Integrative and Comparative Biology, volume 50, number 1, pp. 53–62
doi:10.1093/icb/icq047
SYMPOSIUM
Evolution of the Reproductive Endocrine System in Chordates
Kaoru Kubokawa,1,* Yukiko Tando† and Sonali Roy‡
*Misaki Marine Biological Station, University of Tokyo, Koajiro, Miura 238-0225, Japan; †Jichi Medical University,
Tochigi, 329-0498 Japan; ‡North East Institute of Science and Technology, CSIR, Jorhat 785006, Assam, India
From the symposium ‘‘Insights of Early Chordate Genomics: Endocrinology and Development in Amphioxus,
Tunicates and Lampreys’’ presented at the annual meeting of the Society for Integrative and Comparative Biology,
January 3–7, 2010 at Seattle, Washington.
1
E-mail: [email protected]
Introduction
Many differences are encountered between invertebrates and vertebrates when the evolution of the
endocrine system is considered. For example, vertebrates have a hypothalamus and pituitary gland, and
the interaction between these organs comprises a
unique neuroendocrinal control system, which is referred to as the hypothalamus–pituitary axis. On the
other hand, invertebrates do not have a similar neuroendocrinal control system. The hypothalamus and
pituitary gland secrete distinctive hormones.
Molecules homologous to some of these hormones
have been reported in invertebrates. Therefore, in the
evolutional aspects, the origins of the abovementioned organs have been surveyed in primitive
vertebrates such as hagfish and lamprey, and primitive chordates such as cephalochordates and
urochordates.
In the present review, we assumed that amphioxus
has an endocrine system comparable to the hypothalamus–pituitary–gonadal axis of vertebrates for
controlling reproduction, and we compared these
systems between amphioxus and vertebrates. Here,
we describe (i) the characteristics of thyrostimulin
subunits of amphioxus, including a new result on
the third subunit (Tando and Kubokawa 2009a,
2009b), (ii) the localization of transcripts of thyrostimulin subunit genes and vasotocin (VT) gene, since
VT is important for reproduction in lower vertebrates (Tando 2010), and (iii) the sex steroidogenic
pathway in the amphioxus ovary (Mizuta and
Kubokawa 2007; Mizuta et al. 2008).
Hatschek’s pit
Hatschek’s pit, a unique organ in cephalochordates,
is a small epithelial evagination of the oral cavity that
has a densely ciliated external surface. It is located
under the notochord, and hence, does not come in
contact with the nerve cord, despite a portion of the
nerve cord protruding toward the pit (Gorbman
et al. 2005). Nonetheless, on the basis of anatomical
comparisons, it has long been considered to be
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ß The Author(s) 2010. Published by Oxford University Press.
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Synopsis The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate
clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the
presence an endocrine system comparable to the pituitary–gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have
any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone
consisting of a and b subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a
synteny suggest that amphioxus’ thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The
conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified
in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and
sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and
reproductive functions in the neuroendocrine control system of chordates.
54
Amphioxus has hormones homologous
to vertebrate hormones
The genome sequences of amphioxus were compared
with those of humans by synteny analyses, and resulted
in the conclusion that amphioxus and humans evolved
from a common ancestor (Putnam et al. 2008). This
phylogenetic relationship helped us to find homologies
between the endocrine systems of amphioxus and vertebrates. The study of the evolution of amphioxus thus
provides a good model for understanding the evolution of the endocrine system in chordates.
Urochordates are the closest animals to vertebrates, however, genome analysis of Ciona intestinalis
revealed that the genes encoding endocrine substances were not identical to those of vertebrates
(or to those of many invertebrates) except genes
for gonadotropin-releasing hormones (GnRHs) and
their receptors (GnRHRs) (Sherwood 2006). The
Ciona genome is considered to have discarded the
homologous genes related to the endocrine system
of vertebrates during the evolution from the ancestor
of chordates.
The annotation of the amphioxus’ genome
indicated that candidates for hormone genes in
amphioxus homologous to those of vertebrates
are: thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), GnRHR, thyrostimulin, VT, estrogen receptor (ER), and steroid
receptor (SR) (Holland et al. 2008). Despite the
lack of a GnRH homolog gene, cDNAs for four
GnRHRs have been cloned (Tello and Sherwood
2009). GnRHRs expressed in culture cells were stimulated to increase the accumulation of inositol phosphate with different ratios depending on the type of
receptor. However, amphioxus GnRH or the ligand
of GnRHR remained to be purified from the anterior
part of the nerve cord, which is the candidate for
forebrain homolog.
