during the
cycle of the rat seminiferous epithelium. Endocrinology.
1989;124:987–991.
Kaipia A, Parvinen M, Shimasaki S, Ling N, Toppari J.
Stage-specific cellular regulation of inhibin ␣-subunit
mRNA expression in the rat seminiferous epithelium. Mol
Cell Endocrinol. 1991;82:165–173.
Okuma Y, O’Connor AE, Hayashi T, Loveland KL, de
Kretser DM, Hedger MP. Regulated production of activin
A and inhibin B throughout the cycle of the seminiferous
epithelium in the rat. J Endocrinol. 2006;190:331–340.
Buzzard JJ, Loveland KL, O’Bryan MK, et al. Changes in
circulating and testicular levels of inhibin A and B and
activin A during postnatal development in the rat. Endocrinology. 2004;145:3532–3541.
Majdic G, McNeilly AS, Sharpe RM, Evans LR, Groome
NP, Saunders PT. Testicular expression of inhibin and activin subunits and follistatin in the rat and human fetus and
neonate and during postnatal development in the rat. Endocrinology. 1997;138:2136 –2147.
Pierik FH, Vreeburg JT, Stijnen T, De Jong FH, Weber RF.
Serum inhibin B as a marker of spermatogenesis. J Clin
Endocrinol Metab. 1998;83:3110 –3114.
Jensen TK, Andersson AM, Hjollund NH, et al. Inhibin B
as a serum marker of spermatogenesis: correlation to differences in sperm concentration and follicle-stimulating
hormone levels. A study of 349 Danish men. J Clin Endocrinol Metab. 1997;82:4059 – 4063.
788
Makanji et al
Inhibin at 90, a Historical Review
337. Anderson RA. Clinical studies: inhibin in the adult male.
Mol Cell Endocrinol. 2001;180:109 –116.
338. Hayes FJ, Pitteloud N, DeCruz S, Crowley WF Jr, Boepple
PA. Importance of inhibin B in the regulation of FSH secretion in the human male. J Clin Endocrinol Metab. 2001;
86:5541–5546.
339. Fraser HM, Groome NP, McNeilly AS. Follicle-stimulating hormone-inhibin B interactions during the follicular
phase of the primate menstrual cycle revealed by gonadotropin-releasing hormone antagonist and antiestrogen
treatment. J Clin Endocrinol Metab. 1999;84:1365–1369.
340. Sharpe RM, Turner KJ, McKinnell C, et al. Inhibin B levels
in plasma of the male rat from birth to adulthood: effect of
experimental manipulation of Sertoli cell number. J Androl. 1999;20:94 –101.
341. Itoh M, Kondo M, Kojima C, et al. Inhibin B is the major
form of inhibin secreted from testes in male Japanese macaques (Macaca fuscata). Primates. 2003;44:253–257.
342. Ramaswamy S, Plant TM. Operation of the follicle-stimulating hormone (FSH)-inhibin B feedback loop in the control of primate spermatogenesis. Mol Cell Endocrinol.
2001;180:93–101.
343. Okuma Y, Saito K, O’Connor AE, Phillips DJ, de Kretser
DM, Hedger MP. Reciprocal regulation of activin A and
inhibin B by interleukin-1 (IL-1) and follicle-stimulating
hormone (FSH) in rat Sertoli cells in vitro. J Endocrinol.
2005;185:99 –110.
344. Ramaswamy S, Marshall GR, McNeilly AS, Plant TM.
Evidence that in a physiological setting Sertoli cell number
is the major determinant of circulating concentrations of
inhibin B in the adult male rhesus monkey (Macaca mulatta). J Androl. 1999;20:430 – 434.
345. van Beek RD, Smit M, van den Heuvel-Eibrink MM, et al.
Inhibin B is superior to FSH as a serum marker for spermatogenesis in men treated for Hodgkin’s lymphoma with
chemotherapy during childhood. Hum Reprod. 2007;22:
3215–3222.
346. Kumanov P, Nandipati K, Tomova A, Agarwal A. Inhibin
B is a better marker of spermatogenesis than other hormones in the evaluation of male factor infertility. Fertil
Steril. 2006;86:332–338.
347. Duvilla E, Lejeune H, Trombert-Paviot B, Gentil-Perret A,
Tostain J, Levy R. Significance of inhibin B and anti-Müllerian hormone in seminal plasma: a preliminary study.
Fertil Steril. 2008;89:444 – 448.
348. Stewart J, Turner KJ. Inhibin B as a potential biomarker of
testicular toxicity. Cancer Biomark. 2005;1:75–91.
349. Gaudino R, Cavarzere P, Camilot M, Teofoli F, Zampieri
N, Tatò L. Prepubertal serum inhibin B in cryptorchid infants and in monorchid boys with compensatory testicular
hypertrophy. Fertil Steril. 2008;90:2217–2221.
350. Goulis DG, Tsametis C, Iliadou PK, et al. Serum inhibin B
and anti-Müllerian hormone are not superior to folliclestimulating hormone as predictors of the presence of sperm
in testicular fine-needle aspiration in men with azoospermia. Fertil Steril. 2009;91:1279 –1284.
351. Nowroozi MR, Radkhah K, Ayati M, Jamshidian H, Ranjbaran A, Jabalameli P. Serum inhibin B concentration as
a prognostic factor for prediction of sperm retrieval in testis biopsy of patients with azoospermia. Arch Iran Med.
2008;11:54 –56.
Endocrine Reviews, October 2014, 35(5):747–794
352. Tunc L, Kirac M, Gurocak S, et al. Can serum inhibin B and
FSH levels, testicular histology and volume predict the outcome of testicular sperm extraction in patients with nonobstructive azoospermia? Int Urol Nephrol. 2006;38:
629 – 635.
353. Small CL, Shima JE, Uzumcu M, Skinner MK, Griswold
MD. Profiling gene expression during the differentiation
and development of the murine embryonic gonad. Biol
Reprod. 2005;72:492–501.
354. Loveland KL, Hogarth C, Mendis S, et al. Drivers of germ
cell maturation. Ann N Y Acad Sci. 2005;1061:173–182.
355. Adamopoulos D, Kapolla N, Nicopoulou S, Pappa A,
Koukkou E, Gregoriou A. Assessment of Sertoli cell functional reserve and its relationship to sperm parameters. Int
J Androl. 2003;26:215–225.
