C H A P T E R
2
Regulation of Follicle Formation and
Development by Ovarian Signaling Pathways
Rexxi D. Prasasya
Kelly E. Mayo
Introduction
In mammals, ovarian follicles make up the basic func-
tional unit of the female gonad, the ovary. Within the fol-
licle, the female germ cell, the oocyte, interacts with the
surrounding granulosa cells, and they in turn with thecal
cells, through both physical contact and secreted signals.
Bidirectional communication between the oocyte and the
follicular somatic cells is necessary to ensure fertility
through the eventual ovulation of a fertilizable oocyte
as well as production of hormones by the somatic cells
that are important for overall physiology. The endocrine
control of follicular growth and ovulation by the pituitary
gonadotropins follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) is well recognized from classic
physiology experiments. More recently, the advent of
modern molecular biology techniques and genetic mouse
models has allowed investigations into the cellular
signaling pathways and gene regulatory events that are
responsible for follicle formation and development. As
will become clear early in this chapter, a myriad of over-
lapping and often interacting pathways govern each
specific stage of follicular development. It is perhaps by
design that a built-in redundancy exists in the molecular
control of a physiological process that ultimately deter-
mines the continuation of the species. We begin by focus-
ing our discussion on emerging signaling pathways that
contribute to the least understood stages of follicular
development, the formation and activation of primordial
follicles. Subsequently, we review gonadotropin-
independent and -dependent follicular growth by focus-
ing on several types of dominant local factors that
The Ovary
https://doi.org/10.1016/B978-0-12-813209-8.00002-9
demonstrate the diversity of signaling pathways used
in intrafollicular cell communication.
During embryonic development, primordial germ cells
(PGCs) arise at the proximal epiblast and proceed to pro-
liferate, migrate, and eventually colonize the bipotential
gonad [1]. While mechanisms of sex determination are
beyond the scope of this chapter, it is now known that
the process is somatic cell driven, and ovarian fate is pro-
moted in the absence of the Y chromosome and Sry gene
expression. In the XY individual, the presence of SRY sup-
presses Wnt4/β-catenin pathway signaling (reviewed in
Ref. [2]). Conversely, in the XX individual, the lack of
SRY allows for initiation of pregranulosa cell differentia-
tion as well as continued suppression of the Sertoli cell fate
by an increase in Wnt4/β-catenin signaling (reviewed in
Ref. [2]). At around embryonic day 13.5 (E13.5) in the
mouse or 10 weeks of gestation in the human, a rise in reti-
noic acid (RA) levels within the embryonic ovary induces
the expression of stimulated by retinoic acid gene 8 (Stra8),
which triggers germ cells, or oocytes, to enter meiosis [3,4].
RA in the fetal gonad originates from the mesonephros,
and is actively degraded by the P450 cytochrome enzyme
Cyp26b1 in the embryonic testis, thus preventing early
entry into meiosis in spermatogonia [5]. In contrast,
Cyp26b1 expression is downregulated by E12.5 in fetal
mouse ovaries, thus allowing the rise in RA and initiation
of meiosis [4,5].
Interestingly, meiosis, while necessary for fertility,
does not appear to be required for oocyte differentiation
or the ability of oocytes to interact with granulosa cells to
form follicles. While there is an increased rate of germ
cells loss in Stra8-deficient ovaries, leading to complete
23 © 2019 Elsevier Inc. All rights reserved.
24 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS

germ cell depletion by 6–8 weeks of age, the surviving
germ cells are contained within follicular structures,
which can be ovulated through exogenous hormone stim-
ulation [6]. Moreover, these 2n oocyte-like cells are able to
produce a zona pellucida (ZP) matrix as would bona fide
oocytes [6]. Meiosis will continue to the diplotene stage of
meiotic prophase I, at which it arrests and will not be
resumed until postovulation. Following meiotic arrest,
germ cell clusters, which arise through incomplete cytoki-
nesis of rapidly dividing germ cells, begin interacting with
surrounding pregranulosa cells to form a structure termed
a germ cell syncytia, germ cell cyst, or germ cell nest, which
serves as a precursor to primordial follicles [7]. Pregranu-
losa cells are thought to arise from at least three distinct
origins: somatic cells within the bipotential gonad, the
ovarian surface epithelium, and a structure at the ovary-
mesonephros border termed the rete ovarii [2,8,9].
The number of primordial follicles formed following
completion of germ cell nest breakdown contributes to
establishing the reproductive life span of the individual.
The dynamics of primordial follicle formation and activa-
tion were recently studied by two independent groups
using highly sensitive and temporally inducible reporter
lines to label granulosa cells [10,11]. These studies show
that while some primordial follicles are recruited into
the growing population immediately following their for-
mation, the majority of primordial follicles remain quies-
cent for varying periods of time [10,11]. Mork and
colleagues confirmed that in the mouse, there are at least
two different sources of pregranulosa cells, one migrating
from the mesonephros and one migrating from the sur-
face epithelium [11]. Moreover, migration and the subse-
quent differentiation and association with germ cell nests
by these two distinct types of pregranulosa cells occur
sequentially, resulting in what is now accepted to
be two waves of primordial follicle formation [10,11].
Both studies demonstrate that following birth, primor-
dial follicles with embryonically labeled granulosa cells
(presumably part of the first wave of formation) are
immediately recruited into the growing follicle pool,
some reaching the early antral stage by PND23 [10,11].
Furthermore, these first wave follicles appear to contrib-
ute to fertility in early adulthood, as labeled corpora lutea
(CLs) are detected in PND90 ovaries [10]. These studies
demonstrate that early events such as follicular formation
and activation have direct consequences on fertility dur-
ing adulthood.
CELLULAR SIGNALS REGULATING GERM
CELL NEST BREAKDOWN AND
PRIMORDIAL FOLLICLE FORMATION
A mammalian female is born with a finite number of
oocytes, and as such, the subsequent number of primor-
dial follicles formed will in part determine the fecundity
and reproductive life span of the individual. Fig. 1 illus-
trates the process of follicle formation and development,

FIG. 1 Formation and development of the ovarian follicle. During gestation, migrating primordial germ cells form connected clusters through
incomplete cytokinesis and aggregation. Following colonization of the embryonic ovary, somatic pregranulosa cells will interact with these germ
cell clusters to form germ cell nests. Primordial follicles are formed when pregranulosa cells invade the germ cell clusters to encapsulate single
oocytes within a follicle. This process is accompanied by programmed oocyte death, leaving only about 30% surviving oocytes at the completion
of nest breakdown. Promoters of nest breakdown include paracrine and juxtacrine signaling through Activin, KITL, NGF, and Notch pathway sig-
naling. Conversely, nest breakdown is inhibited by steroid hormones such as estrogen and progesterone. Primordial follicles are gradually recruited
into the growing pool throughout the reproductive lifetime. The extracellular cues that initiate primordial follicle activation remain unclear; however,
PI3K/AKT and mTORC signaling within the oocyte are known to govern its exit from quiescence. The growing follicle population, in turn, provides
negative feedback to the primordial follicle pool through production of AMH. Oocyte activation is also regulated by a network of oocyte-specific
transcription factors that includes NOBOX, LHX8, SOHLH1, and SOHLH2. These transcription factors regulate expression of oocyte-specific genes
including those involved in ZP matrix deposition and Gdf9 and Bmp15, which encode secreted factors that are necessary for continued follicle growth.
As the follicle reaches the multilayer secondary stage, the final somatic cell layer, the theca layer, is specified through activation of Hh signaling. Up to
this stage, follicle development is independent of pituitary gonadotropins. Following formation of the fluid-filled antral cavity, FSH becomes dom-
inant in regulating proliferation and differentiation of granulosa cells toward the preovulatory phenotype working in concert with local paracrine
signals.

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

 CELLULAR SIGNALS REGULATING GERM CELL NEST BREAKDOWN AND PRIMORDIAL FOLLICLE FORMATION
accompanied by major factors that are involved in each
developmental stage. In the XX individual, PGCs
undergo rapid mitotic division as they migrate and colo-
nize the gonad until the initiation of meiosis [12]. This
mitotic division is accompanied by incomplete cytokine-
sis [13–15], resulting in structures termed nests or syncy-
tia where multiple cells are connected by cytoplasmic
bridges, similar to those found in invertebrates [16,17].
There is evidence that organelles, including centrosomes,
Golgi bodies, and mitochondria, are transferred between
sister oocytes through these cytoplasmic bridges, produc-
ing a cloud of organelles termed the Balbiani body within
oocytes that are more likely to survive following cyst res-
olution [18,19]. While there is a strong correlation
between the presence of the Balbiani body and oocyte
survival [18], the cytoplasmic bridges do not appear to
be absolutely necessary for female fertility. This is dem-
onstrated by a knockout model of Tex14, a component
of the cytoplasmic bridges [20]. While Tex14
/
mice
are endowed with reduced numbers of oocytes as com-
pared to littermate controls following completion of germ
cell nest breakdown, their primordial follicle pool is able
to support a normal reproductive life span and full
fertility [20].
Primordial follicles are formed when pregranulosa
cells send projections toward the germ cell nests to encap-
sulate an individual oocyte within a follicle [7]. In the
mouse, germ cell nest breakdown begins shortly before
birth and concludes at about PND6, when more than
80% of germ cells are contained within primordial folli-
cles [7,21]. Only about 30% of oocytes from germ cell
nests survive to become primordial follicles, as a wave
of oocyte death occurs concurrently with the progression
of germ cell nest breakdown [7]. It is hypothesized that
this massive loss of oocytes allows for the optimization
of the number of available pregranulosa cells to the num-
ber of surviving oocytes, and also for the remaining
oocytes to acquire organelles from those that are lost.
Alternatively, oocyte death has also been suggested to
directly facilitate the resolution of germ cell syncytia into
primordial follicles, by creating smaller syncytia follow-
ing selective oocyte lost [7], or as a means to eliminate
oocytes with chromosomal aberrations [22].
There is no consensus on whether germ cell nest break-
down is driven by the germ cells or by the somatic cells. It
is apparent that interactions between properly specified
somatic pregranulosa cells and oocytes are important
for successful nest breakdown and establishment of the
primordial follicle pool. Foxl2, a gene encoding a
winged-helix forkhead gene transcription factor, is one
of the earliest markers identified to be required for gran-
ulosa cell specification [23]. Foxl2
/
mice are infertile due
to incompetency of pregranulosa cells to interact with
germ cells clusters to form follicles [24]. In turn, develop-
mental competency of the oocyte is also important in
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
25
primordial follicle formation. For example, an ovary that
lacks expression of the oocyte-restricted Factor in germ
line alpha (Figla), a basic helix-loop-helix transcription
factor that is required for ZP formation [25], does not
form follicles, leading to eventual demise of oocytes
within disorganized germ cell nests [24]. In the following
sections, we discuss the proposed mechanisms for pro-
grammed germ cell death during the period of nest
breakdown, as well as emerging signaling pathways that
contribute to the formation of primordial follicles.
Molecular Control of Neonatal Oocyte Survival
An experimental tool that has been extensively used to
study signaling pathways during germ cell nest break-
down is the ex vivo culture of neonatal rodent ovaries
[26]. This organotypic culture system, along with genetic
mouse models, has revealed many candidate factors and
signaling pathways that contribute to bidirectional com-
munication between somatic pregranulosa cells and
oocytes during nest breakdown. Several investigations
have explored whether modulation of oocyte survival
can alter the primordial follicle endowment, and thus
the reproductive life span. Of interest is the B cell
lymphoma-2 (BCL-2) family of proteins, whose members
are key regulators of cellular apoptosis. These proteins
promote or inhibit apoptosis by controlling mitochon-
drial outer membrane permeabilization. Deletion of
Bax, a proapoptotic member of the Bcl-2 family, prolongs
reproductive life span by 10–12 months in the mouse due
to a decreased rate of follicular atresia [27]. While it was
initially thought that the primordial follicle pool is not
altered in Bax
/
ovaries, a second study shows an
increase in the absolute number of germ cells, and of pri-
mordial follicles formed, in embryonic and neonatal
Bax
/
ovaries [28]. Surprisingly, a wave of oocyte death
during the period of germ cell nest breakdown is still
observed in Bax
/
ovaries, suggesting that oocyte death
during nest breakdown is Bax-independent.
Additional evidence that suggests alternative mecha-
nisms of apoptotic pathway activation in lieu of BCL-2
family proteins in neonatal oocytes comes from knock-
outs of Puma and Bcl-2-modifying factor (Bmf), both proa-
poptotic proteins [29,30]. Similar to the Bax
/
ovary,
both Puma
/
and Bmf
/
ovaries contain more oocytes
at embryonic and neonatal ages [29,30]. In both models,
however, a wave of germ cell death occurs during the
period of nest breakdown. In the case of Bmf
/
ovaries,
the greater number of germ cells does not result in a
larger endowment of primordial follicles at the conclu-
sion of germ cell nest breakdown [30]. This normalization
in the number of follicles is also observed in an overex-
pression model of the antiapoptotic gene Bcl2 where
the increased numbers of primordial follicles formed
26 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS

are eventually lost, with normalization to that of litter-
mate controls by early adulthood [31]. While the mecha-
nisms behind the initiation of programmed oocyte death
during nest breakdown remain unresolved, it is generally
accepted that oocyte death occurs via apoptosis following
activation of CASPASE-2. Activation of caspases or
cysteine-aspartic proteases leads to cleavage of cellular
proteins and triggers apoptosis. At PND4, Caspase2
/

ovaries contain greater number of primordial follicles,
and thus oocytes, compared to ovaries of littermate con-
trols [32]. Moreover, Caspase2
/
oocytes are shown to
better survive assault from exposure to the chemothera-
peutic agent doxorubicin [32]. While genetic mouse
models have allowed for identification of necessary as
well as dispensable molecules in programmed oocyte
death, the mechanisms by which surviving oocytes are
able to escape apoptosis remain elusive.
The Role of Steroid Hormones in Nest
Breakdown
In rodents, cattle, and macaques, the period of nest
breakdown coincides with a drastic drop in the levels
of steroid hormones to which the newborns or fetuses
are exposed [33–37], leading to the hypothesis that estro-
gen and progesterone act as negative regulators of pri-
mordial follicle assembly (Fig. 2). Using ex vivo cultures
of neonatal rat and mouse ovaries, estradiol and proges-
terone were found to inhibit primordial follicle forma-
tion, resulting in the retention of germ cell nests [38,39].
The effect of progesterone on nest breakdown does not
appear to be due to its conversion to estradiol, since expo-
sure of neonatal ovaries to a nonhydrolyzable form of
progesterone, promegestone, also results in retention of
nests [39]. Estrogen receptor-α (Esr1) is expressed in preg-
ranulosa cells, and estrogen receptor-β (Esr2) is expressed
in both pregranulosa cells and oocytes at birth in mouse
ovaries [40,41]. Using selective agonists and antagonists
for ER-α, ER-β, and the nongenomic, membrane-bound
form of estrogen receptor, Chen and colleague show that
inhibition of germ cell nest breakdown by estrogen can be
carried out by all three forms of estrogen receptor [40].
It is thought that there is a critical window within
which nest breakdown must occur, after which pregranu-
losa cell invasion ceases regardless of whether all oocytes
have been contained in a follicle. Therefore, it is

FIG. 2 Select signals that promote germ cell nest breakdown and primordial follicle formation. The process of germ cell nest breakdown occurs
during midgestation in human and during the neonatal period in the mouse. Experimental evidence has shown that high levels of estrogen and
progesterone inhibit primordial follicle formation. Several signaling pathways that are initiated by growth factors have been shown to promote
primordial follicle formation. Activin stimulates proliferation of pregranulosa cells and promotes Notch signaling in the granulosa cells, which
enhances primordial follicle formation. The Ntrk receptors are localized to both oocytes and pregranulosa cells during the period of nest breakdown
and their ligands are expressed by the pregranulosa cells. Signaling through Ntrks promotes nest breakdown, potentially through upregulation of
Jag1 expression in the oocyte. The juxtacrine signal Notch, which is mediated by the ligand JAG1 in the oocyte and the receptor NOTCH2 in preg-
ranulosa cells, is emerging as an important promoter of nest breakdown and granulosa cell proliferation in the early stages of follicle development.
Signaling through the c-KIT receptor in the oocyte and its ligand KITL in the granulosa cells promotes follicle assembly through its contribution to
programmed germ cell death.