Hypothalamus–pituitary–gonadal axis
TRH, one of hypothalamic hormones found in the
genome, is a member of the hypothalamus–
pituitary–thyroid axis in vertebrates. In amphioxus
larvae, thyroid hormone induces metamorphosis
(Paris and Laudet 2008; Paris et al. 2008a), suggesting that amphioxus has a metabolic pathway of
thyroid hormone similar to that in vertebrates.
However, homologs of genes for thyroidstimulating hormone (TSH) and thyroglobuline
were not found in the genome (Holland et al.
2008).
Amphioxus has two distinct sexes, and breeds
once a year in summer. Effects of sex steroids
in vivo were investigated in the gonads (Fang and
Wang 1984); however, neither peptidergic nor proteinaceous endocrine substances, which controls
reproductive functions of amphioxus, was not detected, even after complete annotation of the
genome sequences. Therefore, it is important to
find these substances in amphioxus, particularly in
terms of investigating the origin of the pituitary
gland and of homologs of glycoprotein hormone
(GPH). The most plausible candidate for ancestral
GPH is thyrostimulin, because only the presence of
thyrostimulin was predicted by analyses of the
genome database.
Amphioxus’ thyrostimulin
Pituitary hormones responsible for the control of
reproductive functions consist of two heterodimeric
glycoproteins named as gonadotropins (GTH). The
survey of the amphioxus genome indicated that only
thyrostimulin is a homolog of pituitary GPHs
(Holland et al. 2008; Tando 2010). Thyrostimulin
is a GPH that has been recently discovered in
humans. It is composed of two subunits similar to
the vertebrate GPHs identified until now. The subunits of thyrostimulin are GPA2 as a subunit, and
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homologous to the pituitary gland. This idea is supported by the following evidences: the cells of
Hatschek’s pit contain at least two types of peptidergic granules of different diameters (Tjoa et al. 1974;
Sahlin 1988); the morphology and developmental
process are similar to those of the Rathke’s pouch
of the vertebrate embryo, that is, the invagination of
the placode of the anterior pituitary in the oral ectoderm (Goorich 1918); the pit is weakly immunostained with antiserum against LHb subunit (Nozaki
and Gorbman 1992); and then amphioxus larvae
show similar expression patterns of genes related to
the development of the pituitary gland, that is, Pit-1
(Candiani and Pestarino 1998), Pitx family genes
(Yasui et al. 2000; Boorman and Shimeld 2002),
and Pax6 (Glardon et al. 1998). Physiologically, extraction of the pit stimulates gonadal development
and spawning in amphioxus (Fang and Wang 1984).
A few researchers have reported unfavorable evidence for the homology between Hatschek’s pit and
the pituitary gland. The rostral region of the anterior
vesicle, which corresponds to the brain in amphioxus, is asymmetrically located on the right side of the
notochord (Ruppart 1997). Furthermore, genome
database analysis of amphioxus revealed no gene
encoding homologs of the pituitary hormone
(Holland et al. 2008).
K. Kubokawa et al.
Reproductive endocrinology in amphioxus
and stimulates intracellular signaling in vitro
(Nakabayashi et al. 2002). Possibly, thyrostimulin
regulates the secretion of thyroid hormone
(Macdonald et al. 2005; Okada 2006), but it is not
a major member of the typical hypothalamus–pituitary–thyroid axis. The function of thyrostimulin is
unclear in invertebrates; however, its presence suggests that it is an ancestral GPH of vertebrates.
The deduced amino-acid sequence of AmpGPA2
includes a putative asparagine-linked (N-linked) glycosylation site at Asn97. In contrast, AmpGPA2LP
has a longer N-terminal amino acid chain than
ampGPA2, but does not have any consensus sites
for N-linked glycosylation (Fig. 1A). AmpGPB5 has
a putative N-linked glycosylation site at Asn121 in
the C-terminal region. Nonchordate GPA2 and GPB5
do not have any glycosylation sites, but fly GPA2
does. Subunits of vertebrate GPHs include one
N-linked glycosylation site in TSH and LH, or two
sites in common a, FSHb, and hCGb (Fig. 1B).