356. von Eckardstein S, Simoni M, Bergmann M, et al. Serum
inhibin B in combination with serum follicle-stimulating
hormone (FSH) is a more sensitive marker than serum FSH
alone for impaired spermatogenesis in men, but cannot
predict the presence of sperm in testicular tissue samples.
J Clin Endocrinol Metab. 1999;84:2496 –2501.
357. Boitani C, Stefanini M, Fragale A, Morena AR. Activin
stimulates Sertoli cell proliferation in a defined period of
rat testis development. Endocrinology. 1995;136:5438 –
5444.
358. Mather JP, Attie KM, Woodruff TK, Rice GC, Phillips
DM. Activin stimulates spermatogonial proliferation in
germ-Sertoli cell cocultures from immature rat testis. Endocrinology. 1990;127:3206 –3214.
359. van Dissel-Emiliani FM, Grootenhuis AJ, de Jong FH, de
Rooij DG. Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology.
1989;125:1899 –1903.
360. Clifton RJ, O’Donnell L, Robertson DM. Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of
inhibin ␤ B-subunit and inhibin B but not the inhibin
␣-subunit. J Endocrinol. 2002;172:565–574.
361. Gaddy-Kurten D, Coker JK, Abe E, Jilka RL, Manolagas
SC. Inhibin suppresses and activin stimulates osteoblastogenesis and osteoclastogenesis in murine bone marrow cultures. Endocrinology. 2002;143:74 – 83.
362. Perrien DS, Akel NS, Edwards PK, et al. Inhibin A is an
endocrine stimulator of bone mass and strength. Endocrinology. 2007;148:1654 –1665.
363. Sun L, Peng Y, Sharrow AC, et al. FSH directly regulates
bone mass. Cell. 2006;125:247–260.
364. Vural F, Vural B, Yucesoy I, Badur S. Ovarian aging and
bone metabolism in menstruating women aged 35–50
years. Maturitas. 2005;52:147–153.
365. Perrien DS, Achenbach SJ, Bledsoe SE, et al. Bone turnover
across the menopause transition: correlations with inhibins and follicle-stimulating hormone. J Clin Endocrinol
Metab. 2006;91:1848 –1854.
366. Hanley NA, Ball SG, Clement-Jones M, et al. Expression of
steroidogenic factor 1 and Wilms’ tumour 1 during early
human gonadal development and sex determination. Mech
Dev. 1999;87:175–180.
367. Ikeda Y, Shen WH, Ingraham HA, Parker KL. Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol. 1994;8:654 – 662.
doi: 10.1210/er.2014-1003
368. Keegan CE, Hammer GD. Recent insights into organogenesis of the adrenal cortex. Trends Endocrinol Metab. 2002;
13:200 –208.
369. Voutilainen R, Erämaa M, Ritvos O. Hormonally regulated inhibin gene expression in human fetal and adult adrenals. J Clin Endocrinol Metab. 1991;73:1026 –1030.
370. Vänttinen T, Liu J, Kuulasmaa T, Kivinen P, Voutilainen
R. Expression of activin/inhibin signaling components in
the human adrenal gland and the effects of activins and
inhibins on adrenocortical steroidogenesis and apoptosis.
J Endocrinol. 2003;178:479 – 489.
371. Wang EY, Ma EY, Woodruff TK. Activin signal transduction in the fetal rat adrenal gland and in human H295R
cells. J Endocrinol. 2003;178:137–148.
372. Nishi Y, Haji M, Tanaka S, et al. Human recombinant
activin-A modulates the steroidogenesis of cultured bovine
adrenocortical cells. J Endocrinol. 1992;132:R1–R4.
373. Spencer SJ, Mesiano S, Lee JY, Jaffe RB. Proliferation and
apoptosis in the human adrenal cortex during the fetal and
perinatal periods: implications for growth and remodeling.
J Clin Endocrinol Metab. 1999;84:1110 –1115.
374. Bohnsack BL, Szabo M, Kilen SM, Tam DH, Schwartz
NB. Follistatin suppresses steroid-enhanced follicle-stimulating hormone release in vitro in rats. Biol Reprod. 2000;
62:636 – 641.
375. McCluggage WG, Burton J, Maxwell P, Sloan JM. Immunohistochemical staining of normal, hyperplastic, and neoplastic adrenal cortex with a monoclonal antibody against
␣ inhibin. J Clin Pathol. 1998;51:114 –116.
376. Munro LM, Kennedy A, McNicol AM. The expression of
inhibin/activin subunits in the human adrenal cortex and
its tumours. J Endocrinol. 1999;161:341–347.
377. Pelkey TJ, Frierson HF Jr, Mills SE, Stoler MH. The ␣
subunit of inhibin in adrenal cortical neoplasia. Mod
Pathol. 1998;11:516 –524.
378. Beuschlein F, Looyenga BD, Bleasdale SE, et al. Activin
induces x-zone apoptosis that inhibits luteinizing hormone-dependent adrenocortical tumor formation in inhibin-deficient mice. Mol Cell Biol. 2003;23:3951–3964.
379. Johnsen IK, Slawik M, Shapiro I, et al. Gonadectomy in
mice of the inbred strain CE/J induces proliferation of subcapsular adrenal cells expressing gonadal marker genes.
J Endocrinol. 2006;190:47–57.
380. Looyenga BD, Hammer GD. Origin and identity of adrenocortical tumors in inhibin knockout mice: implications
for cellular plasticity in the adrenal cortex. Mol Endocrinol. 2006;20:2848 –2863.
381. Looyenga BD, Wiater E, Vale W, Hammer GD. Inhibin-A
antagonizes TGF␤2 signaling by down-regulating cell surface expression of the TGF␤ coreceptor betaglycan. Mol
Endocrinol. 2010;24:608 – 620.
382. Belecky-Adams TL, Scheurer D, Adler R. Activin family
members in the developing chick retina: expression patterns, protein distribution, and in vitro effects. Dev Biol.
1999;210:107–123.
383. Eto Y, Tsuji T, Takezawa M, Takano S, Yokogawa Y,
Shibai H. Purification and characterization of erythroid
differentiation factor (EDF) isolated from human leukemia
cell line THP-1. Biochem Biophys Res Commun. 1987;
142:1095–1103.
384. Murata M, Eto Y, Shibai H, Sakai M, Muramatsu M. Ery-
edrv.endojournals.org
385.
