Annales d’Endocrinologie 71 (2010) 132–143
Journées Klotz 2010
Human ovarian follicular development: From activation of resting
follicles to preovulatory maturation
Croissance folliculaire dans l’ovaire humain : de l’entrée en croissance du follicule primordial
jusqu’à la maturation préovulatoire
A. Gougeon
Inserm U865, Anipath, faculté de médecine Laënnec, 7, rue Guillaume-Paradin, 69372 Lyon cedex 08, France
Available online 2 April 2010
Presented by Jacques Young
Résumé
En intégrant les données morphologiques et endocrinologiques disponibles ainsi que les effets biologiques des diverses molécules synthétisées
par le follicule, nous proposons une vision dynamique de la croissance folliculaire dans l’ovaire humain. La folliculogenèse débute par l’entrée
en phase de croissance des follicules au repos, un processus où le système kit joue un rôle central. Il faut plusieurs mois pour qu’un follicule
en début de croissance atteigne le stade préantral (0,15 mm) et 70 jours supplémentaires pour qu’il atteigne une taille de 2 mm. La croissance de
petits follicules est finement régulée par des interactions entre la follicle-stimulating hormone (FSH) et les diverses molécules synthétisées par la
granulosa, la thèque interne et l’ovocyte. Lorsqu’ils deviennent sélectionnables, les follicules deviennent sensibles aux variations cycliques de FSH
en termes de prolifération des cellules de la granulosa (CGs). Au début de la phase folliculaire, le follicule sélectionné se développe très rapidement
et synthétise de l’œstradiol. Toutefois, la production totale de stéroïdes reste modérée. À partir du milieu de la phase folliculaire, le follicule
préovulatoire synthétise des quantités croissantes d’œstradiol, puis après la décharge ovulante de grandes quantités de progestérone. La capacité
de réponse des CGs aux gonadotrophines, et plus particulièrement à luteinizing hormone (LH), est alors maximale. LH induit la dissociation de la
granulosa et l’expansion du cumulus ainsi que la maturation nucléaire de l’ovocyte. En conclusion, on peut dire qu’au cours de son développement,
la capacité du follicule à répondre aux gonadotrophines augmente en réponse à des facteurs locaux agissant de façon autocrine et/ou paracrine.
© 2010 Publié par Elsevier Masson SAS.
Mots clés : Ovaire ; Follicules ; Ovocyte ; Granulosa ; Thèque interne ; Facteurs de croissance ; Gonadotrophines ; Stéroïdogenèse
Abstract
By integrating morphometrical and endocrinological data, as well as biological effects of various molecules synthesized by the human follicle, we
propose a dynamic view of the follicle growth within the human ovary. Folliculogenesis starts with entry of resting follicles into the growth phase,
a process where the kit system plays a key role. Several months are required for a new growing follicle to reach the preantral stage (0.15 mm), then
70 additional days to reach the size of 2 mm. Early growing follicle growth is regulated by subtle interactions between follicle-stimulating hormone
(FSH) and local factors produced by theca and granulosa cells (GCs), as well as the oocyte. From the time they enter the selectable stage during the
late luteal phase, follicles become sensitive to cyclic changes of FSH in terms of granulosa cell proliferation. During the early follicular phase, the
early selected follicle grows very quickly and estradiol is present in the follicular fluid. However, the total steroid production remains moderate.
From the mid-follicular phase, the preovulatory follicle synthesizes high quantities of estradiol, then after the mid-cycle gonadotropin surge,
very large amounts of progesterone. At this stage of development, the responsiveness of the follicle to gonadotropins is maximum, especially to
luteinizing hormone (LH) that triggers granulosa wall dissociation and cumulus expansion as well as oocyte nuclear maturation. Thus, as the follicle
develops, its responsiveness to gonadotropins progressively increases under the control of local factors acting in an autocrine/paracrine fashion.
© 2010 Published by Elsevier Masson SAS.
Keywords: Ovary; Follicles; Oocyte; Granulosa; Theca interna; Growth factors; Gonadotropins; Steroidogenesis
E-mail address: [email protected].
0003-4266/$ – see front matter © 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ando.2010.02.021
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
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1. Introduction
2.2. Ageing changes
In humans, folliculogenesis starts when resting follicles leave
the ovarian reserve and culminates with the production of a single dominant follicle during each menstrual cycle. This process
can be divided into four main steps: initiation; early follicle growth; selection of one follicle from a pool of selectable
(≥ 2 mm) follicles; and maturation of the preovulatory follicle
(Fig. 1). While morphological and dynamic aspects of human
follicular growth did not substantially change during the past
years, significant progress in the knowledge of factors regulating
ovarian function has been made by different techniques, including in vitro studies, transgenic mice models, microarray analyses
and phenotypic observations of women bearing spontaneous
mutations affecting fertility.
One of the most exciting facts that has recently emerged is
that local factors, especially those produced by the oocyte, play
a critical role in folliculogenesis, including activation of resting
follicles, early growth, and terminal maturation. For example, it
has been demonstrated [1] that growing oocytes accelerate follicular development and strongly suggest that the oocyte may
be operating as a “folliculogenesis clock”. The use of transgenic mice models has provided evidence that mutations in
many genes affect the synthesis of hormones and growth factors
and/or responsiveness of hormones and growth factors, which
in turn indirectly affect folliculogenesis. The data obtained from
transgenic mouse models may provide clues about the reasons
for abnormal reproductive conditions in humans, because multiple genes shown to regulate folliculogenesis and fertility in
mice have been shown to play a role in human reproduction.
However, the data obtained in the mouse must be considered
with caution before extrapolation to humans since they may
be irrelevant because of strong differences existing between
human and mouse folliculogenesis. The array technologies are
also very promising tools to identify which genes are either
down- or up-regulated during a given step of folliculogenesis.
The aim of this article is to remember the changes affecting
the follicular tissues during folliculogenesis and to describe the
regulations that may be operating inside the primate follicle
at the different stages of its development and especially those
that may be relevant for future researches to improve fertility in
humans.
In humans, as in other mammals, the size of the ovarian
reserve decreases drastically with age. At birth, the size of the
ovarian reserve varies between individuals. Although follicular counts have been performed in a small number of newborn
ovaries, it can be proposed, from both counts [4,5] and mathematical extrapolation [6] that each ovary contains between
250,000 and 500,000 healthy resting follicles. In humans, the
rate of follicular depletion might be variable between subjects
and accelerates from approximately 38 years of age onward,
leading to a stock at menopause estimated between less than
100 [6] and 1000 [7] resting follicles per ovary. The discrepancy
between these two studies can be explained by the follicular
classification used by the authors, in which transitory and small
primary follicles were considered either as resting [6] or growing
follicles [7], despite the demonstration that transitory follicles
are still resting follicles in rats [8] and cattle [9].
In all mammalian species, follicles leave the stock of resting
follicles in a continuous stream, either by apoptosis or by entry
into the growth phase.
2. Initiation of follicular growth
2.1. Composition of the ovarian reserve
After birth, the pool of follicles at the resting stage constitutes
the ovarian reserve. It includes primordial, transitory and small
primary follicles (Fig. 2A, B, C). These follicle subtypes differ in
their proportion of flattened and cuboidal granulosa cells (GCs)
and in their diameter, due to differences in the number and size of
GCs, but they do not differ in mean diameter of their oocyte and
its nucleus [2]. They do not express functional gonadotropin
receptors [3] and represent between 91 and 98% of the total
ovarian follicular population.
2.3. Depletion of the pool by atresia
It is now widely accepted that apoptosis, an active energyconsuming process controlled by a number of intracellular
proteins, is mainly responsible for attrition of the ovarian
reserve. Atresia of resting follicles is very difficult to appreciate because of a very quick disappearance of apoptotic oocytes
(Fig. 2E). However, by talking into account the number of growing follicles within the ovaries throughout life [6], it can be
estimated that more than 90% of the ovarian reserve escapes
by apoptosis mainly during infancy and early adulthood. In
most cases, survival or apoptosis results from a balance between
expression of survival (antiapoptotic) and pro-apoptotic factors.
Among these factors, the proteins bcl-2 and bax likely play a
critical role. On the one hand, in bcl-2 deficient mice, decreased
numbers of follicles are present after birth [10], while overexpression of bcl-2 leads to decreased follicular apoptosis [11],
and, on the other hand, bax deficiency leads to prolongation of
ovarian lifespan [12]. However, given the very high number of
proteins involved in apoptosis, bax and bcl-2 are probably not
the only factors responsible for attrition of the human ovarian
reserve. In addition, the factors responsible for the onset of the
apoptotic cascade in resting follicles in physiologic conditions
remain unknown and, therefore require further very exciting
studies.