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

 CELLULAR SIGNALS REGULATING GERM CELL NEST BREAKDOWN AND PRIMORDIAL FOLLICLE FORMATION
postulated that remnants of germ cell nests from delayed
breakdown will ultimately lead to the formation of multi-
oocytic follicles (MOFs). The in vitro findings of the neg-
ative effects of estrogen on nest breakdown are consistent
with in vivo studies where neonatal exposure to the syn-
thetic estrogen diethylstilbestrol (DES) [42,43] or the soy
phytoestrogen genistein [44,45] results in delayed germ
cell nest break down and a higher incidence of MOFs
as compared to control mice. The consequence of MOFs
for fertility remains unclear. Iguchi et al. report reduced
fertilization capacity of oocytes obtained from ovulated
MOFs in a mouse model, but the confounding effects of
early exposure to DES in these oocytes cannot be elimi-
nated [46]. However, at least one report suggests that
using oocytes from MOFs does not lead to any adverse
outcomes in human in vitro fertilization [47].
The Roles of Growth Factors and Somatic Cell-
Cycle Progression During Germ Cell Nest
Breakdown
Several growth factors have been implicated in germ
cell nest breakdown. Activin is a member of the TGF-β
family of proteins that is expressed in granulosa cells of
growing follicles [48]. Treatment of neonatal mice with
activin leads to a 30% increase in the number of primor-
dial follicles, and this is associated with increased prolif-
eration of both granulosa cells, and, to a limited degree,
the germ cells population (Fig. 2) [21]. Similar to several
genetic models of BCL-2 family protein modulation
described earlier, the expanded primordial follicle pool
that is observed in neonatally activin-exposed ovaries is
not maintained into reproductive maturity, a paradigm
that is described as an inherent quorum-sensing mecha-
nism by the ovary [21]. Follistatin (FST) is expressed in
the gonad and functions to bind activin and neutralize
its action [49]. Germ cell nest breakdown is significantly
delayed in a mouse model that selectively expresses a
high-affinity isoform of FST (FST-288) due to an increase
in somatic cell proliferation and a decrease in germ cell
death during the neonatal period [50]. Whether this delay
in germ cell nest breakdown leads to MOFs was not char-
acterized [51]; however, the primordial follicle pool is
expanded in the FST-288 mouse [50]. Despite this, the
FST-288 mouse is subfertile due to accelerated depletion
of the primordial follicle pool in adulthood [52]. Consis-
tent with this genetic mouse model, the addition of FST-
288 to ex vivo ovarian culture system during the period of
germ cell nest breakdown leads to a delay in primordial
follicle formation [53]. FST-288 treatment leads to
reduced granulosa cell proliferation and suppressed
Notch signaling, a pathway that is known to promote
germ cell nest breakdown, in granulosa cells [53]. These
studies illustrate the importance of activin signaling in
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
27
formation and maintenance of the primordial follicle pool
through its influence on granulosa cell proliferation and
likely, germ cell survival.
The importance of receptor tyrosine kinase (RTK) sig-
naling in promoting germ cell nest breakdown is begin-
ning to be elucidated. Kit ligand (KITL) and its
tyrosine-kinase receptor c-KIT are localized to pregranu-
losa cells and oocytes, respectively, during ovarian
embryonic development (Fig. 2) and have been shown
to be necessary for PGC migration [54]. Their expression
remains during the period of nest breakdown and expo-
sure of cultured neonatal mouse ovaries to a c-KIT block-
ing antibody leads to the retention of germ cell nests,
which is hypothesized to result from inhibition of oocyte
death [55]. Furthermore, exposure of neonatal rats to the
small molecule tyrosine kinase inhibitor imatinib, which
targets several RTKs including c-KIT, leads to retention of
germ cell nests and an increased incident of MOFs [56].
Expression of neurotrophic tropomyosin-related
kinase (Ntrk) receptors, a family of RTKs, and their solu-
ble neurotrophin ligands is dynamically regulated during
the period of germ cell nest breakdown in neonatal rat
ovaries [57]. Knockout mouse models of the ligand nerve
growth factor (Ngf) or the receptors Ntrk1 and Ntrk2 lead
to a delay in germ cell nest breakdown and reduction in
the number of primordial follicles formed [58,59]. Further
evidence for the involvement of Ntrk family of receptors
in nest breakdown is demonstrated by the enhancement
of primordial follicle formation observed in neonatal
mouse ovaries that are cultured with connective tissue
growth factor (CTGF), whose expression is upregulated
during the neonatal period [60]. While the receptor by
which CTGF exerts its effects during germ cell nests
breakdown has not been specified, there is evidence of
its ability to activate NTRK1 in human mesenchymal
cell [61].
Additional evidence for an association between regu-
lations of germ cell nest breakdown and cell cycle pro-
gression comes from a knockout model of the cell-cycle
inhibitor p27KipI. A member of the Cip/Kip subfamily,
p27KipI, acts as a cyclin-dependent kinase inhibitor.
p27KipI is localized to somatic cells of primordial folli-
cles, and p27KipI
/
ovaries show an acceleration in germ
cell nest breakdown, reaching completion by PND2 [62].
While proliferation was not directly measured, this result
suggests that the loss of inhibition of somatic cell prolif-
eration facilitates the formation of primordial follicles.
This is consistent with the correlation between granulosa
cell proliferation and germ cell nest breakdown that is
observed following modulation of activin signaling.
The number of primordial follicles formed in p27KipI
/

ovaries is double that of litter-mate controls; however,
the number of follicles eventually normalizes by early
adulthood following massive follicular attrition, consis-
tent with the quorum-sensing model proposed to govern
28 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
follicular dynamic in the ovary [63]. p27KipI also appears
to control primordial follicle maintenance, which we will
discuss in more detail in the subsequent section, as
p27KipI
/
mice are infertile due to a premature depletion
of the primordial follicle pool [62].
Regulation of Germ Cell Nest Breakdown by
Notch Signaling
To form primordial follicles, pregranulosa cells must
invade germ cells clusters and form physical contacts
with the oocyte that will eventually be surrounded and
survive. While direct contact between the first layer of
granulosa cells and the oocyte is initially unimpeded,
ZP matrix deposition following primordial follicle activa-
tion will eventually limit these continuous cell-to-cell con-
tacts. To maintain physical contact with the oocyte,
granulosa cells are able to send cytonemes that traverse
the ZP and form both adherens and gap junctions with
the oocyte plasma membrane, termed transzonal projec-
tions (TZPs) [64]. While initially thought to exclusively
function as a means for granulosa cells to shuttle nutri-
ents in response to the metabolic demands of the oocyte
during development, the presence of TZPs provides a
rationale for investigations into the potential role of
cell-contact-dependent signaling during folliculogenesis.
Among these, the highly conserved Notch juxtacrine sig-
naling pathway has emerged as a key regulator of germ
cell nest breakdown. In mammals, Notch signaling
occurs when one of the five-membrane-bound ligands
(JAGGED1-2, DLL1,3,4) bind to one of the four-
membrane-bound receptors (NOTCH1-4) on an oppos-
ing cell [65]. Ligand binding leads to conformational
changes of the Notch receptor, initiating a proteolytic cas-
cade that will ultimately lead to a γ-secretase-dependent
cleavage and liberation of the Notch receptor intercellular
domain (NICD) [66]. NICD will translocate into the
nucleus and function as a transcriptional coactivator of
Notch target genes upon binding with the obligate cofac-
tor RBPJ [67]. Notch signaling is known to regulate many
cell-fate decisions during development [68], and to con-
trol the proliferation and differentiation of somatic follicle
cells (analogous to somatic pregranulosa cells in mam-
mals) during ovariole development in the Drosophila mel-
anogaster ovary [69].
Notch receptors and ligands are expressed in neonatal
and adult ovaries of mice [70–72]. Using a transgenic
Notch reporter mouse line in which the green fluorescent
protein (Gfp) is expressed in an NICD/RBPJ-dependent
manner [73], Notch active cells are detected as early as
E15.5 in the mouse ovary [74]. By E18.5, Notch activity
is detected in granulosa cells that are actively reorganiz-
ing to encapsulate germ cells clusters [74]. Reporter activ-
ity and Notch receptor expression continue in somatic
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
granulosa cells as follicles are activated and mature.
The oocyte expresses predominantly the ligand JAG1 at
this time.
The importance of functional Notch signaling was
demonstrated using an ex vivo ovarian culture system.
Following a culture period that represents the period of
germ cell nest breakdown, treatment of neonatal mouse
ovaries with the γ-secretase inhibitor, DAPT, leads to
the retention of germ cell nests and formation of fewer
primordial follicles as compared to the vehicle controls
[75,76]. DAPT treatment also leads to suppression of
oocyte-specific transcription factors that are known to
be important for early follicular development such as
newborn ovary homeobox protein (Nobox), Figla, sper-
matogenesis and oogenesis specific basic helix-loop-helix
2 (Sohlh2), and LIM/homeobox protein 8 (Lhx8) [77].
A disintegrin and metalloproteinase 10 (ADAM-10) is
an integral component of the proteolytic cleavage
sequence that leads to the liberation of NICD [78].
In vitro treatment of embryonic ovaries with an ADAM-
10 inhibitor or conditional deletion of ADAM-10 in
somatic pregranulosa cells leads to suppression of Notch
signaling (as identified by reduced level of NICD and
decreased expression of Notch target genes Hey2 and
Hes1) and retention of germ cell nests [79].
In other mouse models, conditional deletion, using the
Cre-loxP system, of the most abundantly expressed Notch
receptor, Notch2, in the pregranulosa cells (N2KO) leads
to decreased numbers of primordial follicles and forma-
tion of MOFs [74,80]. The rate of apoptosing oocytes is
significantly decreased at PND1 in N2KO ovaries, which
is associated with delayed germ cells nests breakdown
[80]. A null mutation in the Notch receptor modifier Luna-
tic fringe (Lfng) also reveals the presence of MOFs and
infertility [81]. Overall, these studies demonstrate the
importance of canonical Notch signaling in the somatic
pregranulosa cells for proper follicular formation.
As expected, conditional deletion of the most abun-
dantly expressed ligand Jag1, from the oocytes (J1KO)
phenocopies, in a slightly more robust manner, the
N2KO line [74]. Jag1 expression within the oocyte was
recently described to be controlled by RAC1, a small
GTPase switch that affects gene transcription via the
STAT3 pathway (Fig. 2) [82]. Inhibition of RAC1 leads
to decreased numbers of primordial follicles formed
and reduced expression of several oocyte specific factors,
including growth differentiation factor-9 (Gdf9), bone
morphogenic protein-15 (Bmp15), Nobox, and Jag1 [82].
MOFs in N2KO and J1KO mice can be extremely large,
containing many oocytes, suggesting that they are indeed
remnants of incompletely broken down germ cells nests
[74]. Both the N2KO [80] and J1KO [74] lines are subfer-
tile, indicating that disruption of critical signals during
follicular formation may lead to adverse fertility out-
comes. Together, these genetic mouse lines support the
 REGULATION OF PRIMORDIAL FOLLICLE ACTIVATION
model that physical contact between oocyte and pregra-
nulosa cells facilitates initiation of Notch signaling, which
in turn acts as a critical mean of bidirectional communi-
cation between the two cell types during follicular assem-
bly (Fig. 2).
It is interesting to note that many of the previously
discussed candidates for regulators of germ cell nest
breakdown appear to function upstream of Notch signal-
ing. Progesterone exposure of embryonic and neonatal
ovaries, which is shown to suppress follicular assembly,
inhibits expression of Jag2, Notch2, and a known Notch
target gene Hey2 [83]. Jag1, Hes1, and Hey2 were identi-
fied to be significantly downregulated in neonatal
Ntrk2
/
ovaries, in which reductions in follicular assem-
bly and granulosa cell proliferation are observed [84].
Additionally, lentiviral-mediated targeted expression of
Jag1 in oocytes of Ntrk2
/
ovaries was shown to rescue
the defects in granulosa cell proliferation [84]. Fig. 2 sum-
marizes known as well as proposed interactions between
different signaling pathways that regulate germ cell nest
breakdown. It will require further exploration to under-
stand whether Notch signaling acts as a potential integra-
tor of signals that promote germ cell nest breakdown in
the mammalian ovary.
REGULATION OF PRIMORDIAL FOLLICLE
ACTIVATION
Throughout the reproductive life span, growth is initi-
ated in a portion of primordial follicles, and they transi-
tion to the activated primary follicle stage.
Morphologically, this is characterized by the transition
of the single layer of squamous granulosa cells into cuboi-
dal cells, as well as deposition of a ZP matrix by the
oocyte [85,86]. In turn, a cohort of these small activated
follicles will be recruited to undergo further growth dur-
ing each reproductive cycle, with their final fates being
either atresia or ovulation. Perhaps one of the least under-
stood aspects of the mammalian ovary is the physiolog-
ical cues that are responsible for initiating the
activation of some, but not all, primordial follicles at
any given moment.
One candidate for a signal regulating primordial folli-
cle activation is the Anti-M€ ullerian hormone (AMH).
A member of the TGF-β superfamily of proteins, AMH
is produced by granulosa cells of growing follicles in
the ovary [87,88]. Clinically, AMH is used as a marker
of ovarian follicular reserve due to the well-characterized
positive correlation between its serum levels and antral
follicle counts [89]. Serum levels of AMH decline with
age, and AMH levels are often found to be low or unde-
tectable in clinical cases of primary ovarian insufficiency
[90,91]. Female mice with a null mutation of Amh are fer-
tile; however, more growing follicles are detected in adult
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
29
Amh
/
ovaries compared to wild-type littermates [92].
By 4 months of age, the number of primordial follicles
is significantly reduced in Amh
/
ovaries, becoming
almost completely depleted by 13 months of age [93].
Despite the increase in the absolute number of growing
follicles, Amh-/- ovaries do not contain an increased pro-
portion of preovulatory follicles due to an enhancement
in the rate of atresia of small preantral follicles [94]. Con-
versely, addition of AMH to ex vivo cultured ovaries leads
to a decrease in the growing follicle population by about
40% [93]. These studies suggest that the growing follicle
population plays a role in regulating the dynamics of acti-
vation of the primordial follicle pool through production
of AMH, which acts as a maintenance signal.
Despite unresolved questions regarding the extracellu-
lar signals initiating activation, studies involving the
intracellular pathways regulation of follicle activation
have been significantly advanced, largely through the
use of mouse genetic models. Fig. 3 summarizes pheno-
types of relevant mouse genetic models that are discussed
in the proceeding sections. In addition to discussing path-
ways intrinsic to the oocyte and their role in primordial
follicle activation, we review below the networks of tran-
scriptional control associated with oocyte and granulosa
cell differentiation during the transition from the primor-
dial to the primary follicle stage.
Intracellular Signaling Pathways That Regulate
Primordial Follicle Activation
Regulation of Oocyte Activation by the PI3K/AKT
Pathway
Perhaps the most intensely investigated pathway in
primordial follicle activation is PI3K/AKT signaling
within the oocyte. A major kinase cascade that can be acti-
vated by RTKs, the PI3K/AKT pathway (Fig. 4) functions
to regulate metabolism, growth, proliferation, and sur-
vival through activation of gene transcription or protein
synthesis. Following ligand binding, some RTKs trans-
duce signals through production of the plasma mem-
brane lipid phosphatidylinositol 3,4,5-triphosphate
(PIP3) by phosphoinositide 3-kinase (PI3K). PIP3 acts as
an anchor for phosphoinositide-dependent kinase
I (PDK1), which will in turn phosphorylate and activate
the serine/threonine kinase AKT. Direct targets of AKT
are numerous and include transcription factors or repres-
sors such as Foxo3 [95]. An important antagonist of
PI3K/AKT signaling is the phosphatase and tensin
homolog (PTEN), which functions to dephosphorylate
PIP3 to phophatydylinositol 4,5-biphosphate (PIP2), thus
abrogating the anchoring of PDK1 to the plasma
membrane.
FOXO3 is a transcriptional repressor that is now
accepted to function as a brake for primordial follicle
30 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
activation. Initial studies revealed premature reproduc-
tive senescence due to unrestricted, global activation of
the primordial follicle pool in a Foxo3-/- mouse line
(Fig. 3) [96]. In wild-type ovaries, FOXO3 is localized to
both nucleus and cytoplasm of oocytes of primordial
and primary follicles [97]. FOXO3 then becomes unde-
tectable in oocytes of secondary follicles and beyond
[97]. Conditional Foxo3 ablation from the oocyte [98]
leads to global primordial follicle activation identical to
that observed in the conventional Foxo3-/- ovary, confirm-
ing its oocyte-centric function [99]. Identification of
FOXO3 as a likely suppressor of oocyte activation
prompted follow-up investigations into the PI3K/AKT
pathway as its potential upstream regulator.
Conditional deletion of the negative regulator of the
PI3K/AKT pathway, Pten, from the oocyte leads to acti-
vation of the entire primordial follicle pool, resulting in its
premature depletion and ovarian failure by early adult-
hood [100]. In a similar model of Pten knockout from
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
FIG. 3 The balance of activating and maintenance cues
in the primordial follicle pool. (A) PI3K/AKT and mTORC
pathway activation, which promotes the exit of primordial
follicles from quiescence, is restrained by molecules such as
Pten and the Tsc1/2 complex. The transcriptional repressor
FOXO3 also serves as a molecular brake for primordial fol-
licle recruitment. Growing follicles secrete AMH that acts
as a maintenance signal for the primordial follicle pool,
most likely acting within the granulosa cells of these pri-
mordial follicles. Balanced activation and maintenance sig-
nals result in cyclic recruitment of primordial follicles
throughout the reproductive lifespan. (B) Genetic models
that abrogate the function of maintenance molecules lead
to unrestrained activation of the PI3K/AKT/mTORC path-
way. This results in activation of the entire primordial fol-
licle pool that leads to exhaustion of the follicular reserve
and premature reproductive senescence. (C) Conversely,
genetic models that prevent activation of the PI3K/AKT/
mTORC pathway lack follicular development beyond the
primordial stage. In these models, a permanent block in pri-
mordial follicle activation will eventually lead to atresia
and premature reproductive senescence.
oocytes, generated using a distinct recombination strat-
egy, it was shown that the resulting unrestrained activa-
tion of the PI3K/AKT pathway leads to
hyperphosphorylation, and thus degradation, of FOXO3
[99]. Interestingly, abrogation of AKT activation through
conditional deletion of Pdk1 in the oocyte also leads to
infertility [101]. In this mouse model, long-term blockade
of primordial follicle growth proved to be detrimental,
eventually resulting in clearance of these nongrowing fol-
licles and depletion of the primordial follicle pool by early
adulthood [101]. These studies show that AKT activation
in the oocyte is both necessary and sufficient for activa-
tion of the primordial follicle. Somewhat counterintui-
tively, however, oocyte-specific constitutive activation
of PI3K (PI3KCA), which leads to hyperactivation of
AKT, does not completely phenocopy the loss of Pten
from the oocyte [102]. By PND50, the primordial follicle
pool is not completely depleted in oocyte-specific Pi3kca
transgenic mice, yet still shows a reduction by about 50%
 REGULATION OF PRIMORDIAL FOLLICLE ACTIVATION 31

FIG. 4 Intraoocyte signaling pathways and regulation of granulosa cell differentiation in primordial follicle activation. Through several genetic
models, activation of the PI3K/AKT and mTOR signaling pathways has been shown to promote the exit of the primordial oocyte from quiescence.
The initiating extracellular cues for these pathways still require further investigation, with RTKs such as cKIT as potential candidates. Granulosa cells
of primordial follicles likely provide such oocyte-activating factor(s). mTOR and the JAK/STAT3 pathways have been shown to promote pregra-
nulosa cell activation, while the transcription factors FOXL2 and GATA4/6 are known regulators of granulosa cell differentiation. Activation of the
PI3K/AKT cascade leads to phosphorylation and inactivation of the transcriptional repressor FOXO3. This allows for expression of genes that
promote follicle activation and growth. AKT activation also inhibits the Tsc1/2 complex, which allows for activation of mTORC resulting in the
promotion of translation of proteins regulating oocyte growth.

compared to wild-type littermates [102]. The slower rate
of global primordial follicle activation in oocyte-specific
Pi3kca transgenic mice may be attributed to the increased
survival rate of follicles at all stages of development [102],
suggesting a secondary role of AKT in maintaining
oocyte and follicle survival.
Findings regarding the involvement of the PI3K/AKT
pathway in primordial follicle activation have led to
explorations into the potential use of pharmacological
modulation of PI3K/AKT in clinical settings. Increas-
ingly improved outcomes of pediatric cancer cases have
led to growing interest in the development of fertility-
preserving regiment against gonadotoxic chemothera-
peutics. At the forefront is the cryopreservation of cortical
ovarian tissues consisting mostly of dormant primordial
follicles, followed by in vitro maturation of follicles and
eventually the recovery of fertilizable oocytes [103,104].
Efficient means of promoting activation of cryopreserved
primordial follicles may greatly improve fertility out-
comes for these cancer survivors. Acute exposure of
neonatal mouse ovaries to a PTEN inhibitor, bpV(pic),
resulted in activation of the PI3K/AKT pathway and
FOXO3 exclusion in oocytes of primordial follicles
[105]. Transplantation of treated ovaries into ovariecto-
mized hosts revealed an enhanced rate of follicular
growth as compared to controls and in vitro fertilization
of the recovered matured oocytes, followed by embryo
transfer, is able to generate viable and fertile offspring
[105,106]. Similarly, treatment of human ovarian cortical
tissues with bpV(pic) followed by xenograft to immune-
deficient mice allows for an increased rate of follicular
development to the preovulatory stage and recovery of
meiotically competent oocytes [105].
Earlier, we discussed the role of KITL and the c-Kit
receptor in regulation of germ cell nest breakdown. Early
observations in ovaries of a naturally occurring mouse
line with a mutation (Steel Panda) that lead to severely
reduced expression of KITL (Kitlg) revealed a block in
follicle development at the primordial and primary
stages [107]. Abrogation of KITL binding to c-KIT using