These facts suggest that an ancestral protein hormone was not glycosylated; however, during the evolution of GPH in chordates, glycosylation sites were
Fig. 1 Alignments of amino acid sequences of thyrostimulin subunits in amphioxus and humans. (A) amphioxus GPA2 (ampGPA2),
amphioxus GPA2-like protein (ampGPA2LP), human a subunit (hGPA1), and human GPA2 (hGPA2) are aligned. (B) amphioxus GPB5
(ampGPB5), human GPB5 (hGPB5), human LHb subunit (hLHb), human FHSb subunit (hFSHb), and human TSHb subunit (hTSHb) are
aligned. Identical Cys are marked with asterisks or numbers under Cys. The numbers indicate the pairs of Cys that form presumed
disulfide bonds and that are necessary for construction of the cystine-knot structure. Putative N-linked glycosylation sites are shown as
inverted squares.
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GPB5 as b subunit (Nakabayashi et al. 2002). They
have the cystine knot motif ‘‘C(X. . .)CXGXC
(X. . ..)C(X. . ..)CXC’’, which is the consensus sequence among GPHs. Three S–S bonds contribute
to form the cystine knot motif (Vitt et al. 2001).
We cloned three homologous genes encoding
GPH subunits from the Japanese amphioxus,
Branchiostoma belcheri. These genes comprise two
a subunits, one of which is a newly found
GPA2-like protein (ampGPA2LP), and one b subunit
(Tando and Kubokawa 2009a, 2009b; Tando 2010).
Homologous genes for thyrostimulin are widely
distributed both in vertebrates and invertebrates, including nematodes and flies (Park et al. 2005; Sudo
et al. 2005). GPA2 and GPB5 genes were expressed
not only in the pituitary, but also in various tissues,
such as the brain, pancreas, placenta, and gonads in
humans, mice, and rats (Hsu et al. 2002;
Nakabayashi et al. 2002; Okada 2006). All GPHs,
including thyrostimulin, are noncovalently associated
with a strong and specific heterodimer of a and b
subunits (Isaacs 1995; Hearn and Gomme 2000). In
vertebrates, thyrostimulin binds to a TSH receptor
55
56
added one by one, and the number of sites increased
subsequently.
Synteny of thyrostimulin subunits in
the genome
syntenies among pufferfish, zebrafish, and amphioxus
showed partial exchanges of gene positions.
The results of synteny analysis between humans
and amphioxus demonstrated that FSHb, LHb, and
TSHb genes arose from GPB5 gene by gene duplication, and then interchromosomal arrangements of
genes may have occurred after two rounds of
whole-genome duplication in the vertebrate lineage.
When compared within the lengths of coding regions, the organization of genes for GPA2 and
GPB5 are well conserved. Different bilaterian taxa,
except for insects, also showed that the organization
of genes for GPA2 and GPB5 are well conserved (Dos
Santos et al. 2009). Taking all these things into consideration, we proposed that thyrostimulin is an ancestral molecule from which vertebrate GPH
diverged (Tando and Kubokawa 2009a, 2009b;
Tando 2010).
According to the genome annotation of B. floridae,
GPA2LP is considered to have three exons like GPA2.
Although amphioxus GPA2 and GPB5 genes are located in tandem in the same scaffold, GPA2LP gene
is located on a different scaffold. The GPA2LP likely
appeared in the amphioxus lineage after separation
from a common ancestor of chordates
Phylogenetic tree
Phylogenetic analysis on the basis of deduced
amino-acid sequences indicated that amphioxus
Fig. 2 Syntenies among GPH subunit genes in amphioxus and humans. Amphioxus GPA2 and GPB5 genes are located in tandem on
scaffold 8. Human GPA2 and FSHb genes are located in tandem on chromosome 11, while GPB5, TSHb, and LHb genes are solely
located on human chromosomes 1, 14, and 15, respectively. Amphioxus scaffold 2 contains ampGPA2LP and human chromosome 6
contains GPA1. The names of the protein family are shown at the top. The numbers in the small rectangular boxes on the scaffold of the
amphioxus genome are the IDs of the family protein described in the genome database. The numbers below the boxes indicate the loci
of genes. Chr: chromosome, Sc: scaffold.
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Conservation of the order of closely linked family
protein genes, or synteny, between GPA2 and GPB5
genes in amphioxus (B. floridae) and GPH subunit
genes in humans showed that amphioxus GPA2 and
GPB5 genes are ancestral to human GPH subunit
genes (Putnam et al. 2008).