386.
387.
388.
389.
390.
391.
392.
393.
394.
395.
396.
397.
398.
399.
789
throid differentiation factor is encoded by the same mRNA
as that of the inhibin ␤ A chain. Proc Natl Acad Sci USA.
1988;85:2434 –2438.
Yu J, Shao LE, Lemas V, et al. Importance of FSH-releasing
protein and inhibin in erythrodifferentiation. Nature.
1987;330:765–767.
Broxmeyer HE, Lu L, Cooper S, Schwall RH, Mason AJ,
Nikolics K. Selective and indirect modulation of human
multipotential and erythroid hematopoietic progenitor cell
proliferation by recombinant human activin and inhibin.
Proc Natl Acad Sci USA. 1988;85:9052–9056.
Hangoc G, Carow CE, Schwall R, Mason AJ, Broxmeyer
HE. Effects in vivo of recombinant human inhibin on myelopoiesis in mice. Exp Hematol. 1992;20:1243–1246.
Petraglia F, Calzà L, Garuti GC, et al. Presence and synthesis of inhibin subunits in human decidua. J Clin Endocrinol Metab. 1990;71:487– 492.
Jones RL, Salamonsen LA, Critchley HO, Rogers PA, Affandi B, Findlay JK. Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes
during the menstrual cycle, in early pregnancy and in
women using progestin-only contraception. Mol Hum Reprod. 2000;6:1107–1117.
Jones RL, Findlay JK, Farnworth PG, Robertson DM,
Wallace E, Salamonsen LA. Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions during decidualization and
trophoblast invasion. Endocrinology. 2006;147:724 –
732.
Jones RL, Salamonsen LA, Zhao YC, Ethier JF, Drummond AE, Findlay JK. Expression of activin receptors, follistatin and betaglycan by human endometrial stromal
cells; consistent with a role for activins during decidualization. Mol Hum Reprod. 2002;8:363–374.
Ciarmela P, Florio P, Toti P, et al. Expression of betaglycan
in pregnant tissues throughout gestation. Eur J Endocrinol. 2003;149:433– 437.
Petraglia F, Vaughan J, Vale W. Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells. Proc Natl Acad Sci USA.
1989;86:5114 –5117.
Muttukrishna S, George L, Fowler PA, Groome NP,
Knight PG. Measurement of serum concentrations of inhibin-A (␣-␤ A dimer) during human pregnancy. Clin Endocrinol (Oxf). 1995;42:391–397.
Wallace EM, Marjono B, Tyzack K, Tong S. First trimester
levels of inhibins and activin A in normal and failing pregnancies. Clin Endocrinol (Oxf). 2004;60:484 – 490.
Muttukrishna S, Bearfield C, Johns J, Jauniaux E. Inhibin,
activin, follistatin, activin receptors and beta-glycan gene
expression in the villous tissue of miscarriage patients. Mol
Hum Reprod. 2004;10:793–798.
Malone FD, Canick JA, Ball RH, et al. First-trimester or
second-trimester screening, or both, for Down’s syndrome.
N Engl J Med. 2005;353:2001–2011.
Wallace EM, Swanston IA, McNeilly AS, et al. Second
trimester screening for Down’s syndrome using maternal
serum dimeric inhibin A. Clin Endocrinol (Oxf). 1996;44:
17–21.
Bersinger NA, Smárason AK, Muttukrishna S, Groome
790
400.
401.
402.
403.
404.
405.
406.
407.
408.
409.
410.
411.
412.
413.
414.
Makanji et al
Inhibin at 90, a Historical Review
NP, Redman CW. Women with preeclampsia have increased serum levels of pregnancy-associated plasma protein A (PAPP-A), inhibin A, activin A and soluble E-selectin. Hypertens Pregnancy. 2003;22:45–55.
Casagrandi D, Bearfield C, Geary J, Redman CW, Muttukrishna S. Inhibin, activin, follistatin, activin receptors
and beta-glycan gene expression in the placental tissue of
patients with pre-eclampsia. Mol Hum Reprod. 2003;9:
199 –203.
Spencer K, Yu CK, Savvidou M, Papageorghiou AT, Nicolaides KH. Prediction of pre-eclampsia by uterine artery
Doppler ultrasonography and maternal serum pregnancyassociated plasma protein-A, free ␤-human chorionic gonadotropin, activin A and inhibin A at 22 ⫹ 0 to 24 ⫹ 6
weeks’ gestation. Ultrasound Obstet Gynecol. 2006;27:
658 – 663.
Ay E, Kavak ZN, Elter K, Gokaslan H, Pekin T. Screening
for pre-eclampsia by using maternal serum inhibin A, activin A, human chorionic gonadotropin, unconjugated estriol, and ␣-fetoprotein levels and uterine artery Doppler in
the second trimester of pregnancy. Aust N Z J Obstet
Gynaecol. 2005;45:283–288.
Madazli R, Kuseyrioglu B, Uzun H, Uludag S, Ocak V.
Prediction of preeclampsia with maternal mid-trimester
placental growth factor, activin A, fibronectin and uterine
artery Doppler velocimetry. Int J Gynaecol Obstet. 2005;
89:251–257.
Florio P, Luisi S, D’Antona D, Severi FM, Rago G, Petraglia F. Maternal serum inhibin A levels may predict pregnancy outcome in women with threatened abortion. Fertil
Steril. 2004;81:468 – 470.
Affolter M, Bellusci S, Itoh N, Shilo B, Thiery JP, Werb Z.
Tube or not tube: remodeling epithelial tissues by branching morphogenesis. Dev Cell. 2003;4:11–18.
Ochoa-Espinosa A, Affolter M. Branching morphogenesis:
from cells to organs and back. Cold Spring Harb Perspect
Biol. 2012;4:pii:a008243.
Ball EM, Risbridger GP. Activins as regulators of branching morphogenesis. Dev Biol. 2001;238:1–12.
Wilcox AJ, Weinberg CR, O’Connor JF, et al. Incidence of
early loss of pregnancy. N Engl J Med. 1988;319:189 –
194.
O’Connor AE, McFarlane JR, Hayward S, Yohkaichiya T,
Groome NP, de Kretser DM. Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study. Hum Reprod. 1999;14:
827– 832.
Woodruff TK, Ackland J, Rahal JO, Schwartz NB, Mayo
KE. Expression of ovarian inhibin during pregnancy in the
rat. Endocrinology. 1991;128:1647–1654.
