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Focus Article
Regulation of germ cell

development by intercellular
signaling in the mammalian
ovarian follicle
Hugh J. Clarke *
Prior to ovulation, the mammalian oocyte undergoes a process of differentiation

within the ovarian follicle that confers on it the ability to give rise to an embryo.
Differentiation comprises two phases—growth, during which the oocyte
increases more than 100-fold in volume as it accumulates macromolecules and
organelles that will sustain early embryogenesis; and meiotic maturation, during
which the oocyte executes the first meiotic division and prepares for the second
division. Entry of an oocyte into the growth phase appears to be triggered when
the adjacent granulosa cells produce specific growth factors. As the oocyte grows,
it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless,
cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projec-
tions (TZPs), enable them to maintain contact-dependent communication with
the oocyte. Through gap junctions located where the TZP tips meet the oocyte
membrane, they provide the oocyte with products that sustain its metabolic
activity and signals that regulate its differentiation. Conversely, the oocyte
secretes diffusible growth factors that regulate proliferation and differentiation of
the granulosa cells. Gap junction-permeable products of the granulosa cells pre-
vent precocious initiation of meiotic maturation, and the gap junctions also ena-
ble oocyte maturation to begin in response to hormonal signals received by the
granulosa cells. Development of the oocyte or the somatic compartment may also
be regulated by extracellular vesicles newly identified in follicular fluid and at
TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte
differentiation thus depends on continuous signaling interactions with the
somatic cells of the follicle. © 2017 Wiley Periodicals, Inc.
How to cite this article:
WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294
INTRODUCTION cells in a structure termed a primordial follicle. Before

ovulation, each oocyte undergoes a process of differen-
N ewborn mammalian females contain an enormous

number—ranging from about 20,000 in the
mouse1 to up to one million in humans2—of oocytes,
tiation to generate an egg that can be fertilized and
develop as an embryo. The oocyte does not undertake
this journey alone. Rather, it relies on support provided
each enclosed by a small number of somatic granulosa by the somatic compartment of the follicle, which pro-
vides nutrients that support its metabolic activity and
*Correspondence to: hugh.clarke@mcgill.ca
signals that regulate its differentiation. The oocyte is
not, however, simply a passive participant in this proc-
Department of Obstetrics and Gynecology, Research Institute of
the McGill University Health Centre, McGill University, Montreal, ess. It also sends signals to the somatic cells that regu-
Canada late their differentiation and help to ensure that they
Conflict of interest: The author has declared no conflicts of interest provide the microenvironment that the oocyte needs as
for this article. it grows and develops. Thus, bidirectional and
Volume 7, January/February 2018 © 2017 Wiley Periodicals, Inc. 1 of 22

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Focus Article wires.wiley.com/devbio
continuous signaling between the oocyte and somatic Growth of the oocyte is accompanied by