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

32 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
antibody ACK2 was shown to block resumption of
growth of primordial follicles in both in vivo and
ex vivo models across several different species
[26,108,109]. In turn, addition of KITL to ex vivo cultured
ovaries promotes primordial follicle activation [109].
c-KIT is a RTK that is localized to the oocyte, and c-KIT
inhibition appears to phenocopy the block to primordial
follicle activation seen in models of PI3K/AKT inhibition
[101]. This suggests that c-KIT activation may be at least
partially responsible for PI3K/AKT activation within the
oocyte during the primordial to primary follicle transi-
tion. Indeed, short-term culture of oocytes that are col-
lected from mouse primordial follicles with KITL leads
to nuclear export, and as a consequent inactivation, of
the transcriptional repressor FOXO3, which is dependent
upon activation of the PI3K/AKT-signaling pathway
[110]. An oocyte-specific Kit gain-of-function mutation
leads to global primordial follicle activation, while
oocyte-specific Kit inactivation leads to an extended block
to follicular development at the primordial stage that
eventually results in global follicular atresia (Fig. 3)
[111]. Hyperactivation of AKT and cytoplasmic localiza-
tion of Foxo3 are observed in oocyte-specific Kit gain-of-
function ovaries, while persistent nuclear localization of
Foxo3 is observed in oocyte-specific Kit-inactivated ova-
ries [111]. While these studies provide strong evidence for
Kit involvement in primordial follicle activation, the fact
remains that only a limited number of primordial follicles
are activated at any given time, regardless of c-Kit expres-
sion in the oocyte population [112]. Therefore, it is highly
likely that additional regulators, perhaps spatially or
temporally modulated cofactors to the c-KIT signaling
pathway, are involved in the process of primordial folli-
cle activation.
Contribution of mTOR Signaling to Primordial
Follicle Activation
In addition to inactivation of Foxo3, the PI3K/AKT
pathway in the oocyte has also been linked to mamma-
lian target of rapamycin (mTOR) signaling in the regula-
tion of primordial follicle activation. The mTOR pathway
(Fig. 4) serves as a regulator of cell metabolism, growth,
proliferation, and survival by integrating diverse extra-
cellular and intracellular cues. Central to mTOR signaling
is the assembly of the large multidomain mTOR com-
plexes (mTORC1 and mTORC2) of serine-threonine
kinases. Instead of induction of gene transcription per
se, mTOR signaling promotes protein synthesis through
activation of components of the translation machinery
following phosphorylation by mTORC. Some targets of
mTORC include the eukaryotic initiation factor
4E-binding protein 1 (4E-BP1) and the p70 ribosomal s6
kinase 1 (S6K1). The ribosomal protein S6 (rpS6) is a sub-
strate of S6K1 and its phosphorylation; thus, rpS6 phos-
phorylation is often used as an experimental indicator
of mTOR pathway activation. The activated PI3K/AKT
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
pathway positively regulates mTORC1 activity through
inactivation of the mTORC1 inhibitors TSC1 (tuberous
sclerosis complex 1) and TSC2.
Characterization of one of the Pten oocyte-specific
knockout mouse lines revealed increased phosphoryla-
tion of rpS6 as compared to wild-type littermates, impli-
cating the involvement of mTOR signaling in the
observed global activation of primordial follicles
(Fig. 3) [100]. Inactivation of mTORC1 through treat-
ment with rapamycin is able to partially preserve the
primordial follicle pool in the oocyte-specific Pten-
knockout ovary, suggesting that functions of PTEN in
the oocyte are partially mediated through mTORC1
[113]. Additional evidence for the importance of the
mTOR pathway in primordial follicle activation comes
from conditional deletion of Tsc1 (OoTsc1
/
) or Tsc2
(OoTsc2
/
) in oocytes resulting in global primordial
follicle activation (Fig. 3) [114,115]. S6K1 and rpS6 are
hyperphosphorylated in OoTsc1
/
or OoTsc2
/
oocytes,
indicating increased activation of the mTOR pathway
[114,115]. Global primordial follicle activation in
OoTsc1
/
ovaries can be attributed entirely to over
activation of mTORC1, since long-term treatment with
rapamycin completely abrogates the observed depletion
of the primordial follicle pool [114]. While mTORC1 in
the oocytes appears to be sufficient for activating
primordial follicles, it does not seem to be necessary,
due to the compensatory potential of the PI3K/AKT
pathway. Regulatory-associated protein of mTORC1
(RPTOR) is a part of the mTORC1 complex and is
required for mTORC1 assembly. Conditional deletion
of Rptor from the oocyte (OoRptor
/
) results in no repro-
ductive phenotypes despite the loss of mTORC1 activity
[116]. AKT is hyperphosphorylated in OoRptor
/

oocytes, suggesting that AKT signaling without mTORC1
activity, presumably through inactivation of FOXO3,
is able to support a normal rate of primordial follicle
activation [116].
Interestingly, mTOR also appears to be important in
maintaining primordial follicle quiescence through sig-
naling within the granulosa cells. Granulosa cell-specific
disruption of Rptor (PfGC-Rptor
/
) leads to a prevalent
block in follicular activation, resulting in very few grow-
ing follicles by juvenile age (Fig. 3) [117]. In contrast,
granulosa cell knockout of the mTORC1 negative regula-
tor Tsc1 (PfGC-Tsc1
/
) leads to global activation of pri-
mordial follicles, a phenotype that can be reversed by a
regimen of rapamycin treatment [117]. Granulosa cells
of PfGC-Tsc1
/
ovaries express elevated levels of KITL,
which leads to hyperactivation of the c-KIT-mediated
PI3K/AKT pathway within oocytes [117]. Indeed, phar-
macological abrogation of the PI3K/AKT pathway is
able to rescue the global primordial follicle activation
phenotype of PfGC-Tsc1
/
ovaries [117], consistent
with findings that have been discussed earlier in this
section. This result suggests that granulosa cell-secreted
 REGULATION OF PRIMORDIAL FOLLICLE ACTIVATION
factors, such as KITL, likely act as initiating cues for
the oocyte to exit dormancy and for the follicle to enter
the growing pool (Fig. 4).
Oocyte-Specific Transcriptional Networks That
Promote Primordial Follicle Activation
Several oocyte-specific transcription factors are impor-
tant in the transition from the primordial to the primary
follicle stage. The search for master regulators of primor-
dial follicle activation has been performed by a combina-
tion of in silico analysis, gene expression studies, and
genetic mouse models. Nobox is one of the first oocyte-
specific homeobox genes identified through in silico
analysis of NCBI UniGene database of a neonatal mouse
ovary cDNA library [118]. Detected in oocytes beginning
at E15.5, high expression of Nobox remains throughout
folliculogenesis. While the males are completely fertile,
Nobox
/
females are infertile due to an almost complete
loss of oocytes by 6 weeks of age [119]. Although primor-
dial follicles are initially observed, no growing follicles
are ever detected in Nobox
/
ovaries, suggesting a failure
in primordial follicle activation. By PND14, Nobox
/

ovaries contain mostly degenerating follicles which
lack oocytes. At the molecular level, Nobox binds to
the promoters of the oocyte specific genes Oct4 and
Gdf9, and expression of these genes is lost in Nobox
/

ovaries [119,120]. This finding is consistent with the
clinical observation of high prevalence of NOBOX
mutations, leading to compromised ability to bind the
Gdf9 promoter, in patients with primary ovarian
insufficiency [121].
The lim-homeobox protein, LHX8 is localized to
oocytes throughout follicle development [122]. While
conventional knockout of Lhx8 results in very similar
phenotypes to those observed in Nobox
/
mice, charac-
terized by failure of primordial follicles to transition into
the growing pool and eventual global atresia by PND7,
conditional oocyte deletion of Lhx8 reveals a more spe-
cific function [123], with global primordial follicle activa-
tion due to hyperactivation of the PI3K/AKT pathway
[124]. LIN28A, a known positive regulator of the AKT/
mTOR pathway, is elevated in the oocyte-specific Lhx8
knockout, and indeed, Lhx8 was found to bind the Lin28a
promoter and suppress its expression [124].
Sohlh1 and Sohlh2 encode basic helix-loop-helix tran-
scription factors that are expressed exclusively in female
germ cells beginning at E15.5 and E12.5, respectively
[122,125,126]. While expression of Sohlh1 and Sohlh2 is
robust in oocytes within germ cell nests and primordial
follicles, their expression diminishes in primary follicles
and beyond, suggesting that their functions are restricted
to early follicular development. Sohlh1 [122] or Sohlh2
[127] knockout females are infertile due to lack of follicu-
lar growth. While the size of the primordial follicle pool in
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
33
Sohlh1
/
and Sohlh2
/
ovaries is comparable to wild-
type littermates, follicles with cuboidal granulosa cells
are never observed, indicating a failure of primordial fol-
licles to transition into the primary stage [122,127].
SOHLH1 and SOHLH2 homodimerize, as well as hetero-
dimerize, and promote expression of oocyte specific
genes such as Nobox and Lhx8 [126,128]. Collectively,
these studies begin to clarify a transcriptional network
that regulates the process whereby primordial follicles
remain dormant, or are activated to form primary
follicles.
Granulosa Cell Differentiation During the
Primordial to Primary Follicle Transition
As discussed in the last section, granulosa cells of pri-
mordial follicle likely provide ligands or extracellular
cues that initiate the intra-oocyte cascades that lead to fol-
licle activation. Recently, activation of the JAK/STAT3
pathway in granulosa cells of primordial follicles was
shown to enhance primordial follicle recruitment [129].
Further analysis on the downstream consequences, and
particularly of the changes in secreted products following
JAK/STAT3 pathway activation, may aid in identifying
additional candidates for primordial oocyte-activating
factors.
Failure of granulosa cells to undergo proper differen-
tiation often leads to unsynchronized development
between the oocyte and granulosa cells, which is detri-
mental to the survival of the follicle. Previously, we dis-
cussed the failure of pregranulosa cells to interact with
germ cell nests and properly form follicles in Foxl2
/

mice [130]. A distinct transgenic line of Foxl2 disruption
(Foxl2lacZ), while appearing to undergo primordial follicle
formation, is infertile due to a block in follicular develop-
ment during the primordial to primary stage transition
[23]. Foxl2lacZ squamous granulosa cells never become
cuboidal, and thus remain nonproliferative [23,85]. Inter-
estingly, in 2-week-old Foxl2lacZ ovaries, Kitl expression is
significantly upregulated in the granulosa cells, resulting
in stimulation of Gdf9 expression in almost all oocytes, a
hallmark of a growing oocyte population, and thus global
follicular activation [131]. Despite the failure of the gran-
ulosa cells to differentiate from the dormant squamous
morphology, oocytes do undergo activation in Foxl2lacZ
ovaries, suggesting a failure of the germ cells and somatic
cells to coordinate their developmental transitions [23].
The importance of proper differentiation of granulosa
cells to follicle survival and primordial to primary stage
transition is further illustrated by a transgenic mouse tar-
geting the gonadal specific transcription factors GATA4
and GATA6. GATA proteins are zinc finger transcrip-
tions factors, and GATA4 and GATA6 are predominantly
expressed in granulosa cells beginning at the embryonic
age in the mammalian ovary and function to regulate
34 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
expression of numerous genes that are important
throughout follicular development, including those
involved in follicular growth and steroid biosynthesis
[132,133]. Deletion of both Gata4 and Gata6 in granulosa
cells leads to a loss of expression of granulosa cell
markers, including Foxl2, indicating compromised gran-
ulosa cell identity [134]. Consistent with the most prom-
inent phenotype of Foxl2lacZ ovaries, granulosa cells do
not transition from a squamous to cuboidal morphology
following Gata4 and Gata6 deletion, leading to a lack of
growing follicles and eventually to clearance of the per-
manently dormant primordial follicle pool through
wide-spread atresia [134]. While currently less well
understood than the expansive oocyte-specific transcrip-
tional network that is implicated in primordial follicle
activation, future investigations into novel FOXL2 and
GATA4/6 targets should provide further important
insights into the concurrent differentiation of follicular
granulosa cells.
CELLULAR SIGNALING DURING
PREANTRAL FOLLICULAR
DEVELOPMENT
The growth of follicles between the primary and antral
stages is accepted to be independent from the pituitary
gonadotropin, FSH. This paradigm is derived from the
unaltered preantral follicular development observed in
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
various models of FSH signaling disruption [135–137].
While gonadotropin-responsive antral follicles are never
observed in genetic models lacking FSH (through abroga-
tion of the β-subunit of FSH) [135] or lacking the FSH
receptor (FSHR) [136,137], the numbers of primordial,
primary, and multilayered preantral follicles are unaf-
fected. This suggests that local signals are predominant
in regulating the development of follicles through the
preantral stages.
The acquisition of additional layers of granulosa cells
during the transition between the primary and the sec-
ondary follicular stages and beyond involves many
diverse, and often highly interacting signaling pathways.
Moreover, during this period, the oocytes undergo size
expansion that needs to be tightly coordinated with the
growth of the granulosa cells. Finally, as follicles reach
the secondary stage, local signals are required to initiate
the recruitment and organization of the thecal cell layer, a
second type of somatic cell that is indispensable for ste-
roidogenesis. Fig. 5 illustrates the diversity of signaling
pathways that are involved in gonadotropin-
independent granulosa cell growth, as well as the multi-
directional communication between the oocyte and
somatic cells during this preantral period. While we rec-
ognize the complexity of many pathways involved in
these preantral developmental events, we first focus dis-
cussion on paracrine signaling through the TGF-β super-
family of peptides, whose members are highly
represented among both oocyte-secreted and somatic
FIG. 5 Regulation of gonadotropin-
independent follicular growth. The
growth of ovarian follicles prior to
antrum formation is independent of
FSH and is governed by various modes
of local signaling. Following activation,
oocytes begin secreting GDF9 and
BMP15, which act on granulosa cells to
promote growth and gene expression.
Under the influence of GDF9, granulosa
cells express the Hh ligand that functions
to specify the final somatic cell layer, the
theca cells. Activin is a mitogen that is
secreted and signals within the granulosa
cell layer. Activin promotes expression of
Esr2 and suppresses expression of
Cyp26b1. This results in increased levels
of ERβ signaling and retinoic acid recep-
tor signaling, both of which are growth
promoting within granulosa cells. Pro-
motion of granulosa cell proliferation is
also achieved through cross-regulation
between the activin and Notch signaling
pathways. Notch signaling occurs
between the oocyte and granulosa cells,
as well as between adjacent
granulosa cells.
 CELLULAR SIGNALING DURING PREANTRAL FOLLICULAR DEVELOPMENT
cell-secreted factors in the ovary (reviewed in Ref. [71]).
In addition, we discuss the role of the juxtacrine Notch
signaling pathway that is important for preantral follicu-
lar growth, in addition to its roles during germ cell nest
breakdown. Finally, we summarize recent findings on
the functions of the highly conserved Hedgehog signal-
ing pathway in regulating the formation of the
thecal cells.
TGF-β Family Signal Transduction in the Ovary
Based on ligand sequence similarity and modes of sig-
nal transduction, the TGF-β superfamily is divided into
two subgroups, the growth differentiation factors/bone
morphogenetic proteins (GDFs/BMPs) and the acti-
vins/TGF-βs. TGF-β superfamily peptides form either
homo- or hetero-dimeric ligands. Activins and TGF-βs
initiate signaling by binding to a serine/threonine kinase
cell surface receptor, known as the type II receptor [138].
Five type-II receptors have been identified in mammals
[139]. The type-II receptor recruits a type-I receptor, also
known as an activin-like kinase (ALK), and activates the
type-1 receptor through trans-phosphorylation [138].
Seven type-1 receptors have been characterized in mam-
mals [139]. In contrast, BMPs bind first to a type-I recep-
tor, followed by recruitment of a type-II receptor, or to a
preformed type-1/type-2 receptor complex on the cell
surface [140]. The signal is transduced intracellularly by
type-1 receptor-mediated phosphorylation of a SMAD
protein (receptor-regulated SMAD -rSMAD), which will
then recruit an obligatory common-mediator SMAD
(coSMAD) [141]. Receptor activation by GDFs/BMPs
activates SMAD1, SMAD5, or SMAD8, while receptor
activation by activins or TGF-β activates SMAD2 or
SMAD3 [142]. One exception to this model is the
ligand GDF9, which utilizes SMAD2/3 in its signal
transduction [143,144] SMAD4 acts as a coSMAD for
receptor activation by both subtypes of ligands [142].
Finally, an rSMAD-coSMAD complex translocates to
the nucleus and acts as a transcriptional activator of tar-
get genes [141]. The SMAD pathway is negatively regu-
lated by inhibitory SMADs (SMAD6, SMAD7) through
prevention of rSMAD activation at the receptor level or
through competitive binding of activated rSMAD to
coSMAD [141].
In granulosa cells, there appears to be significant func-
tional redundancies between SMAD1, 5, and 8, and
between SMAD2 and 3. This is demonstrated by the
absence of fertility defects following disruption of any
of these individual rSmad alleles in female mice
[145,146]. Since conventional knockouts of all but Smad8
result in embryonic lethality, a conditional knockout
approach has been taken to study these pathways in
the ovary. Double conditional knockout of Smad1/5 or tri-
ple conditional knockout of Smad1/5/8 in granulosa cells
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
35
results in infertility due to development of metastatic
granulosa cell tumors by 3 months of age [145]. In con-
trast, the double conditional knockout of Smad2/3 is sub-
fertile, showing decreased numbers of antral follicles and
increased follicular atresia as indicated by a high inci-
dence of ZP remnants in 3-month-old ovaries [146]. In
addition to follicular developmental aberrations, a high
incidence of trapped oocytes in luteinizing follicles and
a reduced expression of cumulus expansion markers
strongly suggest ovulatory defects in Smad2/3 conditional
knockout mice [146]. Granulosa cell conditional knockout
of the coSMAD Smad4 results in similar phenotypes to the
Smad2/3 conditional knockout, presenting with increased
numbers of atretic preantral follicles and ovulatory
defects [147].
Control of Follicular Growth by Oocyte-Secreted
GDF9 and BMP15
Immediately following activation, oocytes of primary
follicles begin to produce GDF9, a secreted TGF-β family
ligand [131,148,149]. Gdf9 null female mice are infertile
due to a block in follicular development at the primary
follicle stage, which appears to result from a failure of
granulosa cell proliferation [131,150,151]. Demise of the
follicles is initiated by degeneration of the oocytes in
Gdf9
/
ovaries, which results in surviving follicular rem-
nants that acquire a luteinized morphology [131]. The
actions of GDF9 appear to be specifically directed toward
somatic cells, since Gdf9
/
oocytes can be successfully
matured in vitro (with addition of the necessary factors)
to resume meiosis [150]. In vivo treatment of PND5 rats
with GDF9 leads to increased activation of primordial fol-
licles and subsequent enhancement of growth of primary
follicles into multilayered preantral follicles [152]. Gdf9
stimulates proliferation in cultured granulosa cells, as
well as growth of cultured primary and secondary folli-
cles [153,154]. The actions of GDF9 on granulosa cells
are most likely achieved through binding with a complex
of the type-I receptor ALK5 [143,155] and the bone mor-
phogenetic protein receptor type II (BMPRII) [156] as dis-
ruption of either leads to abrogation of the proliferative
effects of GDF9 on granulosa cells [143, 155, 156].
BMP15, a TGF-β family ligand highly related to GDF9,
was identified through a homology-based cloning strat-
egy [157]. Like Gdf9, Bmp15 is expressed specifically in
oocytes beginning at the primary follicle stage [157]
and exerts proliferative effects on cultured rat granulosa
cells [158]. Several naturally occurring mutations affect-
ing BMP15 signaling, and as a consequent ovulation rate,
have been characterized in sheep. The Inverdale (FecXI)
mutation is a point mutation that leads to a single amino
acid replacement in the mature Bmp15 protein, while a
distinct point mutation known as the Hanna (FecXH)
mutation leads to the creation of premature stop codon
and thus a truncated Bmp15 protein [159]. Both the FecXI
and FecXH alleles lead to nonfunctional Bmp15, and
36 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
homozygous females are infertile due to a block in follicle
development at the primary stage [159]. Counterintui-
tively, ewes heterozygous for either mutation have an
increased ovulation rate, which leads to a higher inci-
dence of multiple births compared to wild-type ewes
[159]. Consistent with the mitogenic role of Bmp15 in
granulosa cell growth, antral follicles in heterozygous
FecXI and FecXH ewes are undersized; yet, the granulosa
cells of these antral follicles have increased expression of
Lhcgr, rendering them more responsive to the
LH-ovulatory signal [160]. Similarly, Booroola (FecB) ewes
with a mutation that leads to reduced intracellular kinase
activity of the type I BMPRIB (ALK6) receptor are hyper-
prolific, with high rates of multiple births [161]. While the
precise molecular basis of the dependency of ovulation
rate on the level of BMP15 signaling remains unclear, it
has been postulated that the determining factor may be
the ratio of GDF9 to BMP15 in the ovary [162].
Given the similarity in the structures of BMP15 and
GDF9, as well as their expression by the oocyte in a tem-
porally overlapping manner, it is of interest to under-
stand whether they assert their effects on granulosa
cells in any cooperative or redundant manner. When
coexpressed in human embryonic kidney 293T (293T) cells,
recombinant GDF9 and BMP15 are able to form heterodi-
mers, suggesting their potential interaction in vivo
[163,164]. In the mouse model, Bmp15
/
females are sub-
fertile, in contrast to the infertile Gdf9
/
females, present-
ing with only minimal morphological defects in follicular
development, albeit having decreased ovulation and fertil-
ization rates [165]. A more severe fertility defect was
observed in Bmp15
/
Gdf9+/
females compared to
Bmp15
/
females [165]. However, Bmp15
/
Gdf9
/

