FERTILITY AND STERILITY

VOL. 76, NO. 5, NOVEMBER 2001

Copyright 2001 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.

The physiology of folliculogenesis: the role

of novel growth factors
Gregory F. Erickson, Ph.D. and Shunichi Shimasaki, Ph.D.
Department of Reproductive Medicine, School of Medicine, University of California, San Diego,
La Jolla, California

Objective: To assess major physiological events underlying folliculogenesis, including FSH-dependent

dominant follicle (DF) formation, LH/hCG signaling, and the role of novel regulatory molecules in these
developmental processes.
Design: Review of some of the past and recent advances in ovarian biology, focusing attention on [1] two
novel oocyte-derived growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic protein
(BMP-15); and [2] a recently discovered follicular insulin-like growth factor binding protein-4 (IGFBP-4)
protease, pregnancy-associated plasma protein-A (PAPP-A), that can degrade the FSH antagonist IGFBP-4.
Result(s): Oocyte-derived GDF-9 and BMP-15 are obligatory for folliculogenesis and female fertility in
laboratory animals through their ability to stimulate granulosa cell proliferation and modulate FSH-dependent
cytodifferentiation. The expression of these growth factors in human primary oocytes supports the hypothesis
that GDF-9 and BMP-15 could be involved in ovary function in women. Pregnancy-associated plasma
protein-A is a marker for the human dominant follicle and its product the corpus luteum, raising the possibility
that this putative FSH antagonist might regulate FSH bioactivity during folliculogenesis and luteogenesis.
Conclusion(s): Oocyte-derived and granulosa-derived regulatory proteins perform very important functions
in FSH-dependent folliculogenesis. The current challenges are to understand the role of these novel proteins
in ovary physiology and pathophysiology in women. (Fertil Steril 2001;76:9439. 2001 by American
Society for Reproductive Medicine.)
Key Words: FSH, LH, hCG, GDF-9, BMP-15, PAPP-A, folliculogenesis

Received March 5, 2001;
revised and accepted June
28, 2001.
This work was supported
by NIH U54 HD12303 as
part of the Specialized
Cooperative Centers
Program in Reproduction
Research.
Reprint requests: Gregory
F. Erickson, Ph.D.,
Department of
Reproductive Medicine,
School of Medicine,
University of California,
San Diego, 9500 Gilman
Drive, CMME 2058, La
Jolla, California 92093-
0674 (FAX: 858-534-4959;
E-mail: gerickson@ucsd.
edu).
0015-0282/01/$20.00
PII S0015-0282(01)02859-X
Normally, the human ovaries are responsi-
ble for the regular production of a single dom-
inant follicle (DF), which participates in a sin-
gle ovulation each menstrual cycle. After the
egg cumulus complex is secreted, the follicle
wall transforms into an endocrine gland, the
corpus luteum (CL). The CL secretes consid-
erable quantities of progesterone (P4) and es-
tradiol (E2) which in turn act on uterine cells to
promote pregnancy. In this capacity, the cells
within the DF and its product, the CL, are the
basis for female fertility. Given their para-
mount importance, understanding the mecha-
nisms responsible for the control of DF and CL
development is a major goal of reproductive
research.
A fundamental principle of reproductive bi-
ology is that FSH is obligatory for DF forma-
tion. Thus, to understand the processes by
which a DF and ultimately a CL are formed,
one must understand the mechanisms underly-
ing FSH action. Because FSH receptors are
found only on granulosa cells (1, 2), the ques-
tion of how FSH controls follicular develop-
ment must be answered at the level of these
cells. In a broad sense, the mechanistic basis of
FSH action involves evoked changes in partic-
ular effector genes that coordinate cell prolif-
eration, differentiation, and long-term survival
of the granulosa cells (3, 4).
Although FSH is the primary regulator of
DF development, it is now clear that growth
factors (GFs) produced by the follicle itself can
act by autocrine and paracrine mechanisms to
modulate, either amplify or attenuate, FSH ac-
tion. This body of evidence has led to the
proposition that ovarian GFs might be impor-
tant factors that control female fertility, for
better or for worse. In this review, we will
focus on the basis of DF formation in women
and discuss the physiological role of oocyte
GFs and the granulosa IGF system in this pro-
cess. With respect to the latter, we will discuss
a recently discovered insulin-like growth fac-