Amphioxus GPA2 and GPB5 genes are located in
tandem on scaffold 8 in the genome data of B. floridae,
whereas GPA2LP is located on scaffold 2. The genes
of family proteins near GPB5 and GPA2 on scaffold 8
are also conserved in human chromosomes. Human
genes for seven proteins homologous to those on
B. floridae scaffold 8 are located on human
GPB-related chromosomes 1, 11, 14, and 19. These
homologous genes were not found in chromosome 6,
where the GPA1 gene is located. The family genes
that we used in synteny analysis were not found on
scaffold 2, where GPA2LP is located. Thus, our synteny analysis indicates that the orders of genes in
amphioxus and humans are highly conserved on
the chromosomes on which GPB genes are located
(Fig. 2). The gene orders near genes of TSHb,
FSHb, GPA2, and LHb are also well conserved between humans and mice; however, comparisons of
K. Kubokawa et al.
57
Reproductive endocrinology in amphioxus
Fig. 4 Schematic diagram of the evolution of GPHs. Ancestral
GPH may be an homologous molecule to GPH a and b subunits.
Thyrostimulin subunits, GPA2 and GPB5, are found in amphioxus
(Holland et al. 2008), lamprey, and gnathostomes. The ancestral
GPB5 diverged into GTHb subunit in lamprey (Sower et al. 2006)
and then into three GPHb subunits (FSHb, LHb, and TSHb) in
gnathostomes, and additional hCGb in mammals. The ancestral
GPH a subunit diverged into a subunit and GPA2 in vertebrates,
although a subunit of lamprey was not found.
GPA2 and GPA2LP are included in the GPA2 clade
and amphioxus GPB5 is included in the GPB5 clade
(Fig. 3). This result supports our hypothesis that
thyrostimulin is the ancestral molecule of the pituitary hormones and that it diverged in the vertebrate
lineage. An in silico survey showed that both GPA2
and GPB5 are present in metazoan genomes, and
that they are located close to each other in all nonvertebrate species (Dos Santos et al. 2009).
The important animals in the examination of the
evolution of GPH are agnathans. Sower et al. (2006)
and Freamat and Sower (2008) reported that lamprey
have GTHb subunit, thyrostimulin, and two GTH
receptors. They hypothesized that GTH and its
Expression of GPA2, GPA2LP,
and GPB5 genes
Gene expression profiles of GPA2, GPA2LP, and
GPB5 were examined in tissues of adult amphioxus.
First strand cDNAs from the head region, skin, gills
including endostyle, muscles, testes, and ovaries were
investigated using RT–PCR. In vertebrates, the gene
expression patterns of GPA2 and GPB5 in various
tissues such as the brain, gonads, placenta, and pancreas were reported by Nakabayashi et al. (2002),
Hsu et al. (2002), and Okada (2006). The results of
RT–PCR indicated the presence of GPA2 and GPB5
mRNAs in various tissues that we examined, whereas
GPA2LP was expressed only in the testes. This result
suggests that GPA2LP acts locally, seasonally, or with
physiological change. Further investigation of
GPA2LP is required for ascertaining the function of
GPA2LP and the evolution of GPHs.
Amphioxus’ VT
Most vertebrates have two neurohypophysial peptides, the vasopressin family and oxytocin family,
whereas agnathans and invertebrates have only peptide. VT is a member of the vasopressin family,
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Fig. 3 The unrooted molecular phylogenetic tree of GPH subunits constructed using the neighbor-joining method. The numbers indicate bootstrap probabilities in the 1000 replication trials
used to construct the tree. The bar represents an evolutionary
distance calculated by the Poisson correction. The DDBJ/EMBL/
GenBank accession numbers of sequences used for analysis are as
follows: Human GPA1 (J00152); Mouse GPA1 (J00643); Xenopus
GPA1 (L07619); Salmon GPA1 (AB050834); Human GPA2
(AF260739); Mouse GPA2 (AF260740); Puffer GPA2 (Q4S0U2);
Fly GPA2 (AY940435); Sea urchin GPA2 (15344); Human LHb
(NM000894); Mouse LHb (NM008497); Salmon LHb
(AB050836); Human FSHb (NM000510); Mouse FSHb
(NP032071); Salmon FSHb(AB050835); Human TSHb
(NM000549); Mouse TSHb (NM009432); Salmon TSHb
(AF060566); Human GPB5 (AF403430); Mouse GPB5
(NM175644); Fly GPB5 (AF403389); and Sea urchin GPB5
(DN791067).
receptor systems emerged as a link between the neurohormonal and peripheral control levels during the
early stages of gnathostome divergence. They further
proposed that pituitary GPHs diverged just after the
emergence of agnathans with the acquisition of the
pituitary as a unique endocrinal organ in vertebrates.