Petraglia F, Aguzzoli L, Gallinelli A, et al. Hypertension in
pregnancy: changes in activin A maternal serum concentration. Placenta. 1995;16:447– 454.
Petraglia F, De Vita D, Gallinelli A, et al. Abnormal concentration of maternal serum activin-A in gestational diseases. J Clin Endocrinol Metab. 1995;80:558 –561.
Gallinelli A, Gallo R, Genazzani AD, et al. Episodic secretion of activin A in pregnant women. Eur J Endocrinol.
1996;135:340 –344.
Petraglia F, Di Blasio AM, Florio P, et al. High levels of fetal
membrane activin ␤ A and activin receptor IIB mRNAs and
Endocrine Reviews, October 2014, 35(5):747–794
415.
416.
417.
418.
419.
420.
421.
422.
423.
424.
425.
426.
427.
428.
429.
augmented concentration of amniotic fluid activin A in
women in term or preterm labor. J Endocrinol. 1997;154:
95–101.
Woodruff TK, Sluss P, Wang E, Janssen I, Mersol-Barg
MS. Activin A and follistatin are dynamically regulated
during human pregnancy. J Endocrinol. 1997;152:167–
174.
Draper LB, Chong H, Wang E, Woodruff TK. The uterine
myometrium is a target for increased levels of activin A
during pregnancy. Endocrinology. 1997;138:3042–3046.
Petraglia F, Woodruff TK, Botticelli G, et al. Gonadotropin-releasing hormone, inhibin, and activin in human placenta: evidence for a common cellular localization. J Clin
Endocrinol Metab. 1992;74:1184 –1188.
Petraglia F, Anceschi MM, Calzá L, et al. Inhibin and activin in human fetal membranes: evidence for a local effect
on prostaglandin release. J Clin Endocrinol Metab. 1993;
77:542–548.
Lockwood GM, Ledger WL, Barlow DH, Groome NP,
Muttukrishna S. Measurement of inhibin and activin in
early human pregnancy: demonstration of fetoplacental
origin and role in prediction of early-pregnancy outcome.
Biol Reprod. 1997;57:1490 –1494.
Treetampinich C, O’Connor AE, MacLachlan V, Groome
NP, de Kretser DM. Maternal serum inhibin A concentrations in early pregnancy after IVF and embryo transfer
reflect the corpus luteum contribution and pregnancy outcome. Hum Reprod. 2000;15:2028 –2032.
Muttukrishna S, Jauniaux E, Greenwold N, et al. Circulating levels of inhibin A, activin A and follistatin in missed
and recurrent miscarriages. Hum Reprod. 2002;17:3072–
3078.
Hauzman E, Fedorcsák P, Klinga K, et al. Use of serum
inhibin A and human chorionic gonadotropin measurements to predict the outcome of in vitro fertilization pregnancies. Fertil Steril. 2004;81:66 –72.
Goldner TE, Lawson HW, Xia Z, Atrash HK. Surveillance
for ectopic pregnancy–United States, 1970–1989. MMWR
CDC Surveill Summ. 1993;42:73–85.
Seifer DB, Lambert-Messerlian GM, Canick JA, Frishman
GN, Schneyer AL. Serum inhibin levels are lower in ectopic
than intrauterine spontaneously conceived pregnancies.
Fertil Steril. 1996;65:667– 669.
Horne AW, van den Driesche S, King AE, et al. Endometrial inhibin/activin ␤-B subunit expression is related to
decidualization and is reduced in tubal ectopic pregnancy.
J Clin Endocrinol Metab. 2008;93:2375–2382.
Segal S, Gor H, Correa N, Mercado R, Veenstra K, Rivnay
B. Inhibin A: marker for diagnosis of ectopic and early
abnormal pregnancies. Reprod Biomed Online. 2008;17:
789 –794.
Refaat B, Amer S, Ola B, Chapman N, Ledger W. The
expression of activin-␤A- and -␤B-subunits, follistatin, and
activin type II receptors in fallopian tubes bearing an ectopic pregnancy. J Clin Endocrinol Metab. 2008;93:293–
299.
Irving C, Basu A, Richmond S, Burn J, Wren C. Twentyyear trends in prevalence and survival of Down syndrome.
Eur J Hum Genet. 2008;16:1336 –1340.
Wald NJ, Hackshaw AK. Combining ultrasound and bio-
doi: 10.1210/er.2014-1003
430.
431.
432.
433.
434.
435.
436.
437.
438.
439.
440.
441.
442.
443.
444.
445.
chemistry in first-trimester screening for Down’s syndrome. Prenat Diagn. 1997;17:821– 829.
American College of Obstetricians and Gynecologists.
ACOG Practice Bulletin No. 88, December 2007. Invasive
prenatal testing for aneuploidy. Obstet Gynecol. 2007;
110:1459 –1467.
Van Lith JM, Pratt JJ, Beekhuis JR, Mantingh A. Secondtrimester maternal serum immunoreactive inhibin as a
marker for fetal Down’s syndrome. Prenat Diagn. 1992;
12:801– 806.
Lambert-Messerlian GM, Canick JA, Palomaki GE, Schneyer AL. Second trimester levels of maternal serum inhibin A, total inhibin, ␣ inhibin precursor, and activin in
Down’s syndrome pregnancy. J Med Screen. 1996;3:
58 – 62.
Wallace EM, Grant VE, Swanston IA, Groome NP. Evaluation of maternal serum dimeric inhibin A as a first-trimester marker of Down’s syndrome. Prenat Diagn. 1995;
15:359 –362.
Cuckle HS, Holding S, Jones R. Maternal serum inhibin
levels in second-trimester Down’s syndrome pregnancies.
Prenat Diagn. 1994;14:387–390.
Spencer K, Wood PJ, Anthony FW. Elevated levels of maternal serum inhibin immunoreactivity in second trimester
pregnancies affected by Down’s syndrome. Ann Clin
Biochem. 1993;30:219 –220.
Aitken DA, Wallace EM, Crossley JA, et al. Dimeric inhibin A as a marker for Down’s syndrome in early pregnancy. N Engl J Med. 1996;334:1231–1236.
Wald NJ, Densem JW, George L, Muttukrishna S, Knight
PG. Prenatal screening for Down’s syndrome using inhibin-A as a serum marker. Prenat Diagn. 1996;16:143–
153.