943
 FIGURE 1
Chronology of the process of folliculogenesis in human ovaries. (From Gougeon A. Dynamics of follicular growth in the human:
a model from preliminary results. Hum Reprod 1986;1:81.)
Erickson. Physiology of folliculogenesis. Fertil Steril 2001.
tor-4 protease (IGFBP-4ase), pregnancy-associated plasma
protein-A (PAPP-A), in human ovaries.
Physiology of Dominant Follicle Formation
In normal women, the DF originates from a primordial
follicle that was recruited to grow almost 1 year earlier (Fig.
1). Thus, an important principle in human ovarian physiol-
ogy is that folliculogenesis is a very long process (5). Fol-
liculogenesis can be divided into two rather distinct stages:
the gonadotropin-independent (preantral) and gonadotropin-
dependent (antral or Graafian) periods (6). As seen in Figure
1, the gonadotropin-independent and gonadotropin-depen-
dent steps in folliculogenesis occur over a period of about
300 and 50 days, respectively. Locally produced GFs are
critically involved in controlling preantral follicle develop-
ment during the gonadotropin-independent period. After an-
trum formation, the follicle becomes dependent on FSH
stimulation for continued growth and development; how-
ever, it is becoming increasingly clear that long-term ho-
meostasis of developing Graafian follicles also depends on
positive influences evoked by GF-dependent signaling (6).
Given the duration of human folliculogenesis, one can imag-
ine many different potential mechanisms by which GFs
could influence folliculogenesis, including selection and
atresia.
944 Erickson and Shimasaki Physiology of folliculogenesis
The Role of FSH
The concept that FSH is obligatory for DF selection and
development was arrived at almost 60 years ago. In a very
real sense, it represents the cornerstone of our understanding
of ovary physiology. The increase in plasma FSH that occurs
during the late luteal and early follicular phases of the
menstrual cycle is the basis for DF selection in women. The
stringent requirement for this FSH rise in the selection pro-
cess is demonstrated by the fact that in its absence, there is
no DF and no ovulation (7, 8). The physiological conse-
quence of this FSH rise is that a critical threshold concen-
tration of FSH is achieved within the microenvironment of
the chosen follicle. There is a consensus that the threshold
level of FSH results in the expression of E2, which in turn
suppresses plasma FSH levels; this in turn causes the con-
centration of FSH in developing cohort follicles to fall below
threshold levels. It is widely accepted that this FSH with-
drawal phenomenon in cohort follicles is involved in the
massive apoptosis of the granulosa cells that occurs during
atresia. There is evidence that mitosis in cohort follicles can be
markedly stimulated by treatment with hMG during the early
follicular phase (9). One implication of this observation is that
hMG-treatment might increase the number of presumptive DFs
in women by rescuing cohort follicles from atresia.
Vol. 76, No. 5, November 2001
 Evidence from histomorphometric studies suggests that
changes in granulosa mitosis might constitute one mecha- FIGURE 2
nism by which selection occurs (10). Shortly after the mid- Diagram illustrating the two gonadotropin-two cell concept of
luteal phase, the granulosa cells in all cohort follicles appear follicular estradiol production. (From Kettel M, Erickson GF.
to show an increase (approximately twofold) in the rate of Basic and clinical concepts in ovulation induction. In: Rock J,
mitosis. One of the first indications that a DF has been Alverez-Murphy A, eds. Advances in Obstetrics and Gyneco-
ogy. Vol. 1, St. Louis: Mosby Year Book, 1994.)
selected is that the granulosa cells in the chosen follicle
continue proliferating at a fast rate while the rate of prolif-
eration slows in the non-DFs. Because this distinguishing
event appears in the late luteal phase, it is likely that the DF
is selected at this time of the cycle. Given the importance of
FSH in the selection process, it is not unreasonable to as-
sume that the basic mechanism underlying these changes in
granulosa proliferation are functionally related to the rela-
tively high threshold level of FSH in the microenvironment
of the chosen follicle.
A fundamental question concerns the number of potential
selectable follicles in any given cohort. The simple truth is
that we do not know the answer to this basic question. There
is evidence in women that death of the DF or CL leads to the
immediate selection of a new DF (11, 12). This observation
supports the conclusion that human ovaries always contain a
pool of small Graafian (class 4 and 5) follicles (see Fig. 1)
from which another DF can be selected. Although the precise
number is unknown, Gougeon (13, 14) suggests that the
ovaries of normal young women may contain a cohort of
approximately four to six healthy class 4 to 5 follicles. In this
regard, it is likely that the size of the pool is variable, being
correlated with age and ovary reserve.
Considerable effort has been devoted to understanding the
mechanism of FSH action in the DF. The fact that the
granulosa cells are the only cell types known to express FSH
receptors targets them as physiologically important in medi-
ating FSH action in the ovary. The accumulated data from a Erickson. Physiology of folliculogenesis. Fertil Steril 2001.
large number of studies demonstrate that FSH receptor sig-
naling plays a fundamental role in the growth and differen-
tiation of the DF through its ability to promote follicular The Role of LH
fluid formation, cell proliferation, E2 production, and LH Although FSH is the central regulator of DF survival and
receptor expression (3, 6, 15). The temporal pattern and level development, LH/hCG signaling pathways play fundamental
of expression of these FSH-dependent genes are crucial for physiological roles. Physiologically, LH-dependent signal-
the expression of the normal physiological functions as- ing pathways in the theca interstitial cells elicit changes in
cribed to the DF. It should be mentioned that the FSH gene expression that are critical for E2 production (18).
stimulation of LH receptors in the granulosa cells is required Specifically, activation of the LH receptors in theca cells
for LH/hCG to induce ovulation and luteinization (16). A leads directly to the stimulation of high levels of andro-
key feature of the temporal pattern of LH receptor expres- stenedione production. The major physiological significance
sion is that it is suppressed throughout most of folliculogen- of this LH response is to provide aromatase substrate to the
esis. A high level of LH receptor expression is not induced granulosa cells where it is metabolized by P450 aromatase to
in the granulosa cells until the DF reaches the preovulatory E2; this is the two gonadotropin-two cell concept of DF
stage (17). This fact supports the possibility that when LH estrogen biosynthesis (Fig. 2). Because E2 production is
enters the follicular fluid during the late follicular phase it unique to DFs, the level of plasma E2 is a useful marker for
may be an important effector of granulosa function, perhaps monitoring the physiological responses of endogenous or
even replacing FSH as the principle regulator of cytodiffer- exogenous gonadotropins in women.
entiation. There are three additional physiologically important func-