double-knockout females have a fertility defect that is no
worse than that of the Gdf9
/
single mutant [165]. In
cultured mouse granulosa cells, GDF9 and BMP15 were
shown to act synergistically to induce proliferation and
SMAD3-dependent transcription [166]. The importance
of GDF9:BMP15 heterodimers in regulating granulosa
cell function was further demonstrated in a mouse cumulus
cell expansion assay, where they were shown to be 10- to
30-fold more potent than the GDF9 homodimer in
promoting cumulus expansion through activation of the
SMAD2/3 pathway [167].
Several mutations at the BMP15 locus have been asso-
ciated with cases of primary ovarian insufficiency (POI)
in humans [168]. Biochemical investigations into ten of
these POI-associated BMP15 mutations showed that they
resulted in reduced levels of mature BMP15, a decreased
ability of BMP15 to induce SMAD1/5/8-dependent tran-
scription, or a reduced capacity for BMP15 to synergisti-
cally induce proliferation and SMAD3-dependent
transcription in the presence of GDF9 [168]. Similarly,
mutations in the GDF9 locus have also been identified
in cases of POI, including those that lead to aberrations
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
in the GDF9 transcriptional regulatory region [169] and
point mutations that lead to reduced levels of mature
GDF9 or to aberrant mature GDF9 that is less potent in
activating the SMAD2 pathway [170]. These findings sug-
gest that the cumulative effects of BMP15 and GDF9 on
granulosa cell proliferation and gene expression are a
complex function of the two distinct rSmad pathways
that they activate.
Regulation of Granulosa Cell Proliferation by
Granulosa Cell-Derived TGF-β Family Ligands
In addition to GDF9 and BMP15 originating from the
oocyte, granulosa cells also produce multiple TGF-β fam-
ily ligands that act in a paracrine manner to regulate fol-
licular development. There are three mammalian TGF-β
isoforms (TGF-β1–3), and localization studies have iden-
tified their expression in all three follicular cell types
beginning at the preantral or early-antral stages in human
and rodent ovaries [171,172]. Depending on the species,
TGF-β1 can assert stimulatory or inhibitory effects on
granulosa cell proliferation. While in vitro, TGF-β1 pro-
motes rat granulosa cell proliferation, it exerts inhibitory
effect on the proliferation of granulosa cells from cattle,
sheep, and pig ovaries (reviewed in Ref. [171]). In con-
trast, negative regulation of thecal cell steroidogenesis
by TGF-β1 appears to be conserved across these
species [171].
Activins and inhibins are granulosa cell-secreted pep-
tide hormones that were originally identified as a part of
the ovarian feedback loop regulating the release of FSH
by the pituitary gland [71]. Mature activin and inhibin
are composed of dimers of structurally related peptides
connected by a disulfide bond [173]. Dimers of the two
β subunits form mature activins (Activin A—βAβA, Acti-
vin B—βBβB, or Activin AB—βAβB), while heterodimers of
the α and β subunits result in mature inhibins (Inhibin
A—αβA or Inhibin B—αβB). Expression of all three sub-
units can be detected in granulosa cells and is dynami-
cally regulated throughout the rat estrous cycle [48].
Activin exerts its actions through binding to its type-II
receptor (ACTRIIA or ACTRIIB) and transactivation of
ALK2, 4, or 7, followed by activation of SMAD2/3 and
the co-SMAD SMAD4 (reviewed in Ref. [174]). Inhibin
suppresses the actions of activin through competitive
binding with the type-II receptor. Finally, as previously
discussed, the ovary also produces FST, a molecule that
binds to and neutralizes the biological activity of
activin [175].
In vitro, activin exerts proliferative effects on granulosa
cells of preantral follicles isolated from prepubertal mice,
and FST treatment blocks this effect [176]. The effects of
activin on granulosa cell proliferation appear to be age
dependent, possibly due to additional growth signals
by FSH following the onset of puberty. For example, acti-
vin treatment of neonatal mice leads to increased
 CELLULAR SIGNALING DURING PREANTRAL FOLLICULAR DEVELOPMENT
proliferation of granulosa cells and to a limited degree the
germ cells [21]. However, while activin stimulates growth
of preantral follicles isolated from juvenile mice, it
promotes atresia and abrogates FSH-induced growth in
preantral follicles isolated from adult animals [177,178].
Regardless of this age-specific effect, activin appears to
be necessary for female fertility, as animals lacking any
ovarian expression of either of the β subunits are infertile,
showing decreased expression of factors that are associ-
ated with granulosa cell survival and proliferation such
as Kitl, Taf4b, and c-Myc [179]. Consistent with a defi-
ciency in activin signaling, juvenile Smad3
/
ovaries
have decreased numbers of preantral and early antral
follicles, indicating disrupted follicular growth from the
secondary stage and beyond [180].
Overexpression of the inhibin α subunit in transgenic
mice (MT-alpha) results in increased levels of circulating
mature inhibins and decreased levels of mature activins
[181]. Expression of both β subunits is downregulated
in the MT-alpha ovaries, suggesting a certain degree of
autoregulation of gene expression by activin signaling
[181]. Consistent with pharmacological and genetic
models of suppressed activin action discussed above,
MT-alpha female mice are subfertile with decreased num-
bers of mature antral follicles in the ovary [181,182].
While inhibin is not currently known to activate any spe-
cific receptor, the genetic model of inhibin deficiency
Inha
/
has demonstrated the necessity for its presence
in order to restrain and balance the mitogenic effects of
activin on granulosa cells [183]. Inha
/
ovaries are
abnormally large compared to the wild-type littermates
due to granulosa cell hyperproliferation, which eventu-
ally leads to gonadal tumors and infertility in
adulthood [183].
Studies in cultured granulosa cells from preantral
follicles have revealed the potential for cross-talk
between activin and at least two different types of
nuclear receptor signaling pathways in regulating gran-
ulosa cell proliferation (Fig. 5). Activin treatment is
found to induce expression of Esr1 and Esr2 in a
SMAD2/3-dependent manner in proliferating granu-
losa cells [184]. Since estrogen is mitogenic, as well as
pro-survival, in preantral granulosa cells [185–187], it
is likely that activin promotes proliferation at least in
part through enhancement of estrogen receptor signal-
ing. Transcriptomic analysis of activin-treated granu-
losa cells identified Cyp26b1 as downregulated by
activin [188]. A member of the cytochrome P450 family,
the gene product of Cyp26b1 functions to degrade RA
and is expressed in granulosa cells of growing follicles
[188]. RA or Cyp26b1 inhibitor treatments of cultured
granulosa cells leads to an increase in granulosa cell
proliferation in a dose-dependent manner, while inhibi-
tion of retinoic acid receptors abrogates this effect [188].
Besides identifying a novel mitogenic factor in
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
37
granulosa cells, this study further illustrates the exten-
sive network of signals with which activin interacts
during gonadotropin-independent follicle growth.
The Role of Notch Signaling in Preantral Follicle
Development
Earlier, we reviewed evidence for a role of Notch sig-
naling in promoting germ cell nest breakdown. It is now
appreciated that Notch signaling continues to be impor-
tant during follicle growth and development, functioning
to promote granulosa cell proliferation and to coordinate
the development of the oocyte and granulosa cells
(Fig. 5). Inhibition of Notch signaling using the
γ-secretase inhibitors, DAPT or L658,458, leads to
decreased proliferation of cultured granulosa cells or sec-
ondary follicles [189]. Expression of c-Myc, a
proliferation-associated transcription factor, in granulosa
cells is suppressed following Notch inhibition, and resto-
ration of Notch signaling through expression of constitu-
tively active N2ICD leads to recovery of both c-Myc
expression and granulosa cell proliferation [189]. Addi-
tionally, Notch may also regulate granulosa cell prolifer-
ation through interactions with activin signaling. There is
evidence that the Notch target gene Hey2 is coregulated
by activin in a SMAD3-dependent manner in preantral
granulosa cells [190]. Indeed, activin treatment is able
to overcome the effect of DAPT inhibition and restore
proliferation in cultured granulosa cells [190].
In conditional-knockout models of the ligand JAG1
from the oocytes (J1KO) or the receptor NOTCH2 from
granulosa cells (N2KO), defects in proliferation of granu-
losa cells are first observed by PND19, and are associated
with enhanced cell death [74]. J1KO and N2KO ovaries
also present with a phenotype that indicates disruption
of developmental coordination between oocytes and
granulosa cells, characterized by enlarged oocytes that
are contained in very early stage growing follicles (i.e.,
follicles with a single layer of granulosa cells) [74].
Molecularly, this is supported by elevated expression of
the oocyte-specific factors Gdf9, Figla, and Zp3 as com-
pared to wild-type littermates [74].
The Notch target gene Hes1 is expressed in somatic
cells embryonically (by E15.5) and its expression
decreases following birth [71,191]. Conventional, or gran-
ulosa cell conditional, knockouts of Hes1 lead not only to
increased oocyte death but also increased proliferation of
the pregranulosa cells [191]. Both mouse lines are subfer-
tile, indicating that disruption of early follicular develop-
ment results in detrimental effects on fecundity.
Increased expression of the βB subunit (Inhbb) of activin
is observed in both models of Hes1 deficiency [191],
which makes the enhanced pregranulosa cell prolifera-
tion consistent with effects observed in neonatal mice
38 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
treated with exogenous activin [21]. An additional com-
mon phenotype between the two models of Hes1 defi-
ciency is the suppression of c-Kit expression in oocytes
[191]. As covered in earlier sections, KITL/c-KIT signal-
ing is particularly important for direct communication
between granulosa cells and oocytes, and it regulates nest
breakdown and primordial follicle activation in the neo-
natal ovary. Thus, while Notch signaling is mitogenic in
preantral granulosa cells, an additional function is per-
haps to ensure coordinated development between
oocytes and granulosa cells prior to exposure to gonado-
tropins, which will act as the master regulators for follic-
ular development beyond the preantral stage.
Molecular Mechanisms of Theca Cell
Recruitment and Specification
The thecal cell layer forms just outside the basement
membrane surrounding the outermost granulosa cells
when the follicle reaches the secondary stage [192].
Through the use of transgenic reporter mouse lines and
lineage tracing, it was shown that theca cells originate
from one of two sources, the fibroblast-like precursor cells
indigenous to the ovary (Wt1 expressing cells) or mesen-
chymal cells that migrate into the ovary from the meso-
nephros [193,194]. Theca cells function in ovarian
steroidogenesis by providing aromatizable androstenedi-
one to the granulosa cells, where aromatase will convert
the androgen into 17β-estradiol [192]. It was long sus-
pected that preantral follicles secrete certain thecal cell
differentiating factors, as conditioned medium collected
from theca-less preantral follicles stimulated androstene-
dione production in cultured thecal-interstitial cells [195].
This putative theca differentiating factor was thought to
be a small molecule on the order of 20–25 kDa in
size [196].
Recent studies indicate that the developmentally con-
served Hedgehog (Hh) signaling pathway is the chief reg-
ulator of thecal cell specification in the ovary. Hedgehog
signaling is initiated by binding of one of three HH
ligands (Sonic hedgehog—SHH, Indian hedgehog—
IHH, Desert hedgehog—DHH) to the canonical receptor
Patched (PTCH1, 2). Hh binding to PTCH results in dere-
pression of Smoothened (SMO2), an obligate signal trans-
ducer in Hh signaling, and finally the translocation of the
Hh transcriptional effector GLI (GLI1 in thecal cell pre-
cursors) into the nucleus. Ihh and Dhh are expressed by
granulosa cells of growing follicles, while the receptors
Ptch1, Ptch2, and the transcriptional effector Gli1 are only
detected in the surrounding pretheca cells, suggesting
that theca cells are the exclusive target of Hh ligands in
the ovary (Fig. 5) [197].
The thecal layer never forms in ovaries with condi-
tional deletion of both Dhh and Ihh from the granulosa
cells [194]. These animals are infertile, with blocked
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
follicular development at the preantral stage and blunted
steroid production [194]. It has been shown that GDF9
from the oocyte stimulates the expression of Dhh and
Ihh in granulosa cells, and indeed, expression of both
Hh ligands is severely downregulated in Gdf9
/
ovaries
[194]. Theca layer recruitment and differentiation is an
elegant example of how three distinct cell types within
the follicle communicate and regulate each other’s func-
tions through diverse signaling pathways.
REGULATION OF GRANULOSA CELL
PROLIFERATION AND
DIFFERENTIATION IN ANTRAL AND
PREOVULATORY FOLLICLES
In larger multilayer follicles, small fluid filled spaces
are initiated that will eventually converge to form the
antral cavity [198]. A hallmark of antral follicle develop-
ment is the increased abundance of transcripts for the
FSH receptor (Fshr) within the granulosa cells, which
upon translation into FSHR renders the follicles respon-
sive to circulating FSH [199]. Indeed, FSHR signaling is
required for continued development beyond the multi-
layered secondary follicle stage, as antral follicles are
never detected in mice lacking either mature FSH
(Fshb
/
line) [135] or FSHR [136,137]. Following puberty,
FSH functions to prevent early antral follicles from under-
going atresia [200] by regulating both granulosa cell
proliferation and differentiation [201,202]. Under the
influence of FSH, the cell cycle of granulosa cells is short-
ened, requiring a doubling period of only about 24 h as
compared to requiring greater than 7 d during the FSH-
independent period [203]. FSH also functions to induce
gene expression changes in granulosa cells, which differ-
entiates them toward the LH responsive, preovulatory
stage [204,205]. Characteristics of granulosa cell matura-
tion in response to FSH signaling include the expression
of genes involved in steroid biosynthesis, such as
Cyp19a1, Cyp11a1, and Hsd3b1, which leads to increased
production of estradiol and progesterone, and the expres-
sion of the membrane receptor for LH (Lhcgr) [204,206].
The FSHR is a G-protein-coupled receptor (GPCR) that
stimulates synthesis of the second messenger cyclic AMP
(cAMP) when activated [207,208]. The intracellular rise in
cAMP leads to activation of the cAMP-dependent protein
kinase A (PKA) [209], which in turn promotes gene tran-
scription through phosphorylation and activation of sev-
eral transcription factors, including but not limited to, the
cAMP response element-binding protein (CREB)
[210,211] and GATA4 [212,213]. Additionally, PKA
downstream of FSHR activation also modifies chromatin
structure through phosphorylation of histone H3, which
leads to increased nucleosome accessibility [214,215]. It is
now appreciated that to exert maximum mitogenic and
differentiation effects, FSH/FSHR signaling interacts
 REGULATION OF GRANULOSA CELL PROLIFERATION AND DIFFERENTIATION IN ANTRAL AND PREOVULATORY FOLLICLES
and cooperates with diverse local ovarian signals. In this
section, we discuss intraovarian signals that regulate pro-
liferation and differentiation in antral to preovulatory
granulosa cells and their cross talk with FSHR signaling.
For the purposes of clarity and simplicity, we will refer to
granulosa cells as undifferentiated prior exposure to FSH
(including those collected from estrogen stimulated
animals and used in vitro studies) and as differentiating
following activation of FSHR, while appreciating that
these are points along a continuum [216].
Crosstalk Between FSHR Signaling and Kinase
Cascades in Granulosa Cell Proliferation and
Differentiation
Control of Granulosa Cell Proliferation and Gene
Expression by MAPK/ERK Activation Downstream
of FSHR Signaling
The mitogen-activated protein kinases (MAPKs) are a
highly conserved family of serine/threonine protein
kinases that function to relay extracellular cues by way
of kinase cascade activation and influence diverse cellular
39
processes, including proliferation and gene transcription
(Fig. 6) [217]. While originally described as an intracellu-
lar signaling pathway that is downstream of RTKs, the
MAPK pathway is now also known to function down-
stream of GPCR activation [218]. The MAPK/ERK path-
way is active in proliferating cells, including granulosa
cells. ERK1/2 activation is observed in undifferentiated
proliferating granulosa cells in culture [219]. An increase
in intracellular cAMP levels was found to prevent activa-
tion of ERK2 in response to growth factors in human arte-
rial smooth muscle cells, adipocytes, and mouse
fibroblast cell lines [220]. Based on these findings, FSHR
activation and its associated rise in cAMP levels were ini-
tially hypothesized to downregulate ERK1/2 activation
in differentiating granulosa cells. On the contrary, FSH
stimulation of rat granulosa cells in culture leads to an
acute activation of ERK1/2, which occurs in a cAMP-
dependent manner [219]. In addition to proliferation,
activation of the MAPK/ERK pathway following FSH
stimulation is also important for transcription of genes
associated with the preovulatory phenotype, including
positive regulation of the Cyp19a1, Cyp11a1, Inha, Lhcgr,
and Egfr genes [221].