Cuckle HS, Wald NJ, Lindenbaum RH. Maternal serum
␣-fetoprotein measurement: a screening test for Down syndrome. Lancet. 1984;1:926 –929.
Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum
unconjugated oestriol as an antenatal screening test for
Down’s Syndrome. Br J Obstet Gynaecol. 1988;95:334 –
341.
Kipp JL, Lambert-Messerlian G, Eklund E, Rodriguez G,
Demczuk M, Gundogan F. Expression of transcription factors controlling ␣ inhibin gene expression in placental tissues from pregnancies affected by fetal Down syndrome.
Prenat Diagn. 2012;32:302–305.
Sibai BM, Ewell M, Levine RJ, et al. Risk factors associated
with preeclampsia in healthy nulliparous women. The Calcium for Preeclampsia Prevention (CPEP) Study Group.
Am J Obstet Gynecol. 1997;177:1003–1010.
Cnossen JS, ter Riet G, Mol BW, et al. Are tests for predicting pre-eclampsia good enough to make screening viable? A review of reviews and critical appraisal. Acta Obstet Gynecol Scand. 2009;88:758 –765.
Muttukrishna S, Knight PG, Groome NP, Redman CW,
Ledger WL. Activin A and inhibin A as possible endocrine
markers for pre-eclampsia. Lancet. 1997;349:1285–1288.
Kang JH, Farina A, Park JH, et al. Down syndrome biochemical markers and screening for preeclampsia at first
and second trimester: correlation with the week of onset
and the severity. Prenat Diagn. 2008;28:704 –709.
Muttukrishna S, North RA, Morris J, et al. Serum inhibin
edrv.endojournals.org
446.
447.
448.
449.
450.
451.
452.
453.
454.
455.
456.
457.
458.
459.
460.
791
A and activin A are elevated prior to the onset of preeclampsia. Hum Reprod. 2000;15:1640 –1645.
Lambert-Messerlian GM, Palomaki GE, Neveux LM, et al.
Early onset preeclampsia and second trimester serum
markers. Prenat Diagn. 2009;29:1109 –1117.
Levine RJ, Lam C, Qian C, et al. Soluble endoglin and other
circulating antiangiogenic factors in preeclampsia. N Engl
J Med. 2006;355:992–1005.
Wright VC, Chang J, Jeng G, Macaluso M. Assisted reproductive technology surveillance–United States, 2005.
MMWR Surveill Summ. 2008;57:1–23.
Coccia ME, Rizzello F. Ovarian reserve. Ann N Y Acad Sci.
2008;1127:27–30.
Seifer DB, Scott RT Jr, Bergh PA, et al. Women with declining ovarian reserve may demonstrate a decrease in day
3 serum inhibin B before a rise in day 3 follicle-stimulating
hormone. Fertil Steril. 1999;72:63– 65.
Ocal P, Aydin S, Cepni I, et al. Follicular fluid concentrations of vascular endothelial growth factor, inhibin A and
inhibin B in IVF cycles: are they markers for ovarian response and pregnancy outcome? Eur J Obstet Gynecol
Reprod Biol. 2004;115:194 –199.
Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner
AC, Blazar AS, Berk CA. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil
Steril. 1997;67:110 –114.
Creus M, Peñarrubia J, Fábregues F, et al. Day 3 serum
inhibin B and FSH and age as predictors of assisted reproduction treatment outcome. Hum Reprod. 2000;15:2341–
2346.
Welt CK, McNicholl DJ, Taylor AE, Hall JE. Female reproductive aging is marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab. 1999;84:105–
111.
Yong PY, Baird DT, Thong KJ, McNeilly AS, Anderson
RA. Prospective analysis of the relationships between the
ovarian follicle cohort and basal FSH concentration, the
inhibin response to exogenous FSH and ovarian follicle
number at different stages of the normal menstrual cycle
and after pituitary down-regulation. Hum Reprod. 2003;
18:35– 44.
Anderson RA, Nelson SM, Wallace WH. Measuring antiMüllerian hormone for the assessment of ovarian reserve:
when and for whom is it indicated? Maturitas. 2012;71:
28 –33.
Buckler HM, McLachlan RI, MacLachlan VB, Healy DL,
Burger HG. Serum inhibin levels in polycystic ovary syndrome: basal levels and response to luteinizing hormonereleasing hormone agonist and exogenous gonadotropin
administration. J Clin Endocrinol Metab. 1988;66:798 –
803.
Falcone T, Billiar R, Morris D. Serum inhibin levels in
polycystic ovary syndrome: effect of insulin resistance and
insulin secretion. Obstet Gynecol. 1991;78:171–176.
Lambert-Messerlian GM, Hall JE, Sluss PM, et al. Relatively low levels of dimeric inhibin circulate in men and
women with polycystic ovarian syndrome using a specific
two-site enzyme-linked immunosorbent assay. J Clin Endocrinol Metab. 1994;79:45–50.
Pigny P, Cortet-Rudelli C, Decanter C, et al. Serum levels
of inhibins are differentially altered in patients with poly-
792
461.
462.
463.
464.
465.
466.
467.
468.
469.
470.
471.
472.
473.
474.
475.
Makanji et al
Inhibin at 90, a Historical Review
cystic ovary syndrome: effects of being overweight and
relevance to hyperandrogenism. Fertil Steril. 2000;73:
972–977.
Cortet-Rudelli C, Pigny P, Decanter C, et al. Obesity and
serum luteinizing hormone level have an independent and
opposite effect on the serum inhibin B level in patients with
polycystic ovary syndrome. Fertil Steril. 2002;77:281–
287.
Welt CK, Taylor AE, Fox J, Messerlian GM, Adams JM,
Schneyer AL. Follicular arrest in polycystic ovary syndrome is associated with deficient inhibin A and B biosynthesis. J Clin Endocrinol Metab. 2005;90:5582–5587.
Fujiwara T, Sidis Y, Welt C, et al. Dynamics of inhibin
subunit and follistatin mRNA during development of normal and polycystic ovary syndrome follicles. J Clin Endocrinol Metab. 2001;86:4206 – 4215.
Anderson RA, Groome NP, Baird DT. Inhibin A and inhibin B in women with polycystic ovarian syndrome during
treatment with FSH to induce mono-ovulation. Clin Endocrinol (Oxf). 1998;48:577–584.