The two rounds of genome duplication may have
occurred before the lineage of agnathans, after
which two a subunit genes were lost (Fig. 4).
58
Distribution of thyrostimulin and VT
gene transcripts in the nerve cord
The nerve cord of amphioxus lies dorsally on the
notochord, and the apical part is called the anterior
vesicle, which shows distinctive architecture and corresponds to the brain of amphioxus (Ruppart 1997;
Wicht and Lacalli 2005). The anterior vesicle is composed of nerve cells that can be distinguished from
the other parts of the nerve cord (called the intercalated region) and the spinal cord. The dorsal expansion of the central canal is observed in the anterior
vesicle.
Thyrostimulin
Localization of GPA2, GPB5, and GPA2LP mRNAs in
the head region was examined by in situ hybridization (ISH). Signals for GPA2 and GPB5 mRNAs were
detected in several cells in the anterior region of the
nerve cord (Fig. 5A). RT–PCR revealed no signals for
GPA2LP in this region.
GPA2 and GPB5 positive cells are scattered in the
dorso-medial parts of the nerve cord (Fig. 5D and
E). Along the antero-posterior axis, ISH positive cells
are first observable at the level of myomere 3 and
disappear at the level of myomere 8. In lateral view,
several GPA2 or GPB5 expressing cells accumulate in
the close vicinity of particular discrete loci, although
the number of cells is different among the loci.
In transverse view, ISH-positive cells are also located
in the dorsal part along the central canal.
Amphioxus’ VT
Two populations of ampVT positive cells were identified in the anterior vesicle and the medial part of
the intercalated region of the nerve cord (Fig. 5A).
Positive cells in the anterior vesicle form a paired
cluster of small nerve cells, which symmetrically
line the ventral half of the rostral central canal, the
region corresponding to the infundibular organ
(Fig. 5B).
The latter population of ampVT-positive cells consists of heavily stained large cells that have a process
that crosses the central canal. These cells are located
in the rather dorsal intercalated region above the
organs of Hesse, groups of cap-shaped cells that include dark pigments, and range antero-posteriorly
between myomeres 3 and 6. Their perikarya cross
the central canal, and processes arising from cell
bodies run through the neuropil layers on both the
sides of nerve cord (Fig. 5B and C). ISH signals for
ampVT were not detected outside the anterior nerve
cord. Positive cells are considered to be commissural
cells, which are located in the middle of central
canal, and which send axons into the bundle compartments (Bone 1960). The previous study of the
amphioxus nerve cord by Uemura et al. (1994)
showed that cells immunoreactive to vasopressin antiserum were only present in the spinal cord. The
difference in localization of ampVT-positive cells between their study and our present ISH remains to be
clarified.
Anterior nerve cord
As summarized in Fig. 5A, thyrostimulin and VT
gene transcripts in the nerve cord of adult amphioxus are within its anterior part. The location of
ISH-positive cells can be divided into three regions
on the basis of expression pattern and morphological
characters. The first region is the anterior vesicle,
where ampVT mRNA is expressed in the infundibular organ in the ventral part. The second is the posterior part of the intercalated region above
Hatschek’s pit. Two mRNAs encoding ampVT and
thyrostimulin are expressed in this region. The third
is the anterior spinal cord behind Hatschek’s pit.
Thyrostimulin subunit genes are expressed in this
region. These regions contain discrete clusters of endocrine nerve cells and do not have the same spatial
relationships as found in the hypothalamus–pituitary
axis.
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found in nonmammalian vertebrates, and is involved
in the control of reproductive function. A survey of
the genome database suggested that amphioxus has
one homologous gene for the neurohypophysial peptide (Holland et al. 2008). Therefore, we cloned cDNA
encoding amphioxus VT (ampVT) in B. belcheri. The
presumed preprovasotocin is composed of 167
amino-acid residues, and consists of a signal peptide,
nonapeptide hormone, neurophysin and copeptin. A
Leucine-rich core segment, which is present in vertebrate copeptin, not find in amphioxus.