FIG. 6 Integration of FSH and paracrine signals in promoting granulosa cell proliferation and differentiation. Following antrum formation, gran-
ulosa cells proliferate and differentiate into the preovulatory stage through upregulation of Lhcgr and genes important for steroidogenesis in
response to FSH. To achieve the maximal proliferative and differentiation effects of FSH, FSHR signaling interacts with kinase cascades downstream
of RTKs such as EGFR and IGFR and cooperates with Smad-dependent signaling pathways downstream of TGFβ and activin family receptors. Inte-
gration of PKA and PI3K/AKT signaling leads to inactivation of the transcriptional repressor FOXO1 through phosphorylation, which targets it for
degradation. In turn, MAPK/ERK and SMAD2/3 activation provide activating signals for the transcription of granulosa cell differentiation-
associated genes. Notch signaling promotes granulosa cell differentiation through positive regulation of steroid biosynthetic enzyme gene expression
and through suppression of proliferative kinase cascades. Additionally, the Notch NICD is known to coregulate expression of common effectors by
interacting with the SMAD complex. Note that distinct subsets of transcriptional regulatory factors will interact at the promoters of different target
genes, and the schematic illustrates generic and summed examples of such regulatory pathway interactions.

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

40 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
It is still debated whether any specific RTK is involved
in MAPK/ERK pathway activation in FSH-stimulated
granulosa cells. In undifferentiated, proliferating granu-
losa cells from estrogen-treated mice, the MAPK pathway
upstream of ERK1/2 is observed to be constitutively
active [222]. In these granulosa cells, ERK1/2 is continu-
ously dephosphorylated by the MAP kinase phosphatase
3 (MKP3), thus restraining the full activation of the
MAPK/ERK pathway [221]. FSH stimulation and PKA
activation were in turn shown to inhibit MKP3 activity,
and thus function to relieve the inhibition on ERK1/2
[221]. Gene expression changes are in turn dependent
upon phosphorylation of the RNA and DNA binding
protein YB-1 by the activated ERK1/2 [220]. While epi-
dermal growth factor receptor (EGFR) functions promi-
nently in propagating the LH signal during ovulation,
there is also evidence for its modulation downstream of
FSHR signaling. Radioactively labeled epidermal growth
factor (EGF) is observed to bind to immature rat follicles,
and FSH treatment enhances the number of EGF mole-
cules bound, suggesting modulation of Egfr expression
by FSH in differentiating granulosa cells [223]. More
recently, Egfr expression was shown to be strongly down-
regulated in Fshb
/
prepubertal ovaries [224]. Treatment
with FSH was shown to restore Egfr expression (and
eventually the ability to ovulate), confirming modulation
of Egfr by FSHR signaling. In addition to MAPK/ERK
activation downstream of FSHR, a known, but minor,
product of Fshr alternative splicing (growth factor type1
receptor or oFSH-R3) whose activation rapidly leads to
phosphorylation of ERK1/2 instead of the rise in cAMP
level can also be detected in the ovary [225].
Through the use of the TNR mouse line, Notch signal-
ing is observed to remain active in granulosa cells of
antral and preovulatory follicles [226]. Notch signaling
molecules are expressed in these later stage follicles
and there is evidence that their expression is dynamically
regulated by gonadotropins [72]. Of note, is the ligand
Jag1 whose localization shifts from being oocyte-specific
to being present in ovarian somatic cells following expo-
sure to exogenous gonadotropins [226,227]. Disruption of
the Notch ligand Jag1 in cultured granulosa cells collected
from PMSG-primed immature mice leads to a compro-
mised differentiation response, as measured by suppres-
sed steroidogenesis [226]. At the expense of suppressed
differentiation, enhanced proliferation, resembling that of
less mature granulosa cells, is maintained in Jag1-deficient
cells and this is associated with to increased activation of
the MAPK/ERK pathway. Together, these studies demon-
strate that in differentiating granulosa cells, FSH functions
to promote proliferation and differentiation in conjunction
with underlying signals that are initiated by local factors.
Regulation of Granulosa Cell Differentiation and
Proliferation by the PI3K/AKT Pathway
Interest in insulin growth factor receptor (IGFR), a
RTK, signaling in follicle maturation began with the
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
observation that the ligand insulin-like growth factor-1
(Igf1) and Fshr are coexpressed in healthy, steroidogeni-
cally active large antral follicles from mouse [228]. Igf1
null females are infertile, with ovaries lacking follicles
beyond the early antral stage, resembling Fshb
/
animals
[228]. In granulosa cells, activation of the IGF1-R or the
FSHR independently stimulates the PI3K/AKT pathway
(Fig. 6), and coactivation of both receptors leads to syner-
gistic effects on AKT activation [229]. Indeed, inhibition
of the IGF1-R prevents the induction of granulosa cell dif-
ferentiation by FSH, as measured by the lack of an
increase in Cyp19a1 expression and estradiol production
[229]. Additional support for the importance of IGF1-R
signaling in facilitating follicular maturation by FSH
comes from the recently reported Igf1r granulosa cell con-
ditional knockout (IGF1Rgcko) mouse line [230].
IGF1Rgcko females are infertile with ovaries lacking antral
follicles and granulosa cells that are unable to differenti-
ate in response to pregnant mare serum gonadotropin
(PMSG) stimulation, which activates the FSHR, despite
normal levels of Fshr expression compared to wild-type
littermates [230].
Another important interaction between FSHR signal-
ing and a paracrine factor occurs in the regulation of gran-
ulosa cell proliferation by activin. In cultured rat
granulosa cells, the full mitogenic effect of FSH requires
the presence of activin [231]. The apparent explanation
is that activation of both AKT and SMAD2/3 is required
for expression of the cell cycle regulator Cyclin D2 (Ccnd2)
following FSH and activin stimulation (Fig. 6) [231]. It is
now appreciated that FSH activation of the PI3K/AKT
pathway occurs through a PKA-dependent cascade
[232] and targets the transcriptional suppressor Foxo1
[233]. Phosphorylation of Foxo1 leads to its translocation
from the nucleus and directs it for degradation. More
than 60% of FSH-regulated genes have now been identi-
fied to be targets for Foxo1, including those involved in
proliferation, such as Ccnd2, and those involved in steroid
biosynthesis, such as Star, Cyp11a1, and Cyp19a1
[233,234]. Fig. 6 summarizes the integration of FSHR,
RTKs, Notch, and activin signaling pathways in regulat-
ing granulosa cell proliferation and differentiation. These
data demonstrate the importance of, what are often
accepted to be ubiquitous, kinase cascades in integrating
endocrine and paracrine cues to promote the differenti-
ated preovulatory phenotype in granulosa cells.
The Roles of Steroid Hormones in Follicular
Maturation
The ovarian follicle is the main source of sex steroid
hormones (estrogens, progestins, and androgens) that
are indispensable for overall female reproductive physi-
ology. Sex steroids act on many organ systems, including
but not limited to the nervous system, the skeletal system,
the cardiovascular system, metabolism, and last but not
 REGULATION OF GRANULOSA CELL PROLIFERATION AND DIFFERENTIATION IN ANTRAL AND PREOVULATORY FOLLICLES
least the reproductive system. Comprehensive discus-
sions of the molecular mechanism of ovarian steroido-
genesis are found elsewhere in this text and reviewed
in Ref. [235]. The two-cell, two-gonadotropin model of
follicular steroidogenesis [236] is based on localization
of the enzymes required for distinct steps in the steroid
biosynthetic cascade. Theca cells convert cholesterol into
androstenedione under the influence of Lhcgr signaling.
In turn, the hydrolyzable androstenedione is utilized
by granulosa cells as a substrate for estradiol under the
influence of FSHR signaling. Following ovulation and
formation of the corpus luteum, progesterone will become
the main sex steroid produced by the ovary. In this section,
our focus will be on the role of estrogens and androgens
as paracrine factors regulating follicular events throughout
the FSH-dependent and periovulatory-developmental
stages. While we recognize the equally important role of
progesterone, we refer readers to Ref. [237] for a discussion
on the importance of progesterone receptor signaling
during the ovulatory cascade and pregnancy.
Estrogen and Estrogen Receptor Signaling in Antral
and Preovulatory Follicles
Estrogen increases granulosa cell proliferation in pre-
antral follicles [185–187]. Estrogen is also found to
increase the presence of FSHR and LHCGR in granulosa
cells and enhances the rise in cAMP following cholera
toxin stimulation (due to blocked hydrolysis of
G-protein-associated GTP, and thus constitutively acti-
vated adenylate cyclase), suggesting its ability to aug-
ment GPCR/cAMP signaling [238]. Ovaries of mice
lacking aromatase (Cyp19a1; ArKO), an enzyme that is
required for the final conversion of androgens into estra-
diol, lack follicles beyond the early antral stages [187].
The phenotypes of the ArKO mouse, however, appear
to be caused by chronic elevated serum gonadotropin
levels due to the lack of estrogen feedback to the pitui-
tary. Upon exogenous estrogen treatment, follicular
development is able to resume and ovulation is
restored [187].
In the ovary, estrogen exerts its mitogenic and differ-
entiative effects through activation of one of two recep-
tors (ER-α or ER-β) belonging to the nuclear receptor
family of transcription factors. While G protein-coupled
receptor 30 (Gpr30), one of many membrane-bound
forms of estrogen receptor, can be detected in the mam-
malian ovary [239], Gpr30-deficient mice are completely
fertile [240]. Following ligand binding in the cytoplasm,
ERs dimerize and translocate into the nucleus to activate
gene transcription upon recognition of an estrogen-
responsive element (ERE) [241]. Esr1 (ER-α) is expressed
throughout the female reproductive tract, including in
the thecal and interstitial cells of the ovary [242]. The
Esr1-knockout mouse is infertile due to reproductive tract
defects, including thin uteri, lack of preovulatory follicles,
and hemorrhagic cysts in the ovary [242,243]. In contrast,
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
41
ER-β (ESR2) is localized almost exclusively to granulosa
cells, and its expression is dynamically regulated by
gonadotropins [41,244]. Thus, models of Esr2 disruption
have allowed for further understanding of the functions
of estrogen in follicular development.
ER-β-knockout mice are subfertile with ovaries show-
ing a block in progression from early antral follicles to the
large antral and preovulatory stages [245,246]. Granulosa
cells of ER-β-knockout follicles show attenuated
responses to the differentiating effect of FSH, including
suboptimal induction of Cyp19a1—therefore reduced
steroidogenesis—and Lhcgr expression [246]. As a conse-
quent of decreased expression of Lhcgr, ER-β-knockout
follicles are less responsive to ovulatory LH signals as
compared to wild-type controls [247]. These findings sug-
gest that ER-β signaling is important to promote FSH-
regulated granulosa cell differentiation and promotion
of the preovulatory phenotype. Interestingly, ovarian
phenotypes of double-knockout Esr1 and Esr2 mice
(αβERKO) are completely distinct from either single
knockout. αβERKO females are infertile with defective
ovarian follicles that appear to transdifferentiate into
seminiferous tubule-like structures in adulthood [248].
Physiological and Supraphysiological Effects of
Androgens on Follicular Development
While initially thought to function solely as the sub-
strate for estrogen synthesis in the ovary [236], the impor-
tance of androgens and androgen receptor (AR) signaling
in follicular development has now been demonstrated
through the generation of granulosa cell-specific knock-
outs of the AR in mice (ARcKO) [249,250]. Two indepen-
dently generated ARcKO lines are subfertile, with
decreased follicular growth and increased follicular atre-
sia observed starting at the preantral stage [249,250].
Testosterone and dihydrotestosterone (DHT) were
shown to prevent granulosa cell apoptosis and folli-
cular atresia by increasing the expression of miR-125b, a
known suppressor of proapoptotic proteins [251]. Also
in FSH-responsive follicles, androgen increases the level
of granulosa cell Fshr mRNA, thus enhancing the FSH
stimulation of follicle growth [251]. Testosterone or DHT
was also shown to augment FSH-stimulated granulosa
cell expression of Star [252].
Clinically, ovarian hyperandrogenism is a feature of
the relatively common endocrine and metabolic disorder
polycystic ovarian syndrome (PCOS), which often leads
to anovulation [253,254]. At supraphysiological levels,
DHT treatment of prepubertal rats leads to a block in fol-
licular development at the early antral stage [255]. The
abnormally large population of small growing follicles,
where dominant follicle(s) fail to emerge, resemble that
of PCOS ovaries where these small follicles localize to
the ovarian cortex and resemble cysts in diagnostic ultra-
sound examination. High levels of DHT were shown to
inhibit FSH-induced granulosa cell proliferation through
42 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
dampening of PI3K/AKT pathway activation and Cyclin
D1 expression [255]. Excess DHT was also shown to
inhibit proliferation by preventing insulin- and FSH-
stimulated activation of the MAPK/ERK and Cyclin D2
expression in cultured granulosa cells [256,257]. These
findings demonstrate that at both physiological and
supraphysiological levels, androgens influence antral fol-
licular development by modulating both the proliferative
and differentiating effects of FSHR signaling.
Conclusions
It is abundantly clear that intraovarian signaling path-
ways are critical for regulating follicular function during
both the gonadotropin-independent and -dependent
stages of follicle development. Through this review, we
hope to have communicated the diversity by which cells
of the follicle communicate with each other to ensure
coordinated development. It is perhaps not surprising
that reproduction, a necessarily robust process to ensure
the continuation of species, requires highly interacting,
and often overlapping, complex signaling mechanisms.
Disruption of early developmental events that are
gonadotropin-autonomous, such as follicle formation or
control of primordial follicular activation, can directly
determine fertility status. However, FSH and LH alone
are also not sufficient to support fertility, as their actions
are highly dependent upon interactions with underlying
paracrine factors. It is remarkable how diverse these
intrinsic ovarian regulators are, spanning the gamut of
evolutionarily conserved developmental pathways such
as Notch and Hedgehog signaling, the large TGF-β family
of ligands, many RTKs, and nuclear receptor signaling.
The advent of transgenic overexpression and reporter
mice, as well as conditional deletion strategies using the
Cre/LoxP system in the mouse, has allowed for the discov-
ery of molecular mechanisms that contribute to specific
periods of follicular development. In the coming years,
we expect that the maturation of CRISPR/Cas9 genome
editing technology will extend our knowledge of molec-
ular endocrinology, and perhaps allow relevant ques-
tions to be asked in mammals beyond rodents [258].
Moreover, CRISPR/Cas9 technology promises to
increase the efficiency—with respect to timing and
precision—of generation of transgenic and knockout
models. In combination with predictive in silico analysis
and high-throughput-omics studies, we may soon iden-
tify molecular regulators of poorly understood aspects
of follicular development, such as follicle formation and
primordial follicle activation.
Most findings presented in this chapter represent basic
research conducted largely in murine models. We posit
that understanding the molecular basis of how ovarian
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
follicles develop will continue to allow for the identifica-
tion of the genetic and molecular basis of reproductive
disorders, including those of often unknown etiology.
Where relevant, we present examples of clinical findings
that were informed by these basic studies. The increasing
sensitivity and affordability of genome-wide analysis has
made possible the identification of genetic polymor-
phisms in complex, often poorly understood diseases,