Lockwood GM, Muttukrishna S, Groome NP, Matthews
DR, Ledger WL. Mid-follicular phase pulses of inhibin B
are absent in polycystic ovarian syndrome and are initiated
by successful laparoscopic ovarian diathermy: a possible
mechanism regulating emergence of the dominant follicle.
J Clin Endocrinol Metab. 1998;83:1730 –1735.
Norman RJ, Milner CR, Groome NP, Robertson DM. Circulating follistatin concentrations are higher and activin
concentrations are lower in polycystic ovarian syndrome.
Hum Reprod. 2001;16:668 – 672.
Tsigkou A, Luisi S, De Leo V, et al. High serum concentration of total inhibin in polycystic ovary syndrome. Fertil
Steril. 2008;90:1859 –1863.
Shelling AN, Burton KA, Chand AL, et al. Inhibin: a candidate gene for premature ovarian failure. Hum Reprod.
2000;15:2644 –2649.
Welt CK, Hall JE, Adams JM, Taylor AE. Relationship of
estradiol and inhibin to the follicle-stimulating hormone
variability in hypergonadotropic hypogonadism or premature ovarian failure. J Clin Endocrinol Metab. 2005;90:
826 – 830.
Marozzi A, Porta C, Vegetti W, et al. Mutation analysis of
the inhibin ␣ gene in a cohort of Italian women affected by
ovarian failure. Hum Reprod. 2002;17:1741–1745.
Dixit H, Deendayal M, Singh L. Mutational analysis of the
mature peptide region of inhibin genes in Indian women
with ovarian failure. Hum Reprod. 2004;19:1760 –1764.
Dixit H, Rao KL, Padmalatha V, et al. Expansion of the
germline analysis for the INHA gene in Indian women with
ovarian failure. Hum Reprod. 2006;21:1643–1644.
Chand AL, Ooi GT, Harrison CA, Shelling AN, Robertson
DM. Functional analysis of the human inhibin ␣ subunit
variant A257T and its potential role in premature ovarian
failure. Hum Reprod. 2007;22:3241–3248.
Robertson DM, Pruysers E, Jobling T. Inhibin as a diagnostic marker for ovarian cancer. Cancer Lett. 2007;249:
14 –17.
Burger HG, Robertson DM, Cahir N, et al. Characterization of inhibin immunoreactivity in post-menopausal
women with ovarian tumours. Clin Endocrinol (Oxf).
1996;44:413– 418.
Endocrine Reviews, October 2014, 35(5):747–794
476. Petraglia F, Luisi S, Pautier P, et al. Inhibin B is the major
form of inhibin/activin family secreted by granulosa cell
tumors. J Clin Endocrinol Metab. 1998;83:1029 –1032.
477. Robertson DM, Cahir N, Burger HG, Mamers P, Groome
N. Inhibin forms in serum from postmenopausal women
with ovarian cancers. Clin Endocrinol (Oxf). 1999;50:
381–386.
478. Lappöhn RE, Burger HG, Bouma J, Bangah M, Krans M,
de Bruijn HW. Inhibin as a marker for granulosa-cell tumors. N Engl J Med. 1989;321:790 –793.
479. Healy DL, Burger HG, Mamers P, et al. Elevated serum
inhibin concentrations in postmenopausal women with
ovarian tumors. N Engl J Med. 1993;329:1539 –1542.
480. Frias AE Jr, Li H, Keeney GL, Podratz KC, Woodruff TK.
Preoperative serum level of inhibin A is an independent
prognostic factor for the survival of postmenopausal
women with epithelial ovarian carcinoma. Cancer. 1999;
85:465– 471.
481. Do TV, Kubba LA, Antenos M, Rademaker AW, Sturgis
CD, Woodruff TK. The role of activin A and Akt/GSK
signaling in ovarian tumor biology. Endocrinology. 2008;
149:3809 –3816.
482. Hempel N, How T, Dong M, Murphy SK, Fields TA, Blobe
GC. Loss of betaglycan expression in ovarian cancer: role
in motility and invasion. Cancer Res. 2007;67:5231–
5238.
483. Whitehouse C, Solomon E. Current status of the molecular
characterization of the ovarian cancer antigen CA125 and
implications for its use in clinical screening. Gynecol Oncol. 2003;88:S152–S157.
484. Burdette JE, Jeruss JS, Kurley SJ, Lee EJ, Woodruff TK.
Activin A mediates growth inhibition and cell cycle arrest
through Smads in human breast cancer cells. Cancer Res.
2005;65:7968 –7975.
485. Reis FM, Cobellis L, Tameirão LC, et al. Serum and tissue
expression of activin A in postmenopausal women with
breast cancer. J Clin Endocrinol Metab. 2002;87:2277–
2282.
486. Reis FM, Luisi S, Carneiro MM, et al. Activin, inhibin and
the human breast. Mol Cell Endocrinol. 2004;225:77– 82.
487. Anders C, Marcom PK, Peterson B, et al. A pilot study of
predictive markers of chemotherapy-related amenorrhea
among premenopausal women with early stage breast cancer. Cancer Invest. 2008;26:286 –295.
488. Santoro N. The menopausal transition. Am J Med. 2005;
118(suppl 12B):8 –13.
489. Burger HG, Hale GE, Robertson DM, Dennerstein L. A
review of hormonal changes during the menopausal transition: focus on findings from the Melbourne Women’s
Midlife Health Project. Hum Reprod Update. 2007;13:
559 –565.
490. Robertson DM, Burger HG. Reproductive hormones: ageing and the perimenopause. Acta Obstet Gynecol Scand.
2002;81:612– 616.
491. Soules MR, Sherman S, Parrott E, et al. Executive summary: Stages of Reproductive Aging Workshop (STRAW).
Fertil Steril. 2001;76:874 – 878.
492. Gougeon A, Ecochard R, Thalabard JC. Age-related
changes of the population of human ovarian follicles: increase in the disappearance rate of non-growing and early-
doi: 10.1210/er.2014-1003
493.
494.
495.
496.
497.
498.
499.
500.
501.
502.
503.
504.
505.
506.
growing follicles in aging women. Biol Reprod. 1994;50:
653– 663.
Burger HG, Hale GE, Dennerstein L, Robertson DM. Cycle and hormone changes during perimenopause: the key
role of ovarian function. Menopause. 2008;15:603– 612.