FERTILITY & STERILITY 945

 FIGURE 3
In situ hybridization of oocyte growth factor mRNA in human ovaries. (A), GDF-9 mRNA expressed in oocyte of a developing
secondary follicle. (B), BMP-15 mRNA expressed in oocyte of healthy Graafian follicle.
Erickson. Physiology of folliculogenesis. Fertil Steril 2001.
tions of LH/hCG in the DF and CL. First, the ovulatory dose
of LH/hCG is responsible for ovulation and CL formation.
Second, LH is essential for P4 and E2 production by the CL
during the early and midluteal phases of the menstrual cycle.
And third, hCG is obligatory for transforming the CL of the
cycle into the CL of pregnancy.
The Role of the Oocyte
One of the most exciting discoveries in the past few years
is that the process of folliculogenesis is controlled by GFs
secreted by the oocyte (19, 20). To date, five GFs have
been identified in mammalian oocytes: growth differenti-
ation factor-9 (GDF-9) (21), bone morphogenetic pro-
tein-15 (BMP-15/GDF-9B) (22, 23), bone morphogenetic
protein-6 (BMP-6) (24), transforming growth factor-2
(TGF-2) (25), and fibroblast growth factor-8 (FGF-8) (26).
Much of our understanding of oocyte GFs comes from
experiments on the effects of GDF-9 and BMP-15 in mice
and sheep. The concept emerging from this seminal work is
that oocyte-derived GDF-9 and BMP-15 are obligatory for
normal folliculogenesis and female fertility. The clinical
significance of this new concept (Fig. 3) is emphasized by
the presence of GDF-9 and BMP-15 expression in oocytes of
developing human follicles (27). It should be emphasized
that in the ovaries of all mammals examined to date, the
oocyte is the only cell type that expresses these genes (19).
The definitive evidence that GDF-9 and BMP-15 play
critical roles in follicle development and female fertility
comes from loss-of-function studies in laboratory animals.
Experiments with knockout mice have demonstrated that in
the absence of GDF-9, folliculogenesis is blocked at the
primary preantral stage (28). Consequently, there are no
Graafian follicles, no ovulations, and no pregnancies. A
similar phenotype has been reported in homozygous sheep
carrying a naturally occurring X-linked loss-of-function mu-
946 Erickson and Shimasaki Physiology of folliculogenesis
tation in BMP-15 (29). Regarding BMP-15, it is particularly
significant that the heterozygotes exhibit increased ovulation
rates (29). Collectively, these findings support the fascinat-
ing new concept that oocyte GDF-9 and BMP-15 are abso-
lutely essential for folliculogenesis and fertility; and it is
interesting that naturally occurring BMP-15 mutations can
cause both infertility and super ovulations (DF formation) in
a dosage-sensitive manner. In a very real sense, this evidence
supports the idea that the oocyte is the cornerstone underly-
ing the physiological mechanisms that control DF formation
and ovulation quota in mammals.
A fundamental question in understanding the functions of
GDF-9 and BMP-15 in the follicle concern the identification
of the target cells for these GFs. Several lines of evidence
indicate that granulosa cells are target cells. In vivo studies
of GDF-9 deficient mice have provided compelling evidence
for a critical physiological role of GDF-9 in stimulating
granulosa cell proliferation during the gonadotropin-inde-
pendent period, specifically at the stages leading to the
secondary stage (30). The fact that GDF-9 acts directly on
granulosa cells from preantral as well as DFs to stimulate
DNA synthesis provides unequivocal support for this impor-
tant principle (31). The observation that GDF-9 also acts to
stimulate preantral follicle growth in vitro is consistent with
this conclusion (32). Interestingly, BMP-15 has also been
found to be a potent stimulator of granulosa mitosis in vitro
(33). An important aspect of the mitogenic effects of both