including idiopathic cases of infertility. Researchers and
clinicians continue to identify novel infertility-associated
loci, making possible mechanistic studies that are often
informed by an understanding of the diverse signaling
pathways discussed here. It is an exciting era of discov-
ery, with robust opportunities to continue to increase
our understanding of the ovary and its central role in
female reproduction.
SUPPORTING GRANTS
NIH/NICDH P01 HD021921
NIH/NIGMS T32 GM008061
DISCLOSURE SUMMARY
The authors have nothing to disclose
References
[1] Ginsburg M, Snow MH, McLaren A. Primordial germ cells in the
mouse embryo during gastrulation. Development 1990;110
(2):521–8.
[2] Liu C-F, Liu C, Yao HHC. Building pathways for ovary organogen-
esis in the mouse embryo. Curr Top Dev Biol 2010;90:263–90.
[3] Anderson EL, Baltus AE, Roepers-Gajadien HL, Hassold TJ, de
Rooij DG, van Pelt AMM, et al. Stra8 and its inducer, retinoic acid,
regulate meiotic initiation in both spermatogenesis and oogenesis
in mice. Proc Natl Acad Sci U S A 2008;105(39):14976–80.
[4] Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD, Page DC.
Retinoic acid regulates sex-specific timing of meiotic initiation in
mice. Proc Natl Acad Sci U S A 2006;103(8):2474–9.
[5] Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S,
et al. Retinoid signaling determines germ cell fate in mice. Am
Assoc Adv Sci 2006;312(5773):596–600.
[6] Dokshin GA, Baltus AE, Eppig JJ, Page DC. Oocyte differentiation
is genetically dissociable from meiosis in mice. Nat Genet 2013;45
(8):877–83.
[7] Pepling ME, Spradling AC. Mouse ovarian germ cell cysts undergo
programmed breakdown to form primordial follicles. Dev Biol
2001;234(2):339–51.
[8] Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC. Ovarian
surface epithelium: biology, endocrinology, and pathology. Endocr
Rev 2001 Apr;22(2):255–88.
[9] Byskov AG, Lintern-Moore S. Follicle formation in the immature
mouse ovary: the role of the rete ovarii. J Anat 1973;116
(Pt 2):207–17.
 REFERENCES
[10] Zheng W, Zhang H, Gorre N, Risal S, Shen Y, Liu K. Two classes of
ovarian primordial follicles exhibit distinct developmental dynam-
ics and physiological functions. Hum Mol Genet 2014;23(4):920–8.
[11] Mork L, Maatouk DM, McMahon JA, Guo JJ, Zhang P,
McMahon AP, et al. Temporal differences in granulosa cell specifi-
cation in the ovary reflect distinct follicle fates in mice. Biol Reprod
2012;86(2):37.
[12] Hilscher B, Hilscher W, B€ ulthoff-Ohnolz B, Kr€amer U, Birke A,
Pelzer H, et al. Kinetics of gametogenesis. I. Comparative histolog-
ical and autoradiographic studies of oocytes and transitional pros-
permatogonia during oogenesis and prespermatogenesis. Cell
Tissue Res 1974;154(4):443–70.
[13] Pepling ME, Spradling AC. Female mouse germ cells form synchro-
nously dividing cysts. Development 1998 Sep;125(17):3323–8.
[14] Mork L, Tang H, Batchvarov I, Capel B. Mouse germ cell clusters
form by aggregation as well as clonal divisions. Mech Dev 2012
Jan;128(11–12):591–6.
[15] Lei L, Spradling AC. Mouse primordial germ cells produce cysts
that partially fragment prior to meiosis. Development 2013;140
(10):2075–81.
[16] B€uning J. The insect ovary. Springer Science & Business Media;
2012. 1 p.
[17] de Cuevas M, Lilly MA, Spradling AC. Germline cyst formation in
Drosophila. Annu Rev Genet 1997;31(1):405–28.
[18] Lei L, Spradling AC. Mouse oocytes differentiate through organelle
enrichment from sister cyst germ cells. Science 2016;352(6281):95–9.
[19] Pepling ME, Wilhelm JE, O’Hara AL, Gephardt GW, Spradling AC.
Mouse oocytes within germ cell cysts and primordial follicles
contain a Balbiani body. Proc Natl Acad Sci U S A 2007;104
(1):187–92.
[20] Greenbaum MP, Iwamori N, Agno JE, Matzuk MM. Mouse TEX14
is required for embryonic germ cell intercellular bridges but not
female fertility. Biol Reprod 2009;80(3):449–57.
[21] Bristol-Gould SK, Kreeger PK, Selkirk CG, Kilen SM, Cook RW,
Kipp JL, et al. Postnatal regulation of germ cells by activin: the estab-
lishment of the initial follicle pool. Dev Biol 2006;298(1):132–48.
[22] Morita Y, Tilly JL. Oocyte apoptosis: like sand through an hour-
glass. Dev Biol 1999;213(1):1–17.
[23] Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier A-C,
et al. The murine winged-helix transcription factor Foxl2 is required
for granulosa cell differentiation and ovary maintenance.
Development 2004;131(4):933–42.
[24] Uda M, Ottolenghi C, Crisponi L, Garcia JE, Deiana M, Kimber W,
et al. Foxl2 disruption causes mouse ovarian failure by pervasive
blockage of follicle development. Hum Mol Genet 2004;13
(11):1171–81.
[25] Liang L, Soyal SM, Dean J. FIGalpha, a germ cell specific transcrip-
tion factor involved in the coordinate expression of the zona pellu-
cida genes. Development 1997;124(24):4939–47.
[26] Parrott JA, Skinner MK. Kit-ligand/stem cell factor induces pri-
mordial follicle development and initiates folliculogenesis.
Endocrinology 1999;140(9):4262–71.
[27] Perez GI, Robles R, Knudson CM, Flaws JA, Korsmeyer SJ, Tilly JL.
Prolongation of ovarian lifespan into advanced chronological age
by Bax-deficiency. Nat Genet 1999;21(2):200–3.
[28] Greenfeld CR, Pepling ME, Babus JK, Furth PA, Flaws JA. BAX reg-
ulates follicular endowment in mice. Reproduction 2007;133
(5):865–76.
[29] Myers M, Morgan FH, Liew SH, Zerafa N, Gamage TU, Sarraj M,
et al. PUMA regulates germ cell loss and primordial follicle endow-
ment in mice. Reproduction 2014 Aug;148(2):211–9.
[30] Liew SH, Vaithiyanathan K, Cook M, Bouillet P, Scott CL, Kerr JB,
et al. Loss of the proapoptotic BH3-only protein BCL-2 modifying
factor prolongs the fertile life span in female mice. Biol Reprod 2014
Apr;90(4):77.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
43
[31] Flaws JA, Hirshfield AN, Hewitt JA, Babus JK, Furth PA. Effect of
bcl-2 on the primordial follicle endowment in the mouse ovary. Biol
Reprod 2001;64(4):1153–9.
[32] Bergeron L, Perez GI, Macdonald G, Shi L, Sun Y, Jurisicova A, et al.
Defects in regulation of apoptosis in caspase-2-deficient mice.
Genes Dev 1998;12(9):1304–14.
[33] Montano MM, Welshons WV. Saal vom FS. Free estradiol in serum
and brain uptake of estradiol during fetal and neonatal sexual dif-
ferentiation in female rats. Biol Reprod 1995;53(5):1198–207.
[34] Weisz J, Ward IL. Plasma testosterone and progesterone titers of
pregnant rats, their male and female fetuses, and neonatal off-
spring. Endocrinology 1980;106(1):306–16.
[35] Nilsson EE, Skinner MK. Progesterone regulation of primordial fol-
licle assembly in bovine fetal ovaries. Mol Cell Endocrinol 2009;313
(1–2):9–16.
[36] Fortune JE, Yang MY, Muruvi W. In vitro and in vivo regulation of
follicular formation and activation in cattle. Reprod Fertil Dev
2011;23(1):15–22.
[37] Thau R, Lanman JT, Brinson A. Declining plasma progesterone
concentration with advancing gestation in blood from umbilical
and uterine veins and fetal heart in monkeys. Biol Reprod
1976;14(4):507–9.
[38] Kezele P, Skinner MK. Regulation of ovarian primordial follicle
assembly and development by estrogen and progesterone: endo-
crine model of follicle assembly. Endocrinology 2003;144
(8):3329–37.
[39] Chen Y, Jefferson WN, Newbold RR, Padilla-Banks E, Pepling ME.
Estradiol, progesterone, and genistein inhibit oocyte nest break-
down and primordial follicle assembly in the neonatal mouse
ovary in vitro and in vivo. Endocrinology 2007;148(8):3580–90.
[40] Chen Y, Breen K, Pepling ME. Estrogen can signal through multiple
pathways to regulate oocyte cyst breakdown and primordial folli-
cle assembly in the neonatal mouse ovary. J Endocrinol 2009;202
(3):407–17.
[41] Jefferson WN, Couse JF, Banks EP, Korach KS, Newbold RR.
Expression of estrogen receptor beta is developmentally regulated
in reproductive tissues of male and female mice. Biol Reprod
2000;62(2):310–7.
[42] Iguchi T, Fukazawa Y, Uesugi Y, Takasugi N. Polyovular follicles
in mouse ovaries exposed neonatally to diethylstilbestrol in vivo
and in vitro. Biol Reprod 1990;43(3):478–84.
[43] Kim H, Nakajima T, Hayashi S, Chambon P, Watanabe H, Iguchi T,
et al. Effects of diethylstilbestrol on programmed oocyte death and
induction of polyovular follicles in neonatal mouse ovaries. Biol
Reprod 2009;81(5):1002–9.
[44] Jefferson WN, Couse JF, Padilla-Banks E, Korach KS, Newbold RR.
Neonatal exposure to genistein induces estrogen receptor (ER)
alpha expression and multioocyte follicles in the maturing mouse
ovary: evidence for ERbeta-mediated and nonestrogenic actions.
Biol Reprod 2002;67(4):1285–96.
[45] Jefferson W. Neonatal genistein treatment alters ovarian differenti-
ation in the mouse: inhibition of oocyte nest breakdown and
increased oocyte survival. Biol Reprod 2005;74(1):161–8.
[46] Iguchi T, Kamiya K, Uesugi Y, Sayama K, Takasugi N. In vitro
fertilization of oocytes from polyovular follicles in mouse ovaries
exposed neonatally to diethylstilbestrol. In Vivo 1991;
5(4):359–63.
[47] Dandekar PV, Martin MC, Glass RH. Polyovular follicles associ-
ated with human in vitro fertilization. Fertil Steril 1988;49(3):483–6.
[48] Woodruff TK, D’Agostino J, Schwartz NB, Mayo KE. Dynamic
changes in inhibin messenger RNAs in rat ovarian follicles during
the reproductive cycle. Science 1988;239(4845):1296–9.
[49] Welt C, Sidis Y, Keutmann H, Schneyer A. Activins, inhibins, and
follistatins: from endocrinology to signaling. A paradigm for the
new millennium. Exp Biol Med (Maywood) 2002;227(9):724–52.
44 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
[50] Kimura F, Bonomi LM, Schneyer AL. Follistatin regulates germ cell
nest breakdown and primordial follicle formation. Endocrinology
2011;152(2):697–706.
[51] Bristol-Gould S. Establishing and maintaining the mammalian
ovarian follicle pool. ProQuest Information and Learning
Company
[52] Kimura F, Sidis Y, Bonomi L, Xia Y, Schneyer A. The follistatin-288
isoform alone is sufficient for survival but not for normal fertility in
mice. Endocrinology 2010;151(3):1310–9.
[53] Wang Z, Niu W, Wang Y, Teng Z, Wen J, Xia G, et al. Follistatin288
regulates germ cell cyst breakdown and primordial follicle assem-
bly in the mouse ovary. PLoS One 2015;10(6):e0129643.
[54] McCoshen JA, McCallion DJ. A study of the primordial germ cells
during their migratory phase in Steel mutant mice. Experientia
1975;31(5):589–90.
[55] Jones RL, Pepling ME. KIT signaling regulates primordial follicle
formation in the neonatal mouse ovary. Dev Biol 2013;382
(1):186–97.
[56] Asadi-Azarbaijani B, Santos RR, Jahnukainen K, Braber S, van
Duursen MBM, Toppari J, et al. Developmental effects of imatinib
mesylate on follicle assembly and early activation of primordial fol-
licle pool in postnatal rat ovary. Reprod Biol 2017;17(1):25–33.
[57] Dissen GA, Hirshfield AN, Malamed S, Ojeda SR. Expression of
neurotrophins and their receptors in the mammalian ovary is
developmentally regulated: changes at the time of folliculogenesis.
Endocrinology 1995;136(10):4681–92.
[58] Dissen GA, Romero C, Hirshfield AN, Ojeda SR. Nerve growth fac-
tor is required for early follicular development in the mammalian
ovary. Endocrinology 2001;142(5):2078–86.
[59] Kerr B, Garcia-Rudaz C, Dorfman M, Paredes A, Ojeda SR. NTRK1
and NTRK2 receptors facilitate follicle assembly and early follicular
development in the mouse ovary. Reproduction 2009;138
(1):131–40.
[60] Schindler R, Nilsson E, Skinner MK. Induction of ovarian primor-
dial follicle assembly by connective tissue growth factor CTGF.
PLoS One 2010;5(9):e12979.
[61] Wahab NA, Weston BS, Mason RM. Connective tissue growth fac-
tor CCN2 interacts with and activates the tyrosine kinase receptor
TrkA. J Am Soc Nephrol 2005 Feb;16(2):340–51.
[62] Rajareddy S, Reddy P, Du C, Liu L, Jagarlamudi K, Tang W, et al.
p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian
development by suppressing follicle endowment and activation
and promoting follicle atresia in mice. Mol Endocrinol 2007;21
(9):2189–202.
[63] Perez-Sanz J, Arluzea J, Matorras R, González-Santiago N, Bilbao J,
Yeh N, et al. Increased number of multi-oocyte follicles (MOFs) in
juvenile p27Kip1 mutant mice: potential role of granulosa cells.
Hum Reprod 2013;28(4):1023–30.
[64] Li R, Albertini DF. The road to maturation: somatic cell interaction
and self-organization of the mammalian oocyte. Nat. Rev Mol Cell
Biol 2013;14(3):141–52.
[65] Kopan R, Ilagan MXG. The canonical Notch signaling pathway:
unfolding the activation mechanism. Cell 2009;137(2):216–33.
[66] Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tian X, Pan DJ,
et al. A ligand-induced extracellular cleavage regulates gamma-
secretase-like proteolytic activation of Notch1. Mol Cell 2000;
5(2):197–206.
[67] Kovall RA. More complicated than it looks: assembly of Notch
pathway transcription complexes. Oncogene 2008;27(38):5099–109.
[68] Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate
control and signal integration in development. Science 1999;284
(5415):770–6.
[69] Lopez-Schier H, St Johnston D. Delta signaling from the germ line
controls the proliferation and differentiation of the somatic follicle
cells during Drosophila oogenesis. Genes Dev 2001;15(11):
1393–405.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
[70] Johnson J, Espinoza T, McGaughey RW, Rawls A, Wilson-Rawls J.
Notch pathway genes are expressed in mammalian ovarian folli-
cles. Mech Dev 2001;109(2):355–61.
[71] Trombly DJ, Woodruff TK, Mayo KE. Roles for transforming
growth factor beta superfamily proteins in early folliculogenesis.
Semin Reprod Med 2009;27(1):14–23.
[72] Murta D, Batista M, Silva E, Trindade A, Mateus L, Duarte A, et al.
Differential expression of Notch component and effector genes dur-
ing ovarian follicle and corpus luteum development during the oes-
trous cycle. Reprod Fertil Dev 2015;27(7):1038–48.
[73] Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G,
Zhao C, et al. Integration of Notch and Wnt signaling in hematopoi-
etic stem cell maintenance. Nat Immunol 2005;6(3):314–22.
[74] Vanorny DA, Prasasya RD, Chalpe AJ, Kilen SM, Mayo KE. Notch
signaling regulates ovarian follicle formation and coordinates fol-
licular growth. Mol Endocrinol 2014;28(4):499–511.
[75] Trombly DJ, Woodruff TK, Mayo KE. Suppression of Notch signal-
ing in the neonatal mouse ovary decreases primordial follicle for-
mation. Endocrinology 2009;150(2):1014–24.
[76] Terauchi KJ, Shigeta Y, Iguchi T, Sato T. Role of Notch signaling in
granulosa cell proliferation and polyovular follicle induction dur-
ing folliculogenesis in mouse ovary. Cell Tissue Res 2016;365
(1):197–208.
[77] Chen C-L, Fu X-F, Wang L-Q, Wang J-J, Ma H-G, Cheng S-F, et al.
Primordial follicle assembly was regulated by Notch signaling
pathway in the mice. Mol Biol Rep 2014;41(3):1891–9.
[78] Edwards DR, Handsley MM, Pennington CJ. The ADAM metallo-
proteinases. Mol Asp Med 2008;29(5):258–89.
[79] Feng L, Wang Y, Cai H, Sun G, Niu W, Xin Q, et al. ADAM10-Notch
signaling governs the recruitment of ovarian pregranulosa cells
and controls folliculogenesis in mice. J Cell Sci 2016;129(11)
jcs.184267–2212.
[80] Xu J, Gridley T. Notch2 is required in somatic cells for breakdown
of ovarian germ-cell nests and formation of primordial follicles.
BMC Biol BioMed 2013;11(1):13.
[81] Hahn KL, Johnson J, Beres BJ, Howard S, Wilson-Rawls J. Lunatic
fringe null female mice are infertile due to defects in meiotic mat-
uration. Development 2005;132(4):817–28.
[82] Zhao L, Du X, Huang K, Zhang T, Teng Z, Niu W, et al. Rac1 mod-
ulates the formation of primordial follicles by facilitating STAT3-
directed Jagged1, GDF9 and BMP15 transcription in mice. Sci
Rep 2016;6(1):23972.
[83] Guo M, Zhang H, Bian F, Li G, Mu X, Wen J, et al. P4 down-
regulates Jagged2 and Notch1 expression during primordial folli-
culogenesis. Front Biosci 2012;4:2731–44.
[84] Dorfman MD, Kerr B, Garcia-Rudaz C, Paredes AH, Dissen GA,
Ojeda SR. Neurotrophins acting via TRKB receptors activate the
JAGGED1-NOTCH2 cell-cell communication pathway to facilitate
early ovarian development. Endocrinology 2011;152(12):5005–16.
[85] Meredith S, Dudenhoeffer G, Jackson K. Classification of small type
B/C follicles as primordial follicles in mature rats. J Reprod Fertil
2000;119(1):43–8.
[86] Epifano O, Liang LF, Familari M, Moos MC, Dean J. Coordinate
expression of the three zona pellucida genes during mouse oogen-
esis. Development 1995;121(7):1947–56.
[87] Ueno S, Takahashi M, Manganaro TF, Ragin RC, Donahoe PK. Cel-
lular localization of m€ullerian inhibiting substance in the develop-
ing rat ovary. Endocrinology 1989;124(2):1000–6.
[88] Ueno S, Kuroda T, Maclaughlin DT, Ragin RC, Manganaro TF,
Donahoe PK. Mullerian inhibiting substance in the adult rat ovary
during various stages of the estrous cycle. Endocrinology 1989;
125(2):1060–6.
[89] van Rooij IAJ, Broekmans FJM, Velde te ER, Fauser BCJM,
Bancsi LFJMM, de Jong FH, et al. Serum anti-M€ ullerian hormone