2.4. Depletion of the pool by initiation of follicular growth
In humans, the follicles enter the growth phase, continuously
from fetal life to menopause, when the oocyte nucleus reaches
a critical diameter of 19 ␮m [2]. The precise timing of events
leading to activation of resting follicles is not still determined,
but multiple experiments [13] and observations showing that
folliculogenesis is normal up to the preantral stage in women
with follicle-stimulating hormone (FSH) deficiency and in both
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FSH-ß subunit and FSH-receptor knock-out (KO) mice [14–16],
strongly suggest that luteinizing hormone (LH) and FSH are not
directly involved in this process.
The follicles can be blocked at the resting stage for a very
long time, up to 50 years in humans. Consequently, one question
can be addressed: is follicle quiescence due to the action of a
molecule(s) preventing resting follicles from activation?
Anti-Müllerian hormone (AMH) inhibits initiation of follicular growth in neonate rat ovaries [17], an effect that may be
due to inhibition of stimulatory factors [18]. In humans, Carlsson et al. [19] reported an inhibitory effect of AMH on initiation
in cultured ovarian cortical biopsies, however, this finding was
disputed by others [20] who assumed that AMH initiates growth
of primordial follicles in the same culture model. Somatostatin
(SST), a potent inhibitor of cAMP generation in most epithelial
cells [21] is able to inhibit activation of the kit system in the
testis [22], and a SST receptor antagonist can increase spontaneous initiation of follicular growth in cultured ovaries from
neonatal mice [23]. It has also been observed that mice lacking
the transcription factors FOXO3a [24] and FOXL2 [25], which
are present in the human ovary, exhibit an accelerated depletion of the ovarian reserve suggesting that these transcription
factors inhibit initiation. In humans, a mutation of FOXL2 is
associated to premature ovarian failure in patients suffering from
blepharophimosis-ptosis epicanthus inversus syndrome (BPES).
Interestingly, in one BPES patient, the early growing follicles
do not product AMH [26], and in mice lacking FOXL2, AMH
expression is absent, suggesting that these transcription factors
may maintain follicles at the resting stage via AMH.
In mice displaying spontaneous mutations of steel, the
cAMP-responsive [27] gene coding for the kit ligand (KL) [28],
follicular growth is more or less blocked at the primary stage.
This role of the kit system has been experimentally confirmed
by injecting antibodies against c-kit, the receptor for KL, [29]
to neonate mice and by culturing neonate rat ovaries in the presence of either KL or antibodies against c-kit [30]. In humans,
KL mRNA is present in GCs [31], and c-kit in GCs and theca
interna cells (TICs) [32], whereas in the monkey KL and c-kit
proteins are present in primordial follicles [33]. Since AMH has
recently been shown to downregulate c-kit expression in the rat
[18], it appears clearly that the kit system plays a key role, if not
central, in initiation of follicular growth.
Other molecules have been reported able to stimulate initiation of follicular growth. A body of evidence argue in favor of
a role for neurotrophins, a group of molecules including nerve
growth factor (NGF), brain derived neurotrophic factor (BDNF)
and neutrophins in initiation of follicular growth [34,35]. These
observations are relevant for human reproduction since NGF
is produced by both human GCs and TICs [36,37]. The cultured neonatal rat or mouse ovary model was used to show that
other proteins, such as basic fibroblast growth factor 2 (FGF2)
[38], keratinocyte growth factor (KGF) [39], leukemia inhibiting factor (LIF) [40], insulin [41], bone morphogenetic protein-4
(BMP4) [42], platelet-derived growth factor (PDGF) [43] and
activin [44], as well as treatment with androgens in monkeys
[45], can stimulate initiation of follicle growth.
Thus, many factors seem to be involved in initiation of follicular growth, but the way by which they act either to inhibit or to
stimulate this process remains to be determined. Interestingly,
expression of both the c-kit and the FGF receptor (FGFR1) are
suppressed by AMH [18] and, in addition, PDGF [43] and FGF2,
which is present with most of its receptors in human oocytes
and GCs [46,47], might act by stimulating GC-production of
KL [38].
Taken together, the available data strongly suggest that the
kit system plays the central role in the process by which
Fig. 1. Schematic representation of the four stages of human folliculogenesis showing their duration, the size of follicles and the status of GCs in terms of
responsiveness to FSH.
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
135
Fig. 2. Human ovarian follicles. A. Primordial follicle, the oocyte is surrounded by flattened GCs (scale bar = 18 ␮m). B. Intermediary follicle, the oocyte is surrounded
by a mixture of flattened and cuboidal GCs (scale bar = 18 ␮m). C. Small primary follicle, the small oocyte is surrounded by a single layer of cuboidal GCs (scale
bar = 18 ␮m). D. Large primary follicle in which a large oocyte is surrounded by a single layer of cuboidal GCs (scale bar = 20 ␮m). E. Atretic primary follicle (top
right) in which the oocyte has disappeared; a normal primordial follicle (left) is present (scale bar = 30 ␮m). F. Secondary follicle (scale bar = 40 ␮m). G. Preantral
follicle (class 1, 0.15–0.2 mm) (scale bar = 75 ␮m). H. High power micrograph of epithelioid theca cells (white arrow) from a preantral follicle (scale bar = 18 ␮m).
I. Follicle with a small antrum (class 2, 0.2–0.4 mm) (scale bar = 100 ␮m). J. Early antral follicle; the GCs surrounding the ocyte constitute the cumulus oophorus
(class 2) (scale bar = 90 ␮m). K. Small antral follicle (class 3, 0.5–0.9 mm); resting follicles are on right (scale bar = 160 ␮m). L. Small antral follicle (class 4, 1–2 mm)
(scale bar = 300 ␮m). M. Selectable follicle (class 5, 2–5 mm) (scale bar = 530 ␮m).
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A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
Fig. 3. Initiation of follicular growth. The kit system, consisting in KL, synthesized by GCs, and its receptor c-kit, localized at the oocyte cell surface, plays the
central role in the activation process of resting follicles. The kit system can be
inhibited by AMH, which is itself under the positive control of certain transcription factors of the FOX family, and by SST, which might indirectly downregulate
KL production by inhibiting cAMP production by GCs. AMH may also inhibit
some factors, such as FGF2 and KGF that are stimulators of resting follicle activation. The way by which other stimulatory factors, such as BMP4, LIF, Activin
A, insulin, androgens, NGF, NT3. . ., induce initiation of follicular growth are
not yet fully understood.
follicles leave the resting stage to enter the growth phase
(Fig. 3).
3. Follicle growth between initiation and the small
antral stage (2 mm): basal follicular growth
to as epithelioid cells (Fig. 2H). From the time of appearance
of epithelioid cells, the secondary follicle is defined as a preantral follicle (Fig. 2G) and constitutes the first class of growing
follicles in a classification based on morphological aspect and
total number of GCs in each individual follicle [13]. When
small fluid-filled cavities aggregate to form the antrum, the follicle becomes early antral (Fig. 2I) and possesses follicular fluid
having a composition near that of the blood serum. From this
time, the GCs surrounding the oocyte constitute the “cumulus
oophorus” (Fig. 2J). Because of the great importance of multiple interactions between the oocyte and follicular tissues [1],
the GC-oocyte gap-junctions play a critical role during folliculogenesis as demonstrated by the lack of preovulatory follicles in
CX37 null mice [49]. Through accumulation of fluid in the antral
cavity and proliferation of GCs and TICs, the follicle progresses
at an increasing rate through subsequent stages of development
(Fig. 2K, L) until it reaches a size comprised between 2 and
5 mm and becomes a selectable follicle (Fig. 2M). In humans,
the time for early growing follicles to reach the preantral stage
is not known, but has been estimated around 2 to 3 months,
70 additional days being necessary for preantral follicles to reach
a size around 2 mm [50]. This chronology has recently been
validated since a preovulatory follicle was observed 5 months
after orthotopic autotransplantation of frozen ovarian cortical
tissue in a patient suffering from a POF after having undergone
a chemotherapy for Hodgkin’s lymphoma [51].
Thus, at the beginning of the follicle growth, the oocyte
enlarges, TICs proliferate and differentiate and GCs proliferate.
3.2. Oocyte growth
3.1. Morphological aspects
When follicles enter the growth phase, they enlarge, both by
proliferation of GCs and by increase in size of the oocyte. The
first stage of follicular growth in humans is the large primary
follicle (Fig. 2D). At this stage of growth, a network of gapjunctions, intercellular membrane channels that allow nutrients,
inorganic ions, second messengers and small metabolites to pass
from cell to cell, appear in the granulosa layer. Each of these
channels are composed of connexins (CX). CX43 is the most
abundant CX in the ovary and is expressed in the GCs from the
start of folliculogenesis, that is arrested at the primary stage in
CX43 null mice [48].
Progressively, follicles become secondary follicles, i.e. follicles with two or more complete layers of GCs surrounding the
oocyte (Fig. 2F) that are served by one or two arterioles, terminating in an anastomotic network just outside the basal lamina.
The physiological importance of this event is emphasized by the
fact that the follicle becomes directly exposed to factors circulating in the blood. At this stage of development, some stroma
cells near the basal lamina become aligned parallel to each other
and constitute the theca layer. As the follicle enlarges, this theca
stratifies and differentiates in two parts. The outer part, the theca
externa, is composed of cells that do not differ in any respect
from the cells of the undifferentiated theca. In the inner part,
the theca interna, some fibroblast-like precursor cells assume
the appearance of typical steroid-secreting cells also referred
It is during early follicle development that the oocyte grows
at the quickest rate; indeed in humans, its diameter increases
from approximately 40 ␮m in large primary follicles to approximately 100 ␮m in early antral follicles. Beyond this stage of
development, the oocyte diameter increases at a very slow rate
to reach approximately 140 ␮m in the preovulatory follicle. The
oocyte, which might possess FSH receptors [52,53], starts to
produce local factors such as GDF9 and BMP15 that are specific regulators of follicle development. The oocyte growth is
sustained by KL up to the preantral stage only in the presence
of contact communication with GC [54]. However, the oocyte
can control its own growth through GDF9 that downregulates
KL expression in the mouse [55], which, in its turn, downregulates BMP15 expression [56]. In the GDF9-KO mouse model,
where follicles possess no thecal layer and contain an asymmetrical arrangement of atypical GCs, follicular growth is blocked
beyond the secondary stage. This abnormal oocyte growth could
be due to an overexpression of KL in the absence of GDF-9 [57]
leading, among others to a BMP15 inhibition.
3.3. Theca cell proliferation and differentiation
The mechanisms leading to differentiation of stroma cells surrounding primary/secondary follicles into TICs remain unclear.
They may involve the action of GDF-9 [58], KL [59] and epidermal growth factor (EGF) [60], which have been postulated
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
137
Fig. 4. Steroidogenic enzymes in the human ovarian follicle. A, B, C. Immunohistochemical detection of P450c17␣/lyase (red staining) in the theca interna (scale
bar = 50 ␮m). A. Weak immunostaining in the theca interna of 0.5 mm follicle. B. Moderate immunostaining in the theca interna of a selectable follicle. C. Strong
immunostaining in the theca interna of a preovulatory follicle. D and E. Enzymohistochemical detection of 3ß-SDH activity in the human preovulatory follicle (scale
bar = 50 ␮m): before the LH surge 3ß-SDH is only present in TICs (D); after the LH surge (E), a strong 3ß-SDH activity appear in GCs. F. Immunohistochemical
detection of P450 AROMATASE in the GCs of a preovulatory follicle before the LH surge (scale bar = 50 ␮m).
to act as “granulosa-derived theca cell organizers”. LH has been
considered to be the primary hormone regulating the epithelioid
differentiation of TICs [61]. However, IGF-I may be acting as
differentiation factors for TICs in inducing either LH receptors
[62], present in primates in all follicles from the preantral stage
[63], or steroidogenic enzymes [64].
In humans, the theca interna is the primary site of androstenedione (A) synthesis, which is the principal aromatizable
steroid, whereas testosterone (T) is produced in lesser amounts
[65]. The androgen secretion by TICs under LH stimulation
appears to result from activity of enzymes, such as cholesterol side chain cleavage (P450scc), 17␣-hydroxylase/lyase
(P45017␣/lyase ), and 3ß-hydroxysteroid dehydrogenase (3ßHSD). Nevertheless, during basal growth, primate follicles
exhibit slight steroidogenic activity as illustrated by a low
expression of P45017␣/lyase in human TICs (Fig. 4A).
3.4. Granulosa cell proliferation and differentiation
3.4.1. Granulosa cells are weakly responsive to FSH
during basal follicular growth
When early growing follicles become vascularized, they are
directly exposed to factors circulating in the blood, especially
FSH, which is the primary regulator of ovarian folliculogenesis.
However, despite the presence of FSH-receptors on GCs, the
role of FSH in sustaining early follicular growth remains unclear
since these follicles seem to be unresponsive to gonadotropins.
Indeed, follicles smaller than 2 mm do not display any change
of their GC proliferation in response to cyclic changes of FSH
[13], and women undergoing assisted reproductive technology
(ART) had fewer number of oocytes obtained than the number
of selectable follicles (> 2 mm) detected at ultrasonography at
the time when treatment starts [66].
It has been proposed that early growing follicles are FSHindependent since they can reach the selectable stage in the
absence of either bioactive FSH [14,16] or FSH receptor [15].
Moreover, in various pathological (hypogonadotropic hypogonadism, hypophysectomy) situations in humans, selectable
follicles were observed despite extremely low levels of
gonadotropin [13], and, in patients displaying high levels of
gonadotropins, because of a partial inactivation of the FSH
receptor gene, follicles can grow up to a size of several millimetres [67] but cannot reach the ovulatory size. Taken together,
these observations indicate that in presence of very low levels of
FSH activity, folliculogenesis can take place until the selectable
stage (≥ 2 mm).
Many local factors such as activin A [68], EGF [69], TGF-ß
[70], FGF2 [71], hepatocyte growth factor (HGF) [71], GDF-9
[72] and BMP-15 [73] have been shown to stimulate GC proliferation in the absence of FSH. Whether these factors, which
are present in the monkey or human ovary [33,46,74–79], act in
a similar way on early growing human follicles remain, how-
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ever, to be determined. While the role of estrogens remains
unclear during follicular growth in primates [80], androgens sustain early follicular development in monkeys [81]. This effect
of androgens, acting through their receptors present in follicles
[82], can be due to their ability in stimulating GC proliferation
of follicles at all stages of development [83], an effect that might
be mediated by KL, BMP15, GDF9 and HGF [84]. It can therefore be assumed that in absence of FSH, local factors are able
to support early follicle growth. However, many experiments
have demonstrated that FSH acts in synergy with local factors
to enhance follicle growth in laboratory rodents [69,70,85,86].
Finally, taken together, these observations lead to the conclusion
that in conditions of FSH deprivation, the development of early
growing follicles can be sustained by local factors. However,
it appears likely that these factors are less efficient to sustain
growth than they are when they act in synergy with FSH.
3.4.2. Follicles grow at a slow, but increasing, rate during
basal follicular growth
In humans, despite the positive role played by FSH and
local factors on their development, follicles smaller than 2 mm
grow slowly [50]. Inhibitory factors produced by the follicle itself can counteract FSH- and local factor-induced GC
mitotic activity. In rats, activin A, produced by large preantral
– early antral follicles, can inhibit the FSH-induced growth
of smaller follicles (100 – 120 ␮m) [87]. In the mouse ovary,
AMH inhibits the FSH-stimulated follicle growth by repressing
synthesis of some proteins involved in GC proliferation such
as GDF9, BMP15, activin receptor 1, c-kit and TGF-ß2 [88].
These observations suggest that AMH, which have a similar
pattern of expression in the primate ovary, may partly inhibit
GC mitotic activity in humans. They may also explain why
the growth rate of small follicles increases with their diameter [50], indeed, AMH being mainly produced in preantral
and early antral follicles, progressively decreases as the follicle diameter increases [89]. In addition to the decreasing levels
of AMH, the increasing levels of androgens produced by TICs
might explain the increasing growth rate as follicles enlarge
[81].
3.4.3. Granulosa cells are undifferentiated during basal
follicular growth
Another characteristic of early growing follicles is that
their GCs are undifferentiated, i.e. they express neither aromatase, enzymes for synthesis of progestins, nor LH receptors
[63,90,91]. Whereas some local factors promote proliferation
of GCs, they also inhibit their FSH-induced differentiation.
EGF and FGF2 in the rat [92], and GDF9 and BMP15 in the
mouse [93] inhibit FSH receptor mRNA expression, and subsequent GC differentiation during early follicular growth. In
humans, TGF-␣ inhibits FSH-induced aromatase [94]. HGF suppressed the forskolin-induced StAR and progesterone synthesis
by a steroidogenic GC tumor line [95] and KGF inhibits basal
or hormone-stimulated progesterone production by GCs [74].
The high intrafollicular levels of certain IGFBPs decreases the
bioavailability of IGFs, which stimulates both GC proliferation
and differentiation and could be partly responsible for the slow
growth rate of these follicles and their poor GC differentiation
[96].
Taken together, these data show that the TICs, GCs, and the
oocyte of early growing follicles produce factors that not only
sustain their growth in the absence or in the presence of FSH,
but also inhibit, at least partly, FSH-induced GC differentiation.
4. The selectable follicle and selection of the follicle
destined to ovulate
Healthy follicles measuring 2 to 5 mm, referred to as
selectable follicles, are observed at all stages of the menstrual
cycle. During the late luteal phase, their number and quality rise
in humans [97] as in monkeys [98] in response to increasing
peripheral FSH levels following the demise of the cyclic corpus
luteum. At this time, their number is between three and 11 per
ovary in 24- to 33-year-old women [99], but it strongly decreases
with ageing. It is among these follicles that the follicle destined
to ovulate during the subsequent cycle will be selected [97,100].
Whereas early growing follicles are unresponsive to cyclic hormonal changes, selectable follicles are more receptive to these
alterations. Their GC mitotic index strongly increases from the
mid- to the late luteal phase [101] and they are highly responsive to exogenous gonadotropins in terms of GC proliferation
[102]. The selectable follicle is a key step in folliculogenesis.
As it develops very quickly, its metabolic requirements are high,
leading to production of high levels of free radicals requiring
action of detoxifying enzymes. Transgenic mouse deficient in
␥-glutamyl transpeptidase, an enzyme involved in glutathione
metabolism, exhibit a block in folliculogenesis at the mouse preantral stage, that corresponds to the selectable follicle in humans
[103].
However, the intrafollicular concentration of estradiol in
selectable follicles is very low when compared to that of androgens (Table 1). The follicular fluid of selectable follicles contains
high levels of TGF␣ and EGF [78,104], as well as of IGFBPs
[105] that inhibit FSH- [106] and IGFII- [107] induced aromatase, respectively. In addition, they also exhibit high levels
of activin that decreases production of thecal androgens [108].
Nonetheless, the latter could be counteracted, at least partly, by
an increasing number of LH receptors [109], an increasing LH
pulse frequency during the early follicular phase [110] and the
positive effect of GDF-9 on androgen production by TICs [111],
leading to a significant activity of enzymes involved in androgen
production in selectable follicles (Fig. 4B).
In conclusion, it appears that when follicles reach the
selectable stage, their GCs become responsive to FSH in terms
of proliferation, but not in terms of estrogen production.
Every mammalian species is characterized by a fixed number
of follicle(s) that ovulate at each cycle (ovulatory quota). The
term selection has been used by Goodman and Hodgen [112]
to indicate the final adjustment of the cohort of growing follicles to this ovulatory quota. Each growing follicle possesses a
FSH threshold requirement that should be surpassed to ensure
ongoing preovulatory development [113]. It has been suggested
that the selected follicle is the one which is most rapidly growing in response to the intercycle rise in FSH, i.e. the one with
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
139
Table 1
Changes in follicular fluid mean (±SEM) concentrations (ng/mL) of steroids from the selectable to the preovulatory stage (mean concentrations estimated from
[97,137–139]). E2: estradiol; A: androstenedione; T: testosterone; DHT: dihydro-testosterone; 17␣-OHP: 17␣-hydroxyprogesterone; P: progesterone. Preovulatory
follicles: 1: during the mid-follicular phase when E2 plasmatic levels increase; 2: at the time of E2 peak; 3: between the E2 peak and the LH surge; 4: after the LH
surge.
Follicle type
E2
Atretic (1–5 mm)
Selectable
Newly selected
Preovulatory 1
Preovulatory 2
Preovulatory 3
Preovulatory 4
20
15
658
1270
2396
2583
1109
A + T + DHT
±5
±5
± 38
± 161
± 348
± 228
± 142
794
638
487
542
203
287
79
±
±
±
±
±
±
±
90
113
128
176
37
44
21
the lowest FSH threshold [97]. During the late luteal phase in
the oldest ovulating women [114], or at the beginning of the
follicular phase in younger women, the largest healthy follicle
appears to be the selected follicle. It grows at a quicker rate than
do other subordinate follicles [50], contains a detectable level
of FSH [115] and differs substantially from selectable follicles
by its estradiol concentration in the follicular fluid (Table 1).
Taken together, these data strongly suggest that in the selected
follicle, stimulators of both GC proliferation and differentiation
are operating. Since IGFs promote both human GC proliferation
and differentiation as well as production of androgens by theca
cells, the importance of the IGF system in regulating FSH action
has become increasingly apparent during the past decade [107].
Briefly, the IGF binding protein, IGFBP4, a potent inhibitor of
FSH-induced steroid production by human GCs [116], is able
to neutralize the IGF bioactivity, and especially that of IGFII, the main IGF produced by human preovulatory GCs [105].
By stimulating an IGFBP-4 protease, the pregnancy-associated
plasma protein-A (PAPP-A) in humans [117], FSH eliminates
the inhibitory activity of IGFBP4 [118] and indirectly stimulates
its own effect on both GC proliferation and steroidogenesis via
IGF-II action. In conclusion, it can be proposed that the follicle with the lowest FSH threshold will be the first to exhibit an
increased bioactivity of IGF-II, leading to an enhanced growth
and differentiation of its GCs. Being the first to produce estradiol, to differentiate LH receptors on its GCs, to grow despite
decreasing levels of FSH, to inhibit growth of less developed follicles, it will be the first to undergo preovulatory changes, then
to ovulate. The view that the IGF system plays a key role during
selection of preovulatory follicles was confirmed by the IGF-I
KO mouse model in which folliculogenesis unfolds normally up
to the selectable stage then is blocked thereafter [119].
5. Maturation of the preovulatory follicle
From the time it has been selected, the follicle destined
to ovulate greatly enlarges from 6.9 ± 0.5 mm (2 to 5 × 106
GCs) during the early follicular phase, to 18.8 ± 0.5 mm (50
to 100 × 106 GCs) during the late follicular phase. So, during
the early follicular phase, the selected follicle grows at a very
high rate, requiring protection against free radicals. It is likely the
reason for which transgenic mice deficient in superoxide dismutase 1, but not 2, have fewer large antral follicles than wild type
mice [120]. During this final phase of growth, the GCs are sub-
17␣-OHP
P
–
–
713 ±
460 ±
1002 ±
1812 ±
2034 ±
73
130
417
440
1228
2464
7773
318
112
212
142
326
Total
±
±
±
±
±
±
±
13
45
120
74
228
226
643
887
783
2275
2712
4829
6146
10995
jected to marked morphological transformations resulting from
a modulation of the actin cytoskeleton organization [121].
5.1. Preovulatory maturation before the mid-cycle
gonadotropin surge
From the time it has been selected, the follicle destined to ovulate shows very marked changes in its steroidogenic activity. On
the one side, production of androgens by the TICs is enhanced
in response to increasing production of inhibin A [122,123] that
strongly stimulates P450c17␣/lyase (Fig. 4C). On the other side,
aromatase, which is detected only in GCs from follicles ≥ 10 mm
[90] (Fig. 4F), is stimulated by an increasing production of IGFII [124], while NGF stimulates both FSH receptors synthesis and
estradiol production [37]. Taken together, these changes lead to a
maximal intrafollicular estradiol concentration at the time of the
plasma estradiol peak. So, from the early to late follicular phase,
estradiol in human follicular fluid increases from 658 to 2,583
ng/ml (Table 1). At the same time, intrafollicular concentrations
of 17␣-OH progesterone and progesterone increase (Table 1);
these progestins are mainly produced by TICs, since 3ß-HSD
is only faintly present in GCs (Fig. 4D) [125]. As the follicle
matures, the circulating levels of FSH decrease in response to
estradiol and inhibin produced by the follicle itself, and GCs
become able to bind LH [63,109]. Thus, it has been demonstrated
that LH optimizes late folliculogenesis when administered with
recombinant human FSH, by accelerating follicle growth and
reducing both FSH dose requirements and development of small
follicles [126]. This observation supports the view that when LH
enters the follicle during the late follicular phase, it may replace
FSH, as the principle regulator of GC differentiation.
From the mid-follicular phase, the primate preovulatory
follicle becomes a highly vascularized structure [127]. The
considerable increase in thecal blood vascularization occurs
as a result of an active endothelial cell proliferation of thecal
blood capillaries induced by angiogenic factors such as vascular
endothelial growth factor [128].
5.2. Preovulatory maturation after the mid-cycle
gonadotropin surge
After the mid-cycle gonadotropin surge, dramatic metabolic
and morphologic changes occur simultaneously within the preovulatory follicle that switches from an estradiol-producing to
140
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
a progestin-producing structure. Progesterone receptors appear
on GCs whose proliferation is arrested [129], capillaries from
the theca layers invade the granulosa wall and both mural and
cumulus GCs dissociate.
The production of steroids strongly increases, rising from
a mean follicular fluid concentration of 4,800 ng/ml before the
surge, to 11,000 ng/ml after (Table 1). The high levels of progesterone result from additive effects. As a result of the breakdown
of the basal lamina, the cholesterol substrate required by GCs
for progestin production, and which is provided to cells in the
form of lipoprotein-bound cholesterol, can now reach the follicle via the blood supply [121]. After the gonadotropin surge,
both 3ßHSD [91,125] (Fig. 4E) and P450scc [90] appear in GCs,
where the levels of adrenodoxin and steroid acute regulatory protein (StAR) proteins, which allows entrance of cholesterol into
mitochondria, are significantly increased. While the follicular
progestin production strongly increases, that of androgens and
estradiol dramatically drops.
Some hours before ovulation and in response to angiogenic
factors produced by GCs [128], the granulosa wall, which was
avascular before the mid-cycle gonadotropin surge, appears to
be bloody after invasion of blood vessels originating from the
theca.
LH causes a significant decline in gap junctions [121]
leading to dissociation of mural GC and expansion of the
cumulus-oocyte complex (COC) that is a highly specialized
inflammatory-related process, which is obligatory for successful
ovulation and involves activation of a variety of genes [130,131].
COC expansion requires action of local factors produced by
GCs and the oocyte in response to a direct or indirect action
of LH. Some of them can be mentioned: EGF family members such as amphiregulin, epiregulin, which are present in the
human ovary [132], and ß-cellulin, are produced by mural, but
not cumulus GCs in response to LH and are mandatory for COC
expansion and oocyte maturation [133]. Prostaglandin E (PGE)
is also involved, since mice lacking the PGE-R2 do not ovulate because of defects in COC expansion [134]. The release by
the oocyte of both BMP15, whose production may be upregulated by FSH [135], and GDF9, that stimulates, among others,
expression of hyaluronan synthase and cyclo-oxygenase 2 [93],
is associated with cumulus expansion.
After the LH surge, the oocyte resumes its meiosis and progresses from prophase 1 to metaphase 2 at the time of ovulation.
Phenotyping of mice deficient for specific genes [131], and
oocyte gene-expression profiles [136] have recently brought significant contributions in the knowledge of factors regulating both
cytoplasmic and nuclear oocyte maturation.
6. Conclusion
There is no doubt that our knowledge of how follicles develop
in the ovary has greatly increased during the last years. Whereas
the role of FSH and LH as primary triggers of folliculogenesis
remains indisputable, the role played by local factors has become
increasingly apparent during the past decade. Both granulosa and
theca cells, and more surprisingly, the oocyte, produce a variety
of peptides that act broadly to influence gonadotropin action,
either positively or negatively. In this respect, it is fascinating to
note how large is the number of follicular functions that are controlled by a balance between activating and inhibiting factors.
However, despite a better understanding of folliculogenesis in
the rodent ovary, many processes remain poorly understood in
humans, mainly because of the impossibility to make experimental studies. For example, depletion of the ovarian reserve,
either by apoptosis or by activation of resting follicles, as well
as acquisition by GCs of their fully responsiveness to FSH, are,
among others, fascinating issues that constitute exciting goals
to reach for improvement of women’s health and fertility in the
future.
Conflicts of interest
None.
References
[1] Eppig JJ, Wigglesworth K, Pendola FL. The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci U S
A 2002;99:2890–4.
[2] Gougeon A, Chainy GBN. Morphometric studies of small follicles
in ovaries of women at different ages. J Reprod Fertil 1987;81:
433–42.
[3] Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin
Endocrinol Metab 1997;82:3748–51.
[4] Baker TG. Quantitative and cytological study of germ cells in human
ovaries. Proc R Soc Ser B 1963;158:417–33.
[5] Forabosco A, Sforza C, de Pol A, Vizzotto L, Marzona L, Ferrario V.
Morphometric study of the human neonatal ovary. Anat Rec 1991;231:
201–8.
[6] 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-growing follicles in aging women. Biol Reprod
1994;50:653–63.
[7] Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF. Accelerated disappearance of ovarian follicles in mid-life: Implications for
forecasting menopause. Hum Reprod 1992;7:1342–6.
[8] Meredith S, Dudenhoeffer G, Jackson K. Classification of small type
B/C follicles as primordial follicles in mature rats. J Reprod Fertil
2000;119:43–8.
[9] van Wezel IL, Rodgers RJ. Morphological characterization of bovine
primordial follicles and their environment in vivo. Biol Reprod
1996;55:1003–11.
[10] Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL. Ablation of bcl-2
gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology
1995;136:3665–8.
[11] Hsu SY, Lai RJM, Finegold M, Hsueh AJW. Targeted overexpression
of bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis.
Endocrinology 1996;137:4837–43.
[12] Perez GI, Robles R, Knudson CM, Flaws JA, Korsmeyer SJ, Tilly JL.
Prolongation of ovarian lifespan into advanced chronological age bybaxdeficiency. Nat Genet 1999;21:200–3.
[13] Gougeon A. Regulation of ovarian follicular development in primates:
facts and hypotheses. Endocr Rev 1996;17:121–55.
[14] Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hormone
is required for ovarian follicle maturation but not for male fertility. Nat
Genet 1997;15:201–4.
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
[15] Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur
M, et al. Impairing follicle-stimulating hormone (FSH) signaling in vivo:
targeted disruption of the FSH receptor leads to aberrant gametogenesis
and hormonal imbalance. Proc Natl Acad Sci U S A 1998;95:13612–7.
[16] Matthews CH, Borgato S, Beck-Peccoz, Adams M, Tone Y, Gambino
G, et al. Primary amenorrhoea and infertility due to a mutation in the
ß-subunit of follicle-stimulating hormone. Nat Genet 1993;5:83–6.
[17] Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, et al. Anti-Müllerian hormone inhibits initiation of primordial
follicle growth in the mouse ovary. Endocrinology 2002;143:1076–84.
[18] Nilsson E, Rogers N, Skinner MK. Actions of anti-Müllerian hormone
on the ovarian transcriptome to inhibit primordial to primary follicle
transition. Reproduction 2007;134:209–21.
[19] Carlsson IB, Scott JE, Visser JA, Ritvos O, Themmen AP, Hovatta O.
Anti-Müllerian hormone inhibits initiation of growth of human primordial
ovarian follicles in vitro. Hum Reprod 2006;21:2223–7.
[20] Schmidt KL, Kryger-Baggesen N, Byskov AG, Andersen CY. AntiMüllerian hormone initiates growth of human primordial follicles in vitro.
Mol Cell Endocrinol 2005;234:87–93.
[21] Rajkumar K, Kerr DE, Kirkwood RN, Laarveld B. Inhibitory action
of somatostatin-14 on hormone-stimulated cAMP induction in porcine
granulosa and luteal cells. J Endocrinol 1992;134:397–406.
[22] Goddard I, Bauer S, Gougeon A, Lopez F, Giannetti N, Susini C, et
al. Somatostatin inhibits stem cell factor messenger RNA expression
by sertoli cells and stem cell factor-induced DNA synthesis in isolated
seminiferous tubules. Biol Reprod 2001;65:1732–42.
[23] Gougeon A, Delangle A, Arouche N, Stridsberg M, Gotteland JP,
Loumaye E. Kit ligand and the somatostatin receptor antagonist, BIM23627, stimulate in vitro resting follicle growth in the neonatal mouse
ovary. Endocrinology 2010;151:1299–309.
[24] Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor
FOXO3a. Science 2003;301:215–8.
[25] Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier AC,
et al. The murine winged-helix transcription factor FOXL2 is required
for granulosa cell differentiation and ovary maintenance. Development
2004;131:933–42.
[26] Méduri G, Bachelot A, Duflos C, Bständig B, Poirot C, Genestie C, et al.
FOXL2 mutations lead to different ovarian phenotypes in BPES patients:
Case report. Hum Reprod 2010;25:235–43.
[27] Packer AI, Hsu YC, Besmer P, Bachvarova RF. The ligand of the c-kit
receptor promotes oocyte growth. Dev Biol 1994;161:194–205.
[28] Huang EJ, Manova K, Packer AI, Sanchez S, Bachvarova RM, Besmer P.
The murine steel panda mutation affects kit ligand expression and growth
of early ovarian follicles. Dev Biol 1993;157:100–9.
[29] Yoshida H, Takakura N, Kataoka H, Kunisada T, Okamura H, Nishikawa
SI. Stepwise requirement of c-kit tyrosine kinase in mouse ovarian follicle
development. Dev Biol 1997;184:122–37.
[30] Parrott JA, Skinner MK. Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis. Endocrinology
1999;140:4262–71.
[31] Laitinen M, Rutanen EM, Ritvos O. Expression of c-kit ligand mRNAs in
human ovaries and regulation of their steady state levels by gonadotropins
in cultured granulosa-luteal cells. Endocrinology 1995;136:4407–14.
[32] Horie K, Fujita J, Takakura K, Kanzaki H, Suginami H, Iwai M, et al. The
expression of c-kit protein in human adult and fetal tissues. Hum Reprod
1993;8:1955–62.
[33] Gougeon A, Busso D. Morphologic and functional determinants of primordial and primary follicles in the monkey ovary. Mol Cell Endocrinol
2000;163:33–41.
[34] Dissen GA, Garcia-Rudaz C, Ojeda SR. Role of neurotrophic factors in
early ovarian development. Semin Reprod Med 2009;27:24–31.
[35] Nilsson E, Dole G, Skinner MK. Neurotrophin NT3 promotes ovarian primordial to primary follicle transition. Reproduction 2009;138:
697–707.
[36] Abir R, Fisch B, Jin S, Barnnet M, Ben-Haroush A, Felz C, et al. Presence
of NGF and its receptors in ovaries from human fetuses and adults. Mol
Hum Reprod 2005;11:229–36.
141
[37] Salas C, Julio-Pieper M, Valladares M, Pommer R, Vega M, Mastronardi
C, et al. Nerve growth factor-dependent activation of trkA receptors in the
human ovary results in synthesis of follicle-stimulating hormone receptors and estrogen secretion. J Clin Endocrinol Metab 2006;91:2396–403.
[38] Nilsson EE, Skinner MK. Kit ligand and basic fibroblast growth factor
interactions in the induction of ovarian primordial to primary follicle
transition. Mol Cell Endocrinol 2004;214:19–25.
[39] Kezele PR, Nilsson EE, Skinner MK. Keratinocyte growth factor acts as a
mesenchymal factor that promotes ovarian primordial to primary follicle
transition. Biol Reprod 2005;73:967–73.
[40] Nilsson EE, Kezele P, Skinner MK. Leukemia inhibitory factor (LIF)
promotes the primordial to primary follicle transition in rat ovaries. Mol
Cell Endocrinol 2002;188:65–73.
[41] Kezele PR, Nilsson EE, Skinner MK. Insulin but not insulin-like growth
factor-1 promotes the primordial to primary follicle transition. Mol Cell
Endocrinol 2002;192:37–43.
[42] Nilsson EE, Skinner MK. Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development.
Biol Reprod 2003;69:1265–72.
[43] Nilsson EE, Detzel C, Skinner MK. Platelet-derived growth factor
modulates the primordial to primary follicle transition. Reproduction
2006;131:1007–15.
[44] Oktay K, Karlikaya G, Akman O, Ojakian GK, Oktay M. Interaction of
extracellular matrix and activin-A in the initiation of follicle growth in
the mouse ovary. Biol Reprod 2000;63:457–61.
[45] Vendola K, Zhou J, Wang J, Famuyiwa OA, Bievre M, Bondy CA. Androgens promote oocyte insulin-like growth factor I expression and initiation
of follicle development in the primate ovary. Biol Reprod 1999;61:353–7.
[46] Ben-Haroush A, Abir R, Ao A, Jin S, Kessler-Icekson G, Feldberg D, et
al. Expression of basic fibroblast growth factor and its receptors in human
ovarian follicles from adults and fetuses. Fertil Steril 2005;84(Suppl.
2):1257–68.
[47] Yamamoto S, Konishi I, Nanbu K, Komatsu T, Mandai M, Kuroda H,
et al. Immunohistochemical localization of basic fibroblast growth factor
(bFGF) during folliculogenesis in the human ovary. Gynecol Endocrinol
1997;11:223–30.
[48] Ackert CL, Gittens JE, O’Brien MJ, Eppig JJ, Kidder GM. Intercellular communication via connexin 43 gap junctions is required for ovarian
folliculogenesis in the mouse. Dev Biol 2001;233:258–70.
[49] Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice
lacking connexin 37. Nature 1997;385:525–8.
[50] Gougeon A. Rate of follicular growth in the human ovary. In: Rolland
R, van Hall EV, Hillier SG, McNatty KP, Schoemaker J, editors. Follicular maturation and ovulation. Amsterdam: Excerpta Medica; 1982. p.
155–63.
[51] Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al.
Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.
Lancet 2004;364:1405–10.
[52] Meduri G, Charnaux N, Driancourt MA, Combettes L, Granet P, Vannier B, et al. Follicle-stimulating hormone receptors in oocytes? J Clin
Endocrinol Metab 2002;87:2266–76.
[53] Patsoula E, Loutradis D, Drakakis P, Michalas L, Bletsa R, Michalas S.
Messenger RNA expression for the follicle-stimulating hormone receptor
and luteinizing hormone receptor in human oocytes and pre-implantationstage embryos. Fertil Steril 2003;79:1187–93.
[54] Klinger FG, De Felici M. In vitro development of growing oocytes from
fetal mouse oocytes: stage-specific regulation by stem cell factor and
granulosa cells. Dev Biol 2002;244:85–95.
[55] Joyce IM, Clark AT, Pendola FL, Eppig JJ. Comparison of recombinant
growth differentiation factor-9 and oocyte regulation of KIT ligand messenger ribonucleic acid expression in mouse ovarian follicles. Biol Reprod
2000;63:1669–75.
[56] Otsuka F, Shimasaki S. A negative feedback system between oocyte bone
morphogenetic protein 15 and granulosa cell kit ligand: its role in regulating granulosa cell mitosis. Proc Natl Acad Sci U S A 2002;99:8060–5.
[57] Carabatsos MJ, Elvin J, Matzuk MM, Albertini DF. Characterization
of oocyte and follicle development in growth differentiation factor-9deficient mice. Dev Biol 1998;204:373–84.
142
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
[58] Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM.
Growth differentiation factor-9 is required during ovarian early folliculogenesis. Nature 1996;383:531–5.
[59] Ito M, Harada T, Tanikawa M, Fujii A, Shiota G, Terakawa N. Hepatocyte
growth factor and stem cell factor involvement in paracrine interplays
of theca and granulosa cells in the human ovary. Fertil Steril 2001;75:
973–9.
[60] Erickson GF, Case E. Epidermal growth factor antagonizes ovarian thecainterstitial cytodifferentiation. Mol Cell Endocrinol 1983;31:71–6.
[61] Erickson GF, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen
producing cells: a review of structure/function relationships. Endocr Rev
1985;6:371–99.
[62] Magoffin DA, Weitsman SR. Insulin-like growth factor-l regulation of
luteinizing hormone (LH) receptor messenger ribonucleic acid expression and LH-stimulated signal transduction in rat ovarian theca-interstitial
cells. Biol Reprod 1994;51:766–75.
[63] Shima K, Kitayama S, Nakano R. Gonadotropin binding sites in human
ovarian follicles and corpora lutea during the menstrual cycle. Obstet
Gynecol 1987;69:800–6.
[64] Magoffin DA, Weitsman SR. Effect of insulin-like growth factor-1 on
cholesterol side-chain cleavage cytochrome P450 mRNA expression in
ovarian theca-interstitial cells stimulated to differentiate in vitro. Molec
Cell Endocrinol 1993;96:45–51.
[65] Bergh C, Olsson JH, Selleskog U, Hillensjo T. Steroid production in
cultured thecal cell obtained from human ovarian follicles. Hum Reprod
1993;8:519–24.
[66] Jayaprakasan K, Hilwah N, Kendall NR, Hopkisson JF, Campbell BK,
Johnson IR, et al. Does 3D ultrasound offer any advantage in the pretreatment assessment of ovarian reserve and prediction of outcome after
assisted reproduction treatment? Hum Reprod 2007;22:1932–41.
[67] Beau I, Touraine P, Meduri G, Gougeon A, Desroches A, Matuchansky
C, et al. A novel phenotype related to partial loss of function mutations
of the follicle-stimulating hormone receptor. J Clin Invest 1998;102:1–8.
[68] Yokota H, Yamada K, Liu X, Kobayashi J, Abe Y, Mizunuma H, et al.
Paradoxical action of activin A on folliculogenesis in immature and adult
mice. Endocrinology 1997;138:4572–6.
[69] Roy SK. Epidermal growth factor and transforming growth factor-ß modulation of FSH-induced DNA synthesis in hamster preantral and early
antral follicles. Biol Reprod 1993;48:552–7.
[70] Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K, Mizunuma H, et al.
A comparative study on transforming growth factor-beta and activin A
for preantral follicles from adult, immature, and diethylstilbestrol-primed
immature mice. Endocrinology 1999;140:2480–5.
[71] Parrott JA, Vigne JL, Chu BZ, Skinner MK. Mesenchymal-epithelial
interactions in the ovarian follicle involve keratinocyte and hepatocyte
growth factor production by thecal cells and their action on granulosa
cells. Endocrinology 1994;135:569–75.
[72] Hayashi M, McGee EA, Min G, Klein C, Rose UM, van Duin M, et al.
Recombinant growth differentiation factor-9 (GDF-9) enhances growth
and differentiation of cultured early ovarian follicles. Endocrinology
1999;140:1236–44.
[73] Otsuka F, Yao Z, Lee T, Yamamoto S, Erickson GF, Shimasaki S. Bone
morphogenetic protein-15. Identification of target cells and biological
functions. J Biol Chem 2000;275:39523–8.
[74] Osuga Y, Koga K, Tsutsumi O, Yano T, Kugu K, Momoeda M, et al.
Evidence for the presence of keratinocyte growth factor (KGF) in human
ovarian follicles. Endocr J 2001;48:161–6.
[75] Abir R, Fisch B, Zhang XY, Felz C, Kessler-Icekson G, Krissi H, et al.
Keratinocyte growth factor and its receptor in human ovaries from fetuses,
girls and women. Mol Hum Reprod 2009;15:69–75.
[76] Osuga Y, Tsutsumi O, Momoeda M, Okagaki R, Matsumi H, Hiroi H, et
al. Evidence for the presence of hepatocyte growth factor expression in
human ovarian follicles. Mol Hum Reprod 1999;5:703–7.
[77] Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev
1988;9:267–93.
[78] Westergaard LG, Andersen CY. Epidermal growth factor (EGF) in human
preovulatory follicles. Hum Reprod 1989;4:257–60.
[79] Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen
N, Seppa L, et al. Human growth differentiation factor 9 (GDF-9) and its
novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 1999;84:2744–50.
[80] Palter SF, Tavares AB, Hourvitz A, Veldhuis JD, Adashi EY. Are estrogens of import to primate/human ovarian folliculogenesis? Endocr Rev
2001;22:389–424.
[81] Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA. Androgens
stimulate early stages of follicular growth in the primate ovary. J Clin
Invest 1998;101:2622–9.
[82] Hild-Petito S, Brenner RM, Stouffer RL. Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the
menstrual cycle. Biol Reprod 1991;44:561–8.
[83] Weil S, Vendola K, Zhou J, Bondy CA. Androgen and follicle-stimulating
hormone interactions in primate ovarian follicle development. J Clin
Endocrinol Metab 1999;84:2951–6.
[84] Shiina H, Matsumoto T, Sato T, Igarashi K, Miyamoto J, Takemasa S, et
al. Premature ovarian failure in androgen receptor-deficient mice. Proc
Natl Acad Sci U S A 2006;103:224–9.
[85] Hsueh AJ, McGee EA, Hayashi M, Hsu SY. Hormonal regulation
of early follicle development in the rat ovary. Mol Cell Endocrinol
2000;163:95–100.
[86] Nakamura M, Nakamura K, Igarashi S, Tano M, Miyamoto K, Ibuki Y,
et al. Interaction between activin A and cAMP in the induction of FSH
receptor in cultured rat granulosa cells. J Endocrinol 1995;147:103–10.
[87] Mizunuma H, Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K, et al.
Activin from secondary follicles causes small preantral follicles to remain
dormant at the resting stage. Endocrinology 1999;140:37–42.
[88] Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM,
et al. Anti-Müllerian hormone attenuates the effects of FSH on follicle
development in the mouse ovary. Endocrinology 2001;142:4891–9.
[89] Weenen C, Laven JS, Von Bergh AR, Cranfield M, Groome NP, Visser
JA, et al. Anti-Müllerian hormone expression pattern in the human ovary:
potential implications for initial and cyclic follicle recruitment. Mol Hum
Reprod 2004;10:77–83.
[90] Sasano H, Okamoto M, Mason JI, Simpson ER, Mendelson CR, Sasano
N, et al. Immunolocalization of aromatase, 17␣-hydroxylase and sidechain cleavage cytochrome P-450 in the human ovary. J Reprod Fertil
1989;85:163–9.
[91] Sasano H, Mori T, Sasano N, Nagura H, Mason JI. Immunolocalization
of 3ß-hydroxysteroid dehydrogenase in human ovary. J Reprod Fertil
1990;89:743–51.
[92] Tilly JL, LaPolt PS, Hsueh AJW. Hormonal regulation of folliclestimulating hormone receptor messenger ribonucleic acid levels in
cultured rat granulosa cells. Endocrinology 1992;130:1296–302.
[93] Elvin JA, Yan C, Matzuk MM. Oocyte-expressed TGF-beta superfamily
members in female fertility. Mol Cell Endocrinol 2000;159:1–5.
[94] Steinkampf MP, Mendelson CR, Simpson ER. Effects of epidermal
growth factor and insulin-like growth factor I on the levels of mRNA
encoding aromatase cytochrome P-450 of human ovarian granulosa cells.
Mol Cell Endocrinol 1988;59:93–9.
[95] Taniguchi F, Harada T, Deura I, Iwabe T, Tsukihara S, Terakawa N. Hepatocyte growth factor promotes cell proliferation and inhibits progesterone
secretion via PKA and MAPK pathways in a human granulosa cell line.
Mol Reprod Dev 2004;68:335–44.
[96] Monget P, Bondy C. Importance of the IGF system in early folliculogenesis. Mol Cell Endocrinol 2000;163:89–93.
[97] McNatty KP, Hillier SG, van den Boogaard AMJ, Trimbos-Kemper TCM,
Reichert LE, van Hall EV. Follicular development during the luteal phase
of the human menstrual cycle. J Clin Endocrinol Metab 1983;56:1022–31.
[98] Koering MJ. Cyclic changes in ovarian morphology during the menstrual
cycle in Macaca mulatta. Am J Anat 1969;126:73–101.
[99] Pache TD, Wladimiroff JW, de Jong FH, Hop WC, Fauser BCJM. Growth
patterns of non dominant ovarian follicles during the normal menstrual
cycle. Fertil Steril 1990;54:638–42.
[100] Gougeon A, Lefèvre B. Evolution of the diameters of the largest healthy
and atretic follicles during the human menstrual cycle. J Reprod Fertil
1983;69:497–502.
A. Gougeon / Annales d’Endocrinologie 71 (2010) 132–143
[101] Gougeon A. Dynamics of follicular growth in the human: a model from
preliminary results. Hum Reprod 1986;1:81–7.
[102] Gougeon A, Testart J. Influence of human menopausal gonadotropin on
the recruitment of human ovarian follicles. Fertil Steril 1990;54:848–52.
[103] Kumar TR, Wiseman AL, Kala G, Kala SV, Matzuk MM, Lieberman
MW. Reproductive defects in gamma-glutamyl transpeptidase-deficient
mice. Endocrinology 2000;141:4270–7.
[104] Mason HD, Carr L, Leake R, Franks S. Production of transforming growth
factor-alpha by normal and polycystic ovaries. J Clin Endocrinol Metab
1995;80:2053–6.
[105] el-Roeiy A, Chen X, Roberts VJ, Shimasakai S, Ling N, LeRoith D, et al.
Expression of the genes encoding the insulin-like growth factors (IGF-I
and ll), the IGF and insulin receptors, and IGF-binding proteins 1-6 and
the localization of their gene products in normal and polycystic ovary
syndrome ovaries. J Clin Endocr Metab 1994;78:1488–96.
[106] Mason HD, Margara R, Winston RML, Beard RW, Reed MJ, Franks
S. Inhibition of oestradiol production by epidermal growth factor in
human granulosa cells of normal and polycystic ovaries. Clin Endocrinol
1990;33:511–7.
[107] Kwintkiewicz J, Giudice LC. The interplay of insulin-like growth factors,
gonadotropins, and endocrine disruptors in ovarian follicular development and function. Semin Reprod Med 2009;27:43–51.
[108] Nahum R, Thong KJ, Hillier SG. Metabolic regulation of androgen production by human thecal cells in vitro. Hum Reprod 1995;10:75–81.
[109] Kobayashi M, Nakano R, Ooshima A. Immunocytochemical localization
of pituitary gonadotrophins and gonadal steroids confirms the “two-cell,
two-gonadotrophin” hypothesis of steroidogenesis in the human ovary. J
Endocrinol 1990;126:483–8.
[110] Soules MR, Steiner RA, Clifton DK, Cohen NL, Aksel S, Bremner WJ.
Progesterone modulation of pulsatile luteinizing hormone secretion in
normal women. J Clin Endocrinol Metab 1984;58:378–83.
[111] Solovyeva EV, Hayashi M, Margi K, Barkats C, Klein C, Amsterdam A,
et al. Growth differentiation factor-9 stimulates rat theca-interstitial cell
androgen biosynthesis. Biol Reprod 2000;63:1214–8.
[112] Goodman AL, Hodgen GS. The ovarian triad of the primate menstrual
cycle. Recent Prog Horm Res 1983;39:1–73.
[113] Macklon NS, Fauser BC. Follicle development during the normal menstrual cycle. Maturitas 1998;30:181–8.
[114] Klein NA, Battaglia DE, Fujimoto VY, Davis GS, Bremner WJ, Soules
MR. Reproductive aging: accelerated ovarian follicular development
associated with a monotropic follicle-stimulating hormone rise in normal
older women. J Clin Endocrinol Metab 1996;81:1038–45.
[115] McNatty KP. Ovarian follicular development from the onset of luteal
regression in humans and sheep. In: Rolland R, van Hall EV, Hillier SG,
McNatty KP, Schoemaker J, editors. Follicular maturation and ovulation.
Amsterdam: Excerpta Medica; 1982. p. 1–18.
[116] Mason HD, Cwyfan-Hughes S, Holly JM, Franks S. Potent inhibition
of human ovarian steroidogenesis by insulin-like growth factor binding
protein-4 (IGFBP-4). J Clin Endocrinol Metab 1998;83:284–7.
[117] Hourvitz A, Widger AE, Filho FL, Chang RJ, Adashi EY, Erickson GF.
Pregnancy-associated plasma protein-A gene expression in human ovaries
is restricted to healthy follicles and corpora lutea. J Clin Endocrinol Metab
2000;85:4916–20.
[118] Lawrence JB, Oxvig C, Overgaard MT, Sottrup-Jensen L, Gleich GJ, Hays
LG, et al. The insulin-like growth factor (IGF)-dependent IGF binding
protein-4 protease secreted by human fibroblasts is pregnancy-associated
plasma protein-A. Proc Natl Acad Sci U S A 1999;96:3149–53.
[119] Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellvé A, et al. Effect
of an igf1 gene null mutation on mouse reproduction. Mol Endocrinol
1996;10:903–18.
[120] Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM. Ovarian
function in superoxide dismutase 1 and 2 knockout mice. Endocrinology
1998;139:4008–11.
[121] Amsterdam A, Rotmensch S. Structure-function relationships during
granulosa cell differentiation. Endocr Rev 1987;8:309–37.
143
[122] 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:R1–6.
[123] Roberts VJ, Barth S, El-Roeiy A, Yen SSC. 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;77:1402–10.
[124] Hernandez ER, Hurwitz A, Vera A, Pellicer A, Adashi EY, LeRoith
D, et al. Expression of the genes encoding the insulin-like growth factors and their receptors in the human ovary. J Clin Endocrinol Metab
1992;74:419–25.
[125] Gougeon A. Steroid 3ß-ol-dehydrogenase activity in the largest healthy
and atretic follicles in the human ovary during the menstrual cycle. Ann
Biol Anim Biochim Biophys 1977;17:1087–94.
[126] Filicori M, Cognigni GE, Taraborrelli S, Spettoli D, Ciampaglia W,
Tabarelli De Fatis C, et al. Luteinizing hormone activity in menotropins
optimizes folliculogenesis and treatment in controlled ovarian stimulation. J Clin Endocrinol Metab 2001;86:337–43.
[127] Zeleznik AJ, Schuler HM, Reichert Jr LE. Gonadotropin-binding sites
in the rhesus monkey ovary: role of the vasculature in the selective distribution of human chorionic gonadotropin to the preovulatory follicle.
Endocrinology 1981;109:356–62.
[128] Wulff C, Wiegand SJ, Saunders PT, Scobie GA, Fraser HM. Angiogenesis during follicular development in the primate and its inhibition
by treatment with truncated Flt-1-Fc (vascular endothelial growth factor
Trap[A40]). Endocrinology 2001;142:3244–54.
[129] Chaffkin LM, Luciano AA, Peluso JJ. Progesterone as an
autocrine/paracrine regulator of human granulosa cell proliferation. J
Clin Endocrinol Metab 1992;75:1404–8.
[130] Hernandez-Gonzalez I, Gonzalez-Robayna I, Shimada M, Wayne CM,
Ochsner SA, White L, et al. Gene expression profiles of cumulus cell
oocyte complexes during ovulation reveal cumulus cells express neuronal
and immune-related genes: does this expand their role in the ovulation
process? Mol Endocrinol 2006;20:1300–21.
[131] Edson MA, Nagaraja AK, Matzuk MM. The mammalian ovary from
genesis to revelation. Endocr Rev 2009;30:624–712.
[132] Freimann S, Ben-Ami I, Dantes A, Ron-El R, Amsterdam A. EGFlike factor epiregulin and amphiregulin expression is regulated by
gonadotropins/cAMP in human ovarian follicular cells. Biochem Biophys
Res Commun 2004;324:829–34.
[133] Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth
factors as mediators of LH action in the ovulatory follicle. Science
2004;303:682–4.
[134] Hizaki H, Segi E, Sugimoto Y, Hirose M, Saji T, Ushikubi F, et al.
Abortive expansion of the cumulus and impaired fertility in mice lacking
the prostaglandin E receptor subtype EP(2). Proc Natl Acad Sci U S A
1999;96:10501–6.
[135] Gueripel X, Brun V, Gougeon A. Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during
gonadotropin-induced follicular development in the immature mouse and
is associated with cumulus oophorus expansion. Biol Reprod 2006;75:
836–43.
[136] Assou S, Anahory T, Pantesco V, Le Carrour T, Pellestor F, Klein B, et
al. The human cumulus – oocyte complex gene-expression profile. Hum
Reprod 2006;21:1705–19.
[137] Brailly S, Gougeon A, Milgrom E, Bomsel Helmreich O, Papiernik E.
Androgens and progestins in the human ovarian follicle: differences in
the evolution of preovulatory, healthy nonovulatory, and atretic follicles.
J Clin Endocrinol Metab 1981;53:128–34.
[138] Westergaard L. Follicular atresia in relation to oocyte morphology in non-pregnant and pregnant women. J Reprod Fertil 1985;74:
113–8.
[139] Westergaard LG, McNatty KP, Christensen IJ. Steroid concentrations in
fluid from human ovarian antral follicles during pregnancy. J Endocrinol
1985;107:133–6.