Klein NA, Houmard BS, Hansen KR, et al. Age-related
analysis of inhibin A, inhibin B, and activin A relative to the
intercycle monotropic follicle-stimulating hormone rise in
normal ovulatory women. J Clin Endocrinol Metab. 2004;
89:2977–2981.
Robertson DM, Hale GE, Jolley D, Fraser IS, Hughes CL,
Burger HG. Interrelationships between ovarian and pituitary hormones in ovulatory menstrual cycles across reproductive age. J Clin Endocrinol Metab. 2009;94:138 –
144.
Robertson DM, Hale GE, Fraser IS, Hughes CL, Burger
HG. A proposed classification system for menstrual cycles
in the menopause transition based on changes in serum
hormone profiles. Menopause. 2008;15:1139 –1144.
Klein NA, Battaglia DE, Woodruff TK, et al. Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial
growth factor in spontaneous menstrual cycles of normal
women of advanced reproductive age. J Clin Endocrinol
Metab. 2000;85:4520 – 4525.
Reame NE, Wyman TL, Phillips DJ, de Kretser DM, Padmanabhan V. Net increase in stimulatory input resulting
from a decrease in inhibin B and an increase in activin A
may contribute in part to the rise in follicular phase folliclestimulating hormone of aging cycling women. J Clin Endocrinol Metab. 1998;83:3302–3307.
Danforth DR, Arbogast LK, Mroueh J, et al. Dimeric inhibin: a direct marker of ovarian aging. Fertil Steril. 1998;
70:119 –123.
Burger HG, Dudley EC, Hopper JL, et al. The endocrinology of the menopausal transition: a cross-sectional study of
a population-based sample. J Clin Endocrinol Metab.
1995;80:3537–3545.
Burger HG, Cahir N, Robertson DM, et al. Serum inhibins
A and B fall differentially as FSH rises in perimenopausal
women. Clin Endocrinol (Oxf). 1998;48:809 – 813.
Andersson AM, Müller J, Skakkebaek NE. Different roles
of prepubertal and postpubertal germ cells and Sertoli cells
in the regulation of serum inhibin B levels. J Clin Endocrinol Metab. 1998;83:4451– 4458.
Carreau S. Human Sertoli cells produce inhibin in vitro: an
additional marker to assess the seminiferous epithelium
development. Hum Reprod. 1995;10:1947–1949.
Toulis KA, Iliadou PK, Venetis CA, et al. Inhibin B and
anti-Mullerian hormone as markers of persistent spermatogenesis in men with non-obstructive azoospermia: a
meta-analysis of diagnostic accuracy studies. Hum Reprod
Update. 2010;16:713–724.
Yu AW, Shao LE, Frigon NL Jr, Yu J. Detection of functional and dimeric activin A in human marrow microenvironment. Implications for the modulation of erythropoiesis. Ann N Y Acad Sci. 1994;718:285–298; discussion
298 –299.
Kolb BA, Stanczyk FZ, Sokol RZ. Serum inhibin B levels in
edrv.endojournals.org
507.
508.
509.
510.
511.
512.
513.
514.
515.
516.
517.
518.
519.
520.
521.
522.
793
males with gonadal dysfunction. Fertil Steril. 2000;74:
234 –238.
Klingmüller D, Haidl G. Inhibin B in men with normal and
disturbed spermatogenesis. Hum Reprod. 1997;12:2376 –
2378.
Jørgensen N, Liu F, Andersson AM, et al. Serum inhibin-B
in fertile men is strongly correlated with low but not high
sperm counts: a coordinated study of 1,797 European and
US men. Fertil Steril. 2010;94:2128 –2134.
Mitchell V, Boitrelle F, Pigny P, et al. Seminal plasma levels
of anti-Müllerian hormone and inhibin B are not predictive
of testicular sperm retrieval in nonobstructive azoospermia: a study of 139 men. Fertil Steril. 2010;94:2147–2150.
Stenvers KL, Tursky ML, Harder KW, et al. Heart and liver
defects and reduced transforming growth factor ␤2 sensitivity in transforming growth factor ␤ type III receptordeficient embryos. Mol Cell Biol. 2003;23:4371– 4385.
Li Q, Karam SM, Coerver KA, Matzuk MM, Gordon JI.
Stimulation of activin receptor II signaling pathways inhibits differentiation of multiple gastric epithelial lineages.
Mol Endocrinol. 1998;12:181–192.
Kumar TR, Agno J, Janovick JA, Conn PM, Matzuk MM.
Regulation of FSH␤ and GnRH receptor gene expression
in activin receptor II knockout male mice. Mol Cell Endocrinol. 2003;212:19 –27.
Yan W, Burns KH, Matzuk MM. Genetic engineering to
study testicular tumorigenesis. APMIS. 2003;111:174 –
181; discussion 182–183.
Matzuk MM, Finegold MJ, Mishina Y, Bradley A, Behringer RR. Synergistic effects of inhibins and müllerianinhibiting substance on testicular tumorigenesis. Mol Endocrinol. 1995;9:1337–1345.
Mishina Y, Rey R, Finegold MJ, et al. Genetic analysis of
the Müllerian-inhibiting substance signal transduction
pathway in mammalian sexual differentiation. Genes Dev.
1996;10:2577–2587.
Shou W, Woodruff TK, Matzuk MM. Role of androgens
in testicular tumor development in inhibin-deficient mice.
Endocrinology. 1997;138:5000 –5005.
Li Q, Kumar R, Underwood K, et al. Prevention of cachexia-like syndrome development and reduction of tumor
progression in inhibin-deficient mice following administration of a chimeric activin receptor type II-murine Fc
protein. Mol Hum Reprod. 2007;13:675– 683.
Cipriano SC, Chen L, Burns KH, Koff A, Matzuk MM.
Inhibin and p27 interact to regulate gonadal tumorigenesis. Mol Endocrinol. 2001;15:985–996.
Burns KH, Agno JE, Sicinski P, Matzuk MM. Cyclin D2
and p27 are tissue-specific regulators of tumorigenesis in
inhibin ␣ knockout mice. Mol Endocrinol. 2003;17:2053–
2069.
Gold E, Marino FE, Harrison C, Makanji Y, Risbridger G.
Activin-␤(c) reduces reproductive tumour progression and
abolishes cancer-associated cachexia in inhibin-deficient
mice. J Pathol. 2013;229:599 – 607.