levels: a novel measure of ovarian reserve. Hum Reprod 2002;17
(12):3065–71.
 REFERENCES
[90] de Vet A, Laven JSE, de Jong FH, Themmen APN, Fauser BCJM.
Antim€ ullerian hormone serum levels: a putative marker for ovar-
ian aging. Fertil Steril 2002;77(2):357–62.
[91] Meduri G, Massin N, Guibourdenche J, Bachelot A, Fiori O,
Kuttenn F, et al. Serum anti-M€ ullerian hormone expression in
women with premature ovarian failure. Hum Reprod 2007;22(1):
117–23.
[92] Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT,
Grootegoed JA, et al. Control of primordial follicle recruitment
by anti-M€ ullerian hormone in the mouse ovary. Endocrinology
1999;140(12):5789–96.
[93] Durlinger ALL, Gruijters MJG, Kramer P, Karels B, Ingraham HA,
Nachtigal MW, et al. Anti-M€ ullerian hormone inhibits initiation of
primordial follicle growth in the mouse ovary. Endocrinology
2002;143(3):1076–84.
[94] Visser JA, Durlinger ALL, Peters IJJ, van den Heuvel ER, Rose UM,
Kramer P, et al. Increased oocyte degeneration and follicular atresia
during the estrous cycle in Anti-M€ ullerian hormone null mice.
Endocrinology 2007;148(5):2301–8.
[95] Arden KC, Biggs WH. Regulation of the FoxO family of transcrip-
tion factors by phosphatidylinositol-3 kinase-activated signaling.
Arch Biochem Biophys 2002;403(2):292–8.
[96] Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Sup-
pression of ovarian follicle activation in mice by the transcription
factor Foxo3a. Science 2003;301(5630):215–8.
[97] Liu L, Rajareddy S, Reddy P, Du C, Jagarlamudi K, Shen Y, et al.
Infertility caused by retardation of follicular development in mice
with oocyte-specific expression of Foxo3a. Development 2007;134
(1):199–209.
[98] Gallardo T, Shirley L, John GB, Castrillon DH. Generation of a germ
cell-specific mouse transgenic Cre line, Vasa-Cre. Genesis 2007;45
(6):413–7.
[99] John GB, Gallardo TD, Shirley LJ, Castrillon DH. Foxo3 is a PI3K-
dependent molecular switch controlling the initiation of oocyte
growth. Dev Biol 2008;321(1):197–204.
[100] Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, Shen Y,
et al. Oocyte-specific deletion of Pten causes premature activa-
tion of the primordial follicle pool. Science 2008;319(5863):611–3.
[101] Reddy P, Adhikari D, Zheng W, Liang S, H€am€al€ainen T,
Tohonen V, et al. PDK1 signaling in oocytes controls reproductive
aging and lifespan by manipulating the survival of primordial fol-
licles. Hum Mol Genet 2009;18(15):2813–24.
[102] Kim S-Y, Ebbert K, Cordeiro MH, Romero M, Zhu J, Serna VA,
et al. Cell autonomous phosphoinositide 3-kinase activation in
oocytes disrupts normal ovarian function through promoting sur-
vival and overgrowth of ovarian follicles. Endocrinology 2015;156
(4):1464–76.
[103] Hovatta O. Cryopreservation and culture of human ovarian cor-
tical tissue containing early follicles. Eur J Obstet Gynecol Reprod
Biol 2004;113(Suppl 1):S50–4.
[104] Duncan FE, Pavone ME, Gunn AH, Badawy S, Gracia C,
Ginsberg JP, et al. Pediatric and teen ovarian tissue removed for
cryopreservation contains follicles irrespective of age, disease
diagnosis, treatment history, and specimen processing methods.
J Adolesc Young Adult Oncol 2015;4(4):174–83.
[105] Li J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, et al. Activation
of dormant ovarian follicles to generate mature eggs. Proc Natl
Acad Sci U S A 2010;107(22):10280–4.
[106] Adhikari D, Gorre N, Risal S, Zhao Z, Zhang H, Shen Y, et al. The
safe use of a PTEN inhibitor for the activation of dormant mouse
primordial follicles and generation of fertilizable eggs. PLoS One
2012;7(6):e39034.
[107] Huang EJ, Manova K, Packer AI, Sanchez S, Bachvarova RF,
Besmer P. The murine steel panda mutation affects kit ligand
expression and growth of early ovarian follicles. Dev Biol 1993
May;157(1):100–9.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
45
[108] 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(1):122–37.
[109] Hutt KJ, McLaughlin EA, Holland MK. KIT/KIT ligand in mam-
malian oogenesis and folliculogenesis: roles in rabbit and murine
ovarian follicle activation and oocyte growth. Biol Reprod 2006;75
(3):421–33.
[110] Reddy P, Shen L, Ren C, Boman K, Lundin E, Ottander U, et al.
Activation of Akt (PKB) and suppression of FKHRL1 in mouse
and rat oocytes by stem cell factor during follicular activation
and development. Dev Biol 2005;281(2):160–70.
[111] Saatcioglu HD, Cuevas I, Castrillon DH. Control of oocyte reawa-
kening by kit. PLoS Genet 2016;12(8):e1006215.
[112] Horie K, Takakura K, Taii S, Narimoto K, Noda Y, Nishikawa S,
et al. The expression of c-kit protein during oogenesis and early
embryonic development. Biol Reprod 1991;45(4):547–52.
[113] Adhikari D, Risal S, Liu K, Shen Y. Pharmacological inhibition of
mTORC1 prevents over-activation of the primordial follicle pool
in response to elevated PI3K signaling. PLoS One 2013;8(1):
e53810.
[114] Adhikari D, Zheng W, Shen Y, Gorre N, H€am€al€ainen T,
Cooney AJ, et al. Tsc/mTORC1 signaling in oocytes governs
the quiescence and activation of primordial follicles. Hum Mol
Genet 2010;19(3):397–410.
[115] Adhikari D, Flohr G, Gorre N, Shen Y, Yang H, Lundin E, et al.
Disruption of Tsc2 in oocytes leads to overactivation of the entire
pool of primordial follicles. Mol Hum Reprod 2009;15
(12):765–70.
[116] Gorre N, Adhikari D, Lindkvist R, Br€annstr€ om M, Liu K, Shen Y.
mTORC1 Signaling in oocytes is dispensable for the survival of
primordial follicles and for female fertility. PLoS One 2014;9(10)
e110491.
[117] Zhang H, Liu K. Cellular and molecular regulation of the activa-
tion of mammalian primordial follicles: somatic cells initiate folli-
cle activation in adulthood. Hum Reprod Update 2015;21
(6):779–86.
[118] Suzumori N, Yan C, Matzuk MM, Rajkovic A. Nobox is a
homeobox-encoding gene preferentially expressed in primordial
and growing oocytes. Mech Dev 2002;111(1-2):137–41.
[119] Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM.
NOBOX deficiency disrupts early folliculogenesis and oocyte-
specific gene expression. Science 2004;305(5687):1157–9.
[120] Choi Y, Rajkovic A. Characterization of NOBOX DNA binding
specificity and its regulation of Gdf9 and Pou5f1 promoters.
J Biol Chem 2006;281(47):35747–56.
[121] Bouilly J, Bachelot A, Broutin I, Touraine P, Binart N. Novel
NOBOX loss-of-function mutations account for 6.2% of cases in
a large primary ovarian insufficiency cohort. Hum Mutat
2011;32(10):1108–13.
[122] Pangas SA, Choi Y, Ballow DJ, Zhao Y, Westphal H, Matzuk MM,
et al. Oogenesis requires germ cell-specific transcriptional regula-
tors Sohlh1 and Lhx8. Proc Natl Acad Sci U S A 2006;103
(21):8090–5.
[123] Choi Y, Ballow DJ, Xin Y, Rajkovic A. Lim homeobox gene, lhx8, is
essential for mouse oocyte differentiation and survival. Biol
Reprod 2008;79(3):442–9.
[124] Ren Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M,
Lopes R, et al. Lhx8 regulates primordial follicle activation and
postnatal folliculogenesis. BMC Biol 2015;13(1):39.
[125] Ballow DJ, Xin Y, Choi Y, Pangas SA, Rajkovic A. Sohlh2 is a germ
cell-specific bHLH transcription factor. Gene Expr Patterns 2006;6
(8):1014–8.
[126] Shin YH, Ren Y, Suzuki H, Golnoski KJ, Ahn HW, Mico V, et al.
Transcription factors SOHLH1 and SOHLH2 coordinate oocyte
differentiation without affecting meiosis I. J Clin Invest 2017;127
(6):2106–17.
46 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
[127] Choi Y, Yuan D, Rajkovic A. Germ cell-specific transcriptional
regulator sohlh2 is essential for early mouse folliculogenesis
and oocyte-specific gene expression. Biol Reprod 2008;79(6):
1176–82.
[128] Suzuki H, Ahn HW, Chu T, Bowden W, Gassei K, Orwig K, et al.
SOHLH1 and SOHLH2 coordinate spermatogonial differentia-
tion. Dev Biol 2012;361(2):301–12.
[129] Sutherland JM, Keightley RA, Nixon B, Roman SD, Robker RL,
Russell DL, et al. Suppressor of cytokine signaling 4 (SOCS4):
moderator of ovarian primordial follicle activation. J Cell Physiol
2012;227(3):1188–98.
[130] Uda M, Ottolenghi C, Crisponi L, Garcia JE, Deiana M, Kimber W,
Forabosco A, Cao A, Schssinger D, Pilia G. Foxl2 disruption
causes mouse ovarian failure by pervasive blockage of follicle
development. Hum Mol Genet 2004;13(11):1171–81.
[131] Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM.
Growth differentiation factor-9 is required during early ovarian
folliculogenesis. Nature 1996;383(6600):531–5.
[132] Viger RS, Guittot SM, Anttonen M, Wilson DB, Heikinheimo M.
Role of the GATA family of transcription factors in endocrine
development, function, and disease. Mol Endocrinol 2008;22(4):
781–98.
[133] Zaytouni T, Efimenko EE, Tevosian SG. GATA transcription fac-
tors in the developing reproductive system. Adv Genet
2011;76:93–134.
[134] Padua MB, Fox SC, Jiang T, Morse DA, Tevosian SG. Simulta-
neous gene deletion of gata4 and gata6 leads to early disruption
of follicular development and germ cell loss in the murine ovary.
Biol Reprod 2014;91(1):24.
[135] Kumar TR, Wang Y, Lu N, Matzuk MM. Follicle stimulating hor-
mone is required for ovarian follicle maturation but not male fer-
tility. Nat Genet 1997;15(2):201–4.
[136] 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(23):13612–7.
[137] Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG,
Charlton HM. The effect of a null mutation in the follicle-
stimulating hormone receptor gene on mouse reproduction.
Endocrinology 2000;141(5):1795–803.
[138] Massague J. TGF-beta signal transduction. Annu Rev Biochem
1998;67(1):753–91.
[139] Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone mor-
phogenetic protein system in mammalian reproduction. Endocr
Rev 2004;25(1):72–101.
[140] Miyazono K, Kusanagi K, Inoue H. Divergence and convergence
of TGF-beta/BMP signaling. J Cell Physiol 2001;187(3):265–76.
[141] Kaivo-Oja N, Jeffery LA, Ritvos O, Mottershead DG. Smad signal-
ling in the ovary. Reprod Biol 2006;4(1):21.
[142] Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two
major Smad pathways in TGF-beta superfamily signalling. Genes
Cells 2002;7(12):1191–204.
[143] Mazerbourg S, Klein C, Roh J, Kaivo-Oja N, Mottershead DG,
Korchynskyi O, et al. Growth differentiation factor-9 signaling
is mediated by the type I receptor, activin receptor-like kinase 5.
Mol Endocrinol 2004;18(3):653–65.
[144] Roh J-S, Bondestam J, Mazerbourg S, Kaivo-Oja N, Groome N,
Ritvos O, et al. Growth differentiation factor-9 stimulates inhibin
production and activates Smad2 in cultured rat granulosa cells.
Endocrinology 2003;144(1):172–8.
[145] Pangas SA, Li X, Umans L, Zwijsen A, Huylebroeck D,
Gutierrez C, et al. Conditional deletion of Smad1 and Smad5 in
somatic cells of male and female gonads leads to metastatic tumor
development in mice. Mol Cell Biol 2008;28(1):248–57.
[146] Li Q, Pangas SA, Jorgez CJ, Graff JM, Weinstein M, Matzuk MM.
Redundant roles of SMAD2 and SMAD3 in ovarian granulosa
cells in vivo. Mol Cell Biol 2008;28(23):7001–11.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
[147] Pangas SA, Li X, Robertson EJ, Matzuk MM. Premature luteiniza-
tion and cumulus cell defects in ovarian-specific Smad4 knockout
mice. Mol Endocrinol 2006;20(6):1406–22.
[148] McGrath SA, Esquela AF, Lee SJ. Oocyte-specific expression of
growth/differentiation factor-9. Mol Endocrinol 1995;9(1):131–6.
[149] Jaatinen R, Laitinen MP, Vuojolainen K, Aaltonen J, Louhio H,
Heikinheimo K, et al. Localization of growth differentiation
factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA
cloning of rat GDF-9 and its novel homolog GDF-9B. Mol Cell
Endocrinol 1999;156(1–2):189–93.
[150] Carabatsos MJ, Elvin J, Matzuk MM, Albertini DF. Characteriza-
tion of oocyte and follicle development in growth differentiation
factor-9-deficient mice. Dev Biol 1998;204(2):373–84.
[151] Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM. Molecular
characterization of the follicle defects in the growth differentiation
factor 9-deficient ovary. Mol Endocrinol 1999;13(6):1018–34.
[152] Vitt UA, McGee EA, Hayashi M, Hsueh AJ. In vivo treatment with
GDF-9 stimulates primordial and primary follicle progression and
theca cell marker CYP17 in ovaries of immature rats.
Endocrinology 2000;141(10):3814–20.
[153] Vitt UA, Hayashi M, Klein C, Hsueh AJ. Growth differentiation
factor-9 stimulates proliferation but suppresses the follicle-
stimulating hormone-induced differentiation of cultured granu-
losa cells from small antral and preovulatory rat follicles. Biol
Reprod 2000;62(2):370–7.
[154] Cook-Andersen H, Curnow KJ, Su HI, Chang RJ, Shimasaki S.
Growth and differentiation factor 9 promotes oocyte growth at
the primary but not the early secondary stage in three-dimensional
follicle culture. J Assist Reprod Genet 2016;33(8):1067–77.
[155] Fenwick MA, Mora JM, Mansour YT, Baithun C, Franks S,
Hardy K. Investigations of TGF-β signaling in preantral follicles
of female mice reveal differential roles for bone morphogenetic
protein 15. Endocrinology 2013;154(9):3423–36.
[156] Vitt UA, Mazerbourg S, Klein C, Hsueh AJW. Bone morphoge-
netic protein receptor type II is a receptor for growth differentia-
tion factor-9. Biol Reprod 2002;67(2):473–80.
[157] Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM. The
bone morphogenetic protein 15 gene is X-linked and expressed in
oocytes. Mol Endocrinol 1998;12(12):1809–17.
[158] 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(12):8060–5.
[159] Galloway SM, KP MNATTY, Cambridge LM, Laitinen MP,
Juengel JL, Jokiranta TS, et al. Mutations in an oocyte-derived
growth factor gene (BMP15) cause increased ovulation rate and
infertility in a dosage-sensitive manner. Nat Genet 2000;25
(3):279–83.
[160] KP MN, Smith P, Hudson NL, Heath DA, Tisdall DJ, WS O, et al.
Development of the sheep ovary during fetal and early neonatal
life and the effect of fecundity genes. J Reprod Fertil Suppl
1995;49:123–35.
[161] Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA,
et al. Highly prolific Booroola sheep have a mutation in the intra-
cellular kinase domain of bone morphogenetic protein IB receptor
(ALK-6) that is expressed in both oocytes and granulosa cells. Biol
Reprod 2001;64(4):1225–35.
[162] Crawford JL, KP MN. The ratio of growth differentiation factor 9:
bone morphogenetic protein 15 mRNA expression is tightly
co-regulated and differs between species over a wide range of
ovulation rates. Mol Cell Endocrinol 2012;348(1):339–43.
[163] Liao WX, Moore RK, Otsuka F, Shimasaki S. Effect of intracellular
interactions on the processing and secretion of bone morphoge-
netic protein-15 (BMP-15) and growth and differentiation factor-
9. Implication of the aberrant ovarian phenotype of BMP-15
mutant sheep. J Biol Chem 2003;278(6):3713–9.
[164] Mottershead DG, Sugimura S, Al-Musawi SL, Li J-J, Richani D,
White MA, et al. Cumulin, an oocyte-secreted heterodimer of
 REFERENCES
the transforming growth factor-β family, is a potent activator of
granulosa cells and improves oocyte quality. J Biol Chem
2015;290(39):24007–20.
[165] Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, et al.
Synergistic roles of bone morphogenetic protein 15 and growth
differentiation factor 9 in ovarian function. Mol Endocrinol
2001;15(6):854–66.
[166] Mottershead DG, Ritter LJ, Gilchrist RB. Signalling pathways
mediating specific synergistic interactions between GDF9 and
BMP15. Mol Hum Reprod 2012 Mar;18(3):121–8.
[167] Peng J, Li Q, Wigglesworth K, Rangarajan A, Kattamuri C,
Peterson RT, et al. Growth differentiation factor 9: bone mor-
phogenetic protein 15 heterodimers are potent regulators of
ovarian functions. Proc Natl Acad Sci U S A 2013;110(8):
E776–85.
[168] Patiño LC, Walton KL, Mueller TD, Johnson KE, Stocker W,
Richani D, et al. BMP15 mutations associated with primary ovar-
ian insufficiency reduce expression, activity, or synergy with
GDF9. J Clin Endocrinol Metabol 2017;102(3):1009–19.
[169] Norling A, Hirschberg AL, Rodriguez-Wallberg KA, Iwarsson E,
Wedell A, Barbaro M. Identification of a duplication within the
GDF9 gene and novel candidate genes for primary ovarian insuf-
ficiency (POI) by a customized high-resolution array comparative
genomic hybridization platform. Hum Reprod 2014;29
(8):1818–27.
[170] Wang T-T, Ke Z-H, Song Y, Chen L-T, Chen X-J, Feng C, et al.
Identification of a mutation in GDF9 as a novel cause of dimin-
ished ovarian reserve in young women. Hum Reprod 2013;28
(9):2473–81.
[171] Juengel JL, McNATTY KP. The role of proteins of the trans-
forming growth factor-beta superfamily in the intraovarian reg-
ulation of follicular development. Hum Reprod Update 2005;11
(2):143–60.
[172] Christopher B. Immunolocalization of transforming growth
factor-beta1 during follicular development and atresia in the
mouse ovary. Endocr J 2000;47(4):475–80.
[173] Woodruff TK, Meunier H, Jones PB, Hsueh AJ, Mayo KE. Rat
inhibin: molecular cloning of alpha- and beta-subunit comple-
mentary deoxyribonucleic acids and expression in the ovary.