The amino-acid sequence of ampVT is Cys–Tyr–
Ile–Ser–Asn–Cys–Pro–Arg–Gly (NH2). The fourth
amino-acid residue is Ser in ampVT of B. belcheri,
instead of Ile in the genome annotation from
B. floridae (Gwee et al. 2009). The fourth amino-acid
residue is Gln in the vasopressin family peptides including VT, but in isotocin, the oxytocin family peptide in teleosts, the fourth peptide is Ser. Although
complete details are not shown, the fourth
amino-acid residue of neurohypophysial peptides is
thus rather variable even in phylogenetically close
species.
K. Kubokawa et al.
Reproductive endocrinology in amphioxus
59
Sex steroids in amphioxus
Sex steroids are vital to reproductive function in vertebrates. Therefore, we examined sex steroidogenic
enzymes and the sex steroidogenic pathway in amphioxus (Mizuta and Kubokawa 2007; Mizuta et al.
2008). A survey of the genome showed the presence
of genes for other steroidogenic enzymes such as
StAR, 3b-HSD, 5a-reductase, ER, and SR (Holland
et al. 2008). CYP21 and CYP11B were not found in
the genome, indicating absence of the adrenal steroid
pathway found in vertebrates.
In vertebrates, mammals utilize progesterone, testosterone, and estradiol as physiological sex steroids
in reproduction, whereas many teleosts use
11-ketotestosterone as an active androgen, and 17,
20b-dihydroxy-4-pregnen-3-one as an active progestin. In amphioxus, progesterone and estradiol are
major sex steroids in the fully mature ovary and
testis, as measured by radioimmunoassay; however,
testosterone was not detected (Mizuta and Kubokawa
2007).
In vitro conversion of 14C-labeled sex steroids was
examined using extracts from amphioxus’ ovaries
during breeding and nonbreeding seasons.
Metabolized steroids were separated by thin-layer
chromatography (TLC), in which major metabolites
appeared as thick bands on the gel. Then, the metabolites were identified by comparison with the positions of standard steroids on the thin-layer
chromatograph. Progestin, estrogen, and androgen
existed in the 4 pathway and also some 5a-reduced
steroids were metabolized from the labeled substrates. The number and amount of metabolites in
the immature ovaries increased compared to that in
the mature ovaries. The major hormones were considered to be progesterone in the form of progestin,
estrone as estrogen, and 5a-dehydrotestosterone as
androgen. These results indicated that sex steroidogenic enzymes homologous to those in vertebrates
were active in the 4 pathway in amphioxus ovaries.
The levels of expression of several steroidogenic
enzymes before and after spawning were determined
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Fig. 5 Expressions of genes encoding VT and thyrostimulin subunits (GPA2, GPB5) in the anterior part of he nerve cord in amphioxus.
(A) Schematic diagram of the anterior nerve cord drawn from the lateral side from the apical part to myomere 8 (m8). The red
squares show the schematic distribution of GPA2 and GPB5 mRNAs. The blue squares and the blue bar show the distribution of ampVT
mRNA. FE, frontal eye; HP, Hatschek’s pit; m, myomere; and Ro, Rohde cell. Dark dots represent the organs of Hesse. (B) ISH-positive
cells in the anterior vesicle. Note many ISH-positive cells are bilaterally distributed in the ventral portions surrounding the central
canal. (C) ISH signals for ampVT mRNAs in large nerve cells crossing the central canal in the dorso-medial part of the intercalated
region (upper panel, horizontal view; and lower panel, transverse view). (D) Signals for ampGPA2 mRNA in nerve cells near the wall of
the central canal in the anterior nerve cord (transverse view). (E) Signals for ampGPB5 mRNA in a nerve cell in the dorsal portion of
the anterior nerve cord (transverse view). Dark structures in (D) and (E) are the organs of Hesse. Scale bars, 25 mm.