GDF-9 and BMP-15 is that they are FSH independent.
Collectively, these findings indicate a stimulatory role for
GDF-9 and BMP-15 in mitogenic signaling in granulosa
cells throughout the course of folliculogenesis. Thus GDF-9
and BMP-15 appear to be classic granulosa mitogens, at least
in the murine.
GDF-9 and BMP-15 have also been shown to evoke
Vol. 76, No. 5, November 2001
profound changes in FSH-stimulated granulosa cytodifferen-
tiation. Although GDF-9 and BMP-15 alone have minimal
effects on the differentiated state of cultured preantral gran-
ulosa cells, both factors markedly affect FSH action in these
cells (3133). With respect to GDF-9, Vitt et al. (31) dem-
onstrated that GDF-9 inhibits FSH-induced LH-receptor, E2,
and P4 biosynthesis in these cells. Therefore, GDF-9 signal-
ing pathways in undifferentiated granulosa cells appear to be
involved in blocking FSH action. Regarding BMP-15, it has
recently been shown that this GF (like GDF-9) decreases
FSH-induced P4 production by undifferentiated granulosa
cells; however, it is interesting that BMP-15 had no effect on
FSH-induced E2 production (33). These results with
BMP-15 could have physiological relevance in terms of the
selective ability of FSH to promote E2, but not P4, produc-
tion during the follicular phase of the cycle. Importantly, we
have found recently that BMP-15 is a potent inhibitor of
FSH-receptor expression (34). As such, these data are con-
sistent with the hypothesis that oocyte-derived BMP-15 and
GDF-9 may be critical determinants of FSH action during
folliculogenesis.
There is also compelling evidence that GDF-9 is directly
involved in the regulation of the differentiated state of gran-
ulosa cells in DFs. In contrast to undifferentiated granulosa
cells, GDF-9 itself can elicit changes in the differentiated
state of FSH/LH primed granulosa cells in preovulatory
follicles; GDF-9 stimulates the expression of hyaluronan
synthase 2 (HAS2), cyclo-oxygenase 2 (COX-2), and steroid
acute regulatory protein (StAR) (35). The physiological rel-
evance of the GDF-9 stimulated HAS2 and StAR appears to
be associated with cumulus expansion (35) and P4 produc-
tion, respectively. There is also evidence that GDF-9 is
involved in the regulation of FSH action in DFs. Also,
GDF-9 has been found to significantly decrease FSH-stim-
ulated LH-receptor and P4 production by cultured granulosa
cells from preovulatory follicles (31). The physiological
relevance of this GDF-9 effect on FSH action appears to be
linked to the known inhibitory effect of the oocyte on FSH-
induced LH-receptor and P4 expression in the cumulus gran-
ulosa cells of DFs (19, 36). At present, nothing is known
about the role of BMP-15 in regulating the functions of
differentiated granulosa cells. The hypothesis that emerges
from all this work is that the oocyte plays a major role in
determining the way in which FSH acts in the follicle to
regulate granulosa cytodifferentiation.
It was noted in the GDF-9 knockout animals that theca
cell formation was impaired (28). This supported the possi-
bility that GDF-9 may also be an effector of theca-interstitial
cell development. Indeed, a recent study with cultured rat
theca-interstitial cells indicates that GDF-9 amplifies basal
and LH-stimulated androgen biosynthesis (37). This finding
supports the intriguing hypothesis that theca-interstitial cells
are targets for GDF-9, and that oocyte-derived GDF-9 might
be an important determinant of androgen production by
FERTILITY & STERILITY
ovarian follicles. Finally, there are data, albeit limited, sug-
gesting GDF-9 may play a role in oocyte development,
particularly in regulating oocyte size and cortical granule
formation (38). The role of BMP-15 in the development of
the theca and oocyte is unknown.
It will be of great interest to find out how specific GDF-9