Mol Endocrinol 1987;1(8):561–8.
[174] Makanji Y, Zhu J, Mishra R, Holmquist C, Wong WPS,
Schwartz NB, et al. Inhibin at 90: from discovery to clinical appli-
cation, a historical review. Endocr Rev 2014;35(5):747–94.
[175] Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. Acti-
vin-binding protein from rat ovary is follistatin. Science 1990;247
(4944):836–8.
[176] Li R, Phillips DM, Mather JP. Activin promotes ovarian follicle
development in vitro. Endocrinology 1995;136(3):849–56.
[177] Yokota H, Yamada K, Liu X, Kobayashi J, Abe Y,
Mizunuma H, et al. Paradoxical action of activin A on follicu-
logenesis in immature and adult mice. Endocrinology 1997;138
(11):4572–6.
[178] Mizunuma H, Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K,
et al. Activin from secondary follicles causes small preantral folli-
cles to remain dormant at the resting stage. Endocrinology
1999;140(1):37–42.
[179] Pangas SA, Jorgez CJ, Tran M, Agno J, Li X, Brown CW, et al.
Intraovarian activins are required for female fertility. Mol Endo-
crinol 2007;21(10):2458–71.
[180] Tomic D, Brodie SG, Deng C, Hickey RJ, Babus JK, Malkas LH,
et al. Smad 3 may regulate follicular growth in the mouse ovary.
Biol Reprod 2002;66(4):917–23.
[181] McMullen ML, Cho BN, Yates CJ, Mayo KE. Gonadal pathologies
in transgenic mice expressing the rat inhibin alpha-subunit.
Endocrinology 2001;142(11):5005–14.
[182] Cho BN, McMullen ML, Pei L, Yates CJ, Mayo KE. Reproductive
deficiencies in transgenic mice expressing the rat inhibin alpha-
subunit gene. Endocrinology 2001;142(11):4994–5004.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
47
[183] Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-
inhibin is a tumour-suppressor gene with gonadal specificity in
mice. Nature 1992;360(6402):313–9.
[184] Kipp JL, Kilen SM, Woodruff TK, Mayo KE. Activin regulates
estrogen receptor gene expression in the mouse ovary. J Biol Chem
2007;282(50):36755–65.
[185] Rao MC, Midgley AR, Richards JS. Hormonal regulation of ovar-
ian cellular proliferation. Cell 1978;14(1):71–8.
[186] Bendell JJ, Dorrington J. Estradiol-17 beta stimulates DNA synthe-
sis in rat granulosa cells: action mediated by transforming growth
factor-beta. Endocrinology 1991;128(5):2663–5.
[187] Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG,
Jones ME, et al. An age-related ovarian phenotype in mice with
targeted disruption of the Cyp 19 (aromatase) gene.
Endocrinology 2000;141(7):2614–23.
[188] Kipp JL, Golebiowski A, Rodriguez G, Demczuk M, Kilen SM,
Mayo KE. Gene expression profiling reveals Cyp26b1 to be an
activin regulated gene involved in ovarian granulosa cell prolifer-
ation. Endocrinology 2011;152(1):303–12.
[189] Zhang C-P, Yang J-L, Zhang J, Li L, Huang L, Ji S-Y, et al. Notch
signaling is involved in ovarian follicle development by regulating
granulosa cell proliferation. Endocrinology 2011;152(6):2437–47.
[190] Sun X-F, Sun X-H, Cheng S-F, Wang J-J, Feng Y-N, Zhao Y, et al.
Interaction of the transforming growth factor-β and Notch signal-
ing pathways in the regulation of granulosa cell proliferation.
Reprod Fertil Dev 1873;28(12):3.
[191] Manosalva I, González A, Kageyama R. Hes1 in the somatic cells
of the murine ovary is necessary for oocyte survival and matura-
tion. Dev Biol 2013;375(2):140–51.
[192] Magoffin DA. Ovarian theca cell. Int J Biochem Cell Biol 2005
Jul;37(7):1344–9.
[193] Honda A, Hirose M, Hara K, Matoba S, Inoue K, Miki H, et al. Iso-
lation, characterization, and in vitro and in vivo differentiation of
putative thecal stem cells. Proc Natl Acad Sci U S A 2007;104
(30):12389–94.
[194] Liu C, Peng J, Matzuk MM. Yao HHC. Lineage specification of
ovarian theca cells requires multicellular interactions via oocyte
and granulosa cells. Nat Commun 2015;6:6934.
[195] Magoffin DA, Magarelli PC. Preantral follicles stimulate luteiniz-
ing hormone independent differentiation of ovarian theca-
interstitial cells by an intrafollicular paracrine mechanism.
Endocrine 1995;3(2):107–12.
[196] Magarelli PC, Zachow RJ, Magoffin DA. Developmental and hor-
monal regulation of rat theca-cell differentiation factor secretion in
ovarian follicles. Biol Reprod 1996;55(2):416–20.
[197] Wijgerde M, Ooms M, Hoogerbrugge JW, Grootegoed JA.
Hedgehog signaling in mouse ovary: Indian hedgehog and
desert hedgehog from granulosa cells induce target gene ex-
pression in developing theca cells. Endocrinology 2005;146(8):
3558–66.
[198] Edson MA, Nagaraja AK, Matzuk MM. The mammalian ovary
from genesis to revelation. Endocr Rev 2009;30(6):624–712.
[199] Camp TA, Rahal JO, Mayo KE. Cellular localization and hormonal
regulation of follicle-stimulating hormone and luteinizing hor-
mone receptor messenger RNAs in the rat ovary. Mol Endocrinol
1991;5(10):1405–17.
[200] Chun SY, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJ.
Hormonal regulation of apoptosis in early antral follicles: follicle-
stimulating hormone as a major survival factor. Endocrinology
1996;137(4):1447–56.
[201] Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH,
Falender AE, et al. Novel signaling pathways that control ovarian
follicular development, ovulation, and luteinization. Recent Prog
Horm Res 2002;57:195–220.
[202] Hunzicker-Dunn M, Maizels ET. FSH signaling pathways in
immature granulosa cells that regulate target gene expression:
branching out from protein kinase A. Cell Signal 2006;18
(9):1351–9.
48 2. REGULATION OF FOLLICLE FORMATION AND DEVELOPMENT BY OVARIAN SIGNALING PATHWAYS
[203] Hirshfield AN. Development of follicles in the mammalian ovary.
Int Rev Cytol 1991;124:43–101.
[204] Richards JS. Maturation of ovarian follicles: actions and interac-
tions of pituitary and ovarian hormones on follicular cell differen-
tiation. Physiol Rev 1980;60(1):51–89.
[205] Hsueh AJ, Rauch R. Ovarian Kaleidoscope database: ten years
and beyond. Biol Reprod 2012;86(6):192.
[206] Hsueh AJ, Adashi EY, Jones PB, Welsh TH. Hormonal regulation
of the differentiation of cultured ovarian granulosa cells. Endocr
Rev 1984;5(1):76–127.
[207] Knecht M, Amsterdam A, Catt K. The regulatory role of cyclic
AMP in hormone-induced of granulosa cell differentiation.
J Biol Chem 1981;256(20):10628–33.
[208] Knecht M, Ranta T, Catt KJ. Granulosa cell differentiation in vitro:
induction and maintenance of follicle-stimulating hormone recep-
tors by adenosine 30 ,50 -monophosphate. Endocrinology 1983;113
(3):949–56.
[209] Taylor SS, Knighton DR, Zheng J, Eyck Ten LF, Sowadski JM.
cAMP-dependent protein kinase and the protein kinase family.
Faraday Discuss 1992;93:143–52.
[210] Mukherjee A, Park-Sarge OK, Mayo KE. Gonadotropins induce
rapid phosphorylation of the 30 ,50 -cyclic adenosine monopho-
sphate response element binding protein in ovarian granulosa
cells. Endocrinology 1996;137(8):3234–45.
[211] Carlone DL, Richards JS. Functional interactions, phosphoryla-
tion, and levels of 30 ,50 -cyclic adenosine monophosphate-
regulatory element binding protein and steroidogenic factor-1
mediate hormone-regulated and constitutive expression of aro-
matase in gonadal cells. Mol Endocrinol 1997;11(3):292–304.
[212] Tremblay JJ, Viger RS. Transcription factor GATA-4 is activated
by phosphorylation of serine 261 via the cAMP/protein kinase
a signaling pathway in gonadal cells. J Biol Chem 2003;278
(24):22128–35.
[213] Tremblay JJ, Viger RS. GATA factors differentially activate multi-
ple gonadal promoters through conserved GATA regulatory ele-
ments. Endocrinology 2001;142(3):977–86.
[214] DeManno DA, Cottom JE, Kline MP, Peters CA, Maizels ET,
Hunzicker-Dunn M. Follicle-stimulating hormone promotes his-
tone H3 phosphorylation on serine-10. Mol Endocrinol 1999;13
(1):91–105.
[215] Salvador LM, Park Y, Cottom J, Maizels ET, Jones JC, Schillace RV,
et al. Follicle-stimulating hormone stimulates protein kinase
A-mediated histone H3 phosphorylation and acetylation leading
to select gene activation in ovarian granulosa cells. J Biol Chem
2001;276(43):40146–55.
[216] Hunzicker-Dunn M, Mayo K. Chapter 20—gonadotropin signal-
ing in the ovary. In: Plant TM, Zeleznik AJ, editors. Knobil and
Neill’s physiology of reproduction. 4th ed. Elsevier; 2014.
p. 895–946.
[217] Marshall CJ. Specificity of receptor tyrosine kinase signaling: tran-
sient versus sustained extracellular signal-regulated kinase activa-
tion. Cell 1995;80(2):179–85.
[218] Dikic I, Blaukat A. Protein tyrosine kinase-mediated pathways in
G protein-coupled receptor signaling. Cell Biochem Biophys
1999;30(3):369–87.
[219] Das S, Maizels ET, DeManno D, St Clair E, Adam SA, Hunzicker-
Dunn M. A stimulatory role of cyclic adenosine 30 ,50 -
monophosphate in follicle-stimulating hormone-activated
mitogen-activated protein kinase signaling pathway in rat ovar-
ian granulosa cells. Endocrinology 1996;137(3):967–74.
[220] Burgering BM, Bos JL. Regulation of Ras-mediated signalling:
more than one way to skin a cat. Trends Biochem Sci 1995;20(1):
18–22.
[221] Donaubauer EM, Hunzicker-Dunn ME. Extracellular signal-
regulated kinase (ERK)-dependent phosphorylation of Y-box-
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
binding protein 1 (YB-1) enhances gene expression in granulosa
cells in response to follicle-stimulating hormone (FSH). J Biol
Chem 2016;291(23):12145–60.
[222] Cottom J, Salvador LM, Maizels ET, Reierstad S, Park Y, Carr DW,
et al. Follicle-stimulating hormone activates extracellular signal-
regulated kinase but not extracellular signal-regulated kinase
kinase through a 100-kDa phosphotyrosine phosphatase. J Biol
Chem 2003;278(9):7167–79.
[223] Fujinaga H, Yamoto M, Shikone T, Nakano R. FSH and LH
up-regulate epidermal growth factor receptors in rat granulosa
cells. J Endocrinol 1994;140(2):171–7.
[224] El-Hayek S, Demeestere I, Clarke HJ. Follicle-stimulating hor-
mone regulates expression and activity of epidermal growth fac-
tor receptor in the murine ovarian follicle. Proc Natl Acad Sci U S
A 2014;111(47):16778–83.
[225] Babu PS, Krishnamurthy H, Chedrese PJ, Sairam MR. Activation
of extracellular-regulated kinase pathways in ovarian granulosa
cells by the novel growth factor type 1 follicle-stimulating hor-
mone receptor. Role in hormone signaling and cell proliferation.
J Biol Chem 2000;275(36):27615–26.
[226] Prasasya RD, Mayo KE. Notch signaling regulates differentiation
and steroidogenesis in female mouse ovarian granulosa cells.
Endocrinology 2018;159(1):184–98.
[227] Wang J, Liu S, Peng L, Dong Q, Bao R, Lv Q, et al. Notch signaling
pathway regulates progesterone secretion in murine luteal cells.
Reprod Sci 2015;22(10):1243–51.
[228] Zhou J, Kumar TR, Matzuk MM, Bondy C. Insulin-like growth
factor I regulates gonadotropin responsiveness in the murine
ovary. Mol Endocrinol 1997;11(13):1924–33.
[229] Zhou P, Baumgarten SC, Wu Y, Bennett J, Winston N, Hirshfeld-
Cytron J, et al. IGF-I signaling is essential for FSH stimulation of
AKT and steroidogenic genes in granulosa cells. Mol Endocrinol
2013;27(3):511–23.
[230] Baumgarten SC, Armouti M, Ko C, Stocco C. IGF1R expression in
ovarian granulosa cells is essential for steroidogenesis, follicle sur-
vival, and fertility in female mice. Endocrinology 2017;158
(7):2309–18.
[231] Park Y, Maizels ET, Feiger ZJ, Alam H, Peters CA, Woodruff TK,
et al. Induction of cyclin D2 in rat granulosa cells requires FSH-
dependent relief from FOXO1 repression coupled with positive
signals from Smad. J Biol Chem 2005;280(10):9135–48.
[232] Hunzicker-Dunn ME, Lopez-Biladeau B, Law NC, Fiedler SE,
Carr DW, Maizels ET. PKA and GAB2 play central roles in the
FSH signaling pathway to PI3K and AKT in ovarian granulosa
cells. Proc Natl Acad Sci U S A 2012;109(44):E2979–88.
[233] Herndon MK, Law NC, Donaubauer EM, Kyriss B, Hunzicker-
Dunn M. Forkhead box O member FOXO1 regulates the majority
of follicle-stimulating hormone responsive genes in ovarian gran-
ulosa cells. Mol Cell Endocrinol 2016;434:116–26.
[234] Liu Z, Rudd MD, Hernandez-Gonzalez I, Gonzalez-Robayna I,
Fan H-Y, Zeleznik AJ, et al. FSH and FOXO1 regulate genes in
the sterol/steroid and lipid biosynthetic pathways in granulosa
cells. Mol Endocrinol 2009;23(5):649–61.
[235] Miller WL, Auchus RJ. The molecular biology, biochemistry, and
physiology of human steroidogenesis and its disorders. Endocr
Rev 2011;32(1):81–151.
[236] Hillier SG, Whitelaw PF, Smyth CD. Follicular oestrogen synthe-
sis: the “two-cell, two-gonadotrophin” model revisited. Mol Cell
Endocrinol 1994;100(1–2):51–4.
[237] Conneely OM, Mulac-Jericevic B, Lydon JP, De Mayo FJ.
Reproductive functions of the progesterone receptor isoforms: les-
sons from knock-out mice. Mol Cell Endocrinol 2001;179
(1–2):97–103.
[238] Knecht M, Darbon JM, Ranta T, Baukal AJ, Catt KJ. Estrogens
enhance the adenosine 30 ,50 -monophosphate-mediated induction
 REFERENCES
of follicle-stimulating hormone and luteinizing hormone recep-
tors in rat granulosa cells. Endocrinology 1984;115(1):41–9.
[239] Wang C, Prossnitz ER, Roy SK. Expression of G protein-coupled
receptor 30 in the hamster ovary: differential regulation by gonad-
otropins and steroid hormones. Endocrinology 2007;148
(10):4853–64.
[240] Otto C, Fuchs I, Kauselmann G, Kern H, Zevnik B, Andreasen P,
et al. GPR30 does not mediate estrogenic responses in reproduc-
tive organs in mice. Biol Reprod 2009;80(1):34–41.
[241] Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Sch€ utz G,
Umesono K, et al. The nuclear receptor superfamily: the second
decade. Cell 1995;83(6):835–9.
[242] Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS,
Smithies O. Alteration of reproductive function but not prenatal
sexual development after insertional disruption of the mouse
estrogen receptor gene. Proc Natl Acad Sci U S A 1993;90
(23):11162–6.
[243] Schomberg DW, Couse JF, Mukherjee A, Lubahn DB, Sar M,
Mayo KE, et al. Targeted disruption of the estrogen receptor-alpha
gene in female mice: characterization of ovarian responses and
phenotype in the adult. Endocrinology 1999;140(6):2733–44.
[244] Britt KL, Findlay JK. Regulation of the phenotype of ovarian
somatic cells by estrogen. Mol Cell Endocrinol 2003;202(1–2):11–7.
[245] Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF,
et al. Generation and reproductive phenotypes of mice lacking
estrogen receptor beta. Proc Natl Acad Sci U S A 1998;95
(26):15677–82.
[246] Couse JF, Yates MM, Deroo BJ, Korach KS. Estrogen receptor-beta
is critical to granulosa cell differentiation and the ovulatory
response to gonadotropins. Endocrinology 2005;146(8):3247–62.
[247] Deroo BJ, Rodriguez KF, Couse JF, Hamilton KJ, Collins JB,
Grissom SF, et al. Estrogen receptor beta is required for optimal
cAMP production in mouse granulosa cells. Mol Endocrinol
2009;23(7):955–65.
[248] Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, et al.
Postnatal sex reversal of the ovaries in mice lacking estrogen
receptors alpha and beta. Science 1999;286(5448):2328–31.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
49
[249] Sen A, Hammes SR. Granulosa cell-specific androgen receptors
are critical regulators of ovarian development and function. Mol
Endocrinol 2010;24(7):1393–403.
[250] Walters KA, Middleton LJ, Joseph SR, Hazra R, Jimenez M,
Simanainen U, et al. Targeted loss of androgen receptor signaling
in murine granulosa cells of preantral and antral follicles causes
female subfertility. Biol Reprod 2012;87(6):151.
[251] Sen A, Prizant H, Light A, Biswas A, Hayes E, Lee H-J, et al.
Androgens regulate ovarian follicular development by increasing
follicle stimulating hormone receptor and microRNA-125b
expression. Proc Natl Acad Sci U S A 2014;111(8):3008–13.
[252] Laird M, Thomson K, Fenwick M, Mora J, Franks S, Hardy K.
Androgen stimulates growth of mouse preantral follicles
in vitro: interaction with follicle-stimulating hormone and with
growth factors of the TGFβ superfamily. Endocrinology
2017;158(4):920–35.
[253] Franks S. Polycystic ovary syndrome. N Engl J Med 1995;333
(13):853–61.
[254] Ehrmann DA. Polycystic ovary syndrome. N Engl J Med 2005;352
(12):1223–36.
[255] Chen M-J, Chou C-H, Chen S-U, Yang W-S, Yang Y-S, Ho H-N.
The effect of androgens on ovarian follicle maturation: dihydro-
testosterone suppress FSH-stimulated granulosa cell proliferation
by upregulating PPARγ-dependent PTEN expression. Sci Rep
2015;5(1):18319.
[256] Kayampilly PP, Menon KMJ. Dihydrotestosterone inhibits
insulin-stimulated cyclin D2 messenger ribonucleic acid expres-
sion in rat ovarian granulosa cells by reducing the phosphoryla-
tion of insulin receptor substrate-1. Endocrinology 2006;147
(1):464–71.
[257] Kayampilly PP, Menon KMJ. AMPK activation by dihydrotestos-
terone reduces FSH-stimulated cell proliferation in rat granulosa
cells by inhibiting ERK signaling pathway. Endocrinology
2012;153(6):2831–8.
[258] Wang Y, Du Y, Shen B, Zhou X, Li J, Liu Y, et al. Efficient gener-
ation of gene-modified pigs via injection of zygote with Cas9/
sgRNA. Sci Rep 2015;5(1):8256.