60
The ancestral neuroendocrine
system versus the hypothalamus–
pituitary–gonadal axis
When hormonal substances are compared between
amphioxus and vertebrates, many endocrine substances seem to emerge in the hypothalamus–
pituitary–gonadal axis in vertebrates (Fig. 6). The
Fig. 6 Diagram showing the hypothetical endocrine control
system of reproduction in amphioxus and vertebrates. Since
amphioxus does not have the pituitary gland or its homolog, the
system is composed of the nerve cord and the gonad;
neuroendocrinal substances such as thyrostimulin and vasotocin
might directly control reproductive function. Gonadal steroids
also function in regulating the reproductive phenomena. In
contrast, vertebrates have the hypothalamus–pituitary–gonadal
axis that controls reproductive functions by way of GnRH, GTH,
and sex steroids.
lack of pituitary hormone homologs in amphioxus
indicates that it has no functional organ corresponding to the pituitary gland. However, our present
study has presented a line of evidence that thyrostimulin is a candidate for the ancestral pituitary hormone. Furthermore, we showed that the homolog of
VT is the only neurohypophysial hormone-like peptide in amphioxus. Genes encoding thyrostimulin
and amphioxus VT are expressed in the same loci
in the nerve cord near Hatschek’s pit. Thyrostimulin
gene is also expressed in the anterior part of the
spinal, whereas VT gene is expressed in the infundibular organ. Consequently, we conclude that
Hatschek’s pit has different roles from the vertebrate
pituitary gland, although it can be a secretory organ.
Functionally and not morphologically, the organ corresponding to the pituitary gland is a part of the
nerve cord divided into a few discrete portions,
each of which may play different roles in amphioxus.
Amphioxus utilizes sex steroids that are synthesized in the gonadal steroidogenic pathway like vertebrates. The endocrine control of the synthesis and
release of sex steroids should exist in amphioxus,
probably through the neuroendocrine control
system in the nerve cord.
The phylogenetically significant position of amphioxus suggests that the endocrine organs and the
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by quantitative real-time PCR. The spawning animals
were sampled in a tall aquarium, when individual
animals swam up to release eggs. The expressions
of the genes encoding CYP11A, CYP17, CYP19,
and 17b-HSD were examined. The expression level
of CYP11A and CYP19 genes decreased at spawning,
while that of CYP17 and 17b-HSD did not significantly change. CYP11A acts on the cholesterol metabolite at the start of sex steroidogenesis, and
CYP19 catalyzes the conversion from androgen to
estrogen. Therefore, progesterone and estrone
appear to play important roles in the maturation
of ovaries and in the spawning of eggs.
Recently, the evolution of structures and the presumed function of sex steroid receptors were demonstrated in two species of amphioxus, B. floridae
(Holland et al. 2008; Bridgham et al. 2008; Paris
et al. 2008b; Baker and Chang 2009) and B. belcheri
(Katsu et al. 2010); hereafter, the two species are
distinguished as Bf and Bb, respectively. Two types
of steroid receptors in these species are ER, an ortholog of the vertebrate ERs, and SR, an ortholog of
vertebrate receptors for androgens, progestins, and
corticosteroids. The overall sequence identity of
amino-acid residues between BfER and BbER is
90% and that between BfSR and BbSR is 82%.
Bridgham et al. (2008) reported that the function
of BfER was not clear, since it could not bind estrogens including estradiol (Paris et al. 2008b). The
ligand bound to ER of amphioxus could be a steroid,
which we attempted to identify (Roy et al. unpublished data). Bridgham et al. (2008) showed that
BfER was a negative regulator of BfSR, which was
activated by estrogens, and recognized the
estrogen-response element (ERE) in DNA. BfSR
was activated by the transcription of the luciferase
reporter gene treated with estrogen. Katsu et al.
(2010) showed similar results: BbER repressed the
activation of BbSR. They further showed that BbSR
bound to both ERE and androgen response element.
The above-mentioned finding does not contradict
the report of a 3D model of BfSR bound with E2
(Baker and Chang 2009). Furthermore, they showed
that a point mutation on the 3D model of BfSR
increased the affinity of testosterone for BfSR.
K. Kubokawa et al.
Reproductive endocrinology in amphioxus
present endocrine cascades in vertebrates were established in the linage of vertebrates. Neuroanatomical
studies may clarify the nature of neuroendocrinal function in amphioxus, and identify
the origin of this part of the vertebrate endocrine
system.
Acknowledgments
We thank Dr Takanobu Mizuta for his support of
the experiments on steroids. The authors gratefully
acknowledge Dr Linda Holland and Dr Stacia Sower
for organizing this thematic symposium, and the
financial support provided for it by the National
Science Foundation and the Society for Integrative
and Comparative Biology.
Funding
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