and BMP-15 signaling pathways lead to activation and sup-
pression of particular effector genes in human preantral and
DF follicles and ultimately to fertility and infertility in
women.
The IGF System: The Role of PAPP-A
The GFs produced by the granulosa cells are also in-
volved in many aspects of follicle growth and development.
One of the best studied is the IGF system (39, 40). A
functional link between the IGF system and FSH action has
been demonstrated by the finding that IGFBP-4 is a potent
inhibitor of FSH-induced estradiol production by murine
(39, 41) and human (42) granulosa cells. Although the mech-
anism underlying the IGFBP-4 inhibition of FSH action
remains to be elucidated, it appears to involve neutralization
of intrinsic IGF bioactivity, such as IGF-1 and II in the
murine and human, respectively (41, 43, 44). The physio-
logical significance of this work is demonstrated by the
finding that the gene encoding IGFBP-4 is expressed in
atretic follicles in vivo (45 47). Together, these findings
have implicated IGFBP-4 (through its FSH antagonist activ-
ities) in regulatory pathways that lead to follicle atresia.
Recently, much attention has been focused on several
proteases that degrade IGFBP-4 into fragments that are de-
void of FSH-inhibitory activity (48). An important role for
FSH in stimulating the production a specific IGFBP-4 pro-
tease by rat granulosa cells has been reported (49). Because
IGFBP-4ase could indirectly enhance FSH-mediated cell
signaling, there has been great interest in identifying the
nature of this key molecule. Recently, Lawrence et al. (50)
identified pregnancy-associated plasma protein-A (PAPP-A)
as an IGFBP-4ase. This protein is a member of the metzincin
family of metalloproteases and appears to exist as a dimer
with a molecular mass of 400 kd. The existence of
PAPP-A (51) in human follicular fluid fits the hypothesis
that PAPP-A may be responsible for the IGFBP-4 protease
activity known to be present in follicular fluid (52). If true,
then it is possible that PAPP-A is a critical determinant of
FSH-dependent folliculogenesis in women.
Recent evidence indicates that the gene encoding
PAPP-A is expressed in human ovaries, being restricted to
granulosa cells in healthy Graafian follicles (DF) and gran-
ulosa lutein cells of healthy CL (53). The restricted pattern of
PAPP-A expression in normal human ovaries suggests that
PAPP-A may be a functional marker of the DF and its
product, the CL. Although the physiological function of
ovarian PAPP-A remains to be identified, the data are con-
sistent with the hypothesis that the regulated expression of
947
PAPP-A might play a role in controlling survival, growth,
and/or differentiation of the DF and CL by inactivating the
gonadotropin antagonist IGFBP-4.
CONCLUSION
There is no doubt that FSH, LH, and hCG play critical
roles in directing the development, differentiation, and func-
tion of DF and CL in women. The FSH signaling pathways
play an obligatory role in DF selection and survival; LH/
hCG receptor signaling pathways in the theca-interstitial and
granulosa cells play a fundamental role in the production of
aromatase substrate and ovulation, respectively; and hCG
transforms the CL of the cycle into the CL of pregnancy. It
is becoming evident that the oocyte and granulosa cells
produce a variety of GFs that act broadly to influence go-
nadotropin action in positive and negative ways. Obviously,
alterations in the expression and actions of oocyte and gran-
ulosa GFs have far reaching consequences for female fertil-
ity. The current challenges are to understand how oocyte-
derived GDF-9 and BMP-15 and granulosa/CL-derived
PAPP-A exert control of ovary functions and how these
actions are integrated into the overall pattern of physiology
and pathophysiology during the life of a woman.
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