DePaolo LV, Bicsak TA, Erickson GF, Shimasaki S, Ling
N. Follistatin and activin: a potential intrinsic regulatory
system within diverse tissues. Proc Soc Exp Biol Med.
1991;198:500 –512.
He WW, Gustafson ML, Hirobe S, Donahoe PK. Developmental expression of four novel serine/threonine kinase
794
523.
524.
525.
526.
527.
528.
529.
530.
531.
532.
533.
534.
Makanji et al
Inhibin at 90, a Historical Review
receptors homologous to the activin/transforming growth
factor-␤ type II receptor family. Dev Dyn. 1993;196:133–
142.
Matsuzaki K, Xu J, Wang F, McKeehan WL, Krummen L,
Kan M. A widely expressed transmembrane serine/threonine kinase that does not bind activin, inhibin, transforming growth factor ␤, or bone morphogenic factor. J Biol
Chem. 1993;268:12719 –12723.
Alexander JM, Bikkal HA, Zervas NT, Laws ER Jr, Klibanski A. Tumor-specific expression and alternate splicing
of messenger ribonucleic acid encoding activin/transforming growth factor-␤ receptors in human pituitary adenomas. J Clin Endocrinol Metab. 1996;81:783–790.
Wang H, Tsang BK. Nodal signalling and apoptosis. Reproduction. 2007;133:847– 853.
Anderson RA, Cambray N, Hartley PS, McNeilly AS. Expression and localization of inhibin ␣, inhibin/activin ␤A
and ␤B and the activin type II and inhibin beta-glycan receptors in the developing human testis. Reproduction.
2002;123:779 –788.
Tuuri T, Erämaa M, Hildén K, Ritvos O. The tissue distribution of activin ␤A- and ␤B-subunit and follistatin messenger ribonucleic acids suggests multiple sites of action for
the activin-follistatin system during human development.
J Clin Endocrinol Metab. 1994;78:1521–1524.
Walsh S, Metter EJ, Ferrucci L, Roth SM. Activin-type II
receptor B (ACVR2B) and follistatin haplotype associations with muscle mass and strength in humans. J Appl
Physiol. 2007;102:2142–2148.
Yndestad A, Ueland T, Øie E, et al. Elevated levels of activin A in heart failure: potential role in myocardial remodeling. Circulation. 2004;109:1379 –1385.
Ohga E, Matsuse T, Teramoto S, Ouchi Y. Activin receptors are expressed on human lung fibroblast and activin A
facilitates fibroblast-mediated collagen gel contraction.
Life Sci. 2000;66:1603–1613.
Martins da Silva SJ, Bayne RA, Cambray N, Hartley PS,
McNeilly AS, Anderson RA. Expression of activin subunits
and receptors in the developing human ovary: activin A
promotes germ cell survival and proliferation before primordial follicle formation. Dev Biol. 2004;266:334 –345.
van Schaik RH, Wierikx CD, Timmerman MA, et al. Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues. Br J
Cancer. 2000;82:112–117.
Bondestam J, Kaivo-oja N, Kallio J, et al. Engagement of
activin and bone morphogenetic protein signaling pathway
Smad proteins in the induction of inhibin B production in
ovarian granulosa cells. Mol Cell Endocrinol. 2002;195:
79 – 88.
Sidis Y, Fujiwara T, Leykin L, Isaacson K, Toth T, Sch-
Endocrine Reviews, October 2014, 35(5):747–794
535.
536.
537.
538.
539.
540.
541.
542.
543.
544.
545.
neyer AL. Characterization of inhibin/activin subunit, activin receptor, and follistatin messenger ribonucleic acid in
human and mouse oocytes: evidence for activin’s paracrine
signaling from granulosa cells to oocytes. Biol Reprod.
1998;59:807– 812.
Roberts VJ, Barth S, el-Roeiy A, Yen SS. Expression of
inhibin/activin system messenger ribonucleic acids and
proteins in ovarian follicles from women with polycystic
ovarian syndrome. J Clin Endocrinol Metab. 1994;79:
1434 –1439.
Wada M, Shintani Y, Kosaka M, Sano T, Hizawa K, Saito
S. Immunohistochemical localization of activin A and follistatin in human tissues. Endocr J. 1996;43:375–385.
Haddad G, Penabad JL, Bashey HM, et al. Expression of
activin/inhibin subunit messenger ribonucleic acids by gonadotroph adenomas. J Clin Endocrinol Metab. 1994;79:
1399 –1403.
Penabad JL, Bashey HM, Asa SL, et al. Decreased follistatin gene expression in gonadotroph adenomas. J Clin Endocrinol Metab. 1996;81:3397–3403.
Chen CC, Johnson PA. Expression of inhibin ␣ and inhibin/activin ␤ A subunits in the granulosa layer of the large
preovulatory follicles of the hen. Biol Reprod. 1996;55:
450 – 454.
Di Loreto C, Reis FM, Cataldi P, et al. Human mammary
gland and breast carcinoma contain immunoreactive inhibin/activin subunits: evidence for a secretion into cystic
fluid. Eur J Endocrinol. 1999;141:190 –194.
La Rosa S, Uccella S, Billo P, Facco C, Sessa F, Capella C.
Immunohistochemical localization of ␣- and ␤A-subunits
of inhibin/activin in human normal endocrine cells and
related tumors of the digestive system. Virchows Arch.
1999;434:29 –36.
La Rosa S, Uccella S, Marchet S, Capella C, Lloyd RV.
Localization of inhibins and activins in normal endocrine
cells and endocrine tumors of the gut and pancreas: an
immunohistochemical and in situ hybridization study.
J Histochem Cytochem. 2004;52:217–225.
Jeruss JS, Santiago JY, Woodruff TK. Localization of activin and inhibin subunits, receptors and SMADs in the
mouse mammary gland. Mol Cell Endocrinol. 2003;203:
185–196.
Pangas SA, Rademaker AW, Fishman DA, Woodruff TK.
Localization of the activin signal transduction components
in normal human ovarian follicles: implications for autocrine and paracrine signaling in the ovary. J Clin Endocrinol Metab. 2002;87:2644 –2657.
Wang H, Jiang JY, Zhu C, Peng C, Tsang BK. Role and
regulation of nodal/activin receptor-like kinase 7 signaling
pathway in the control of ovarian follicular atresia. Mol
Endocrinol. 2006;20:2469 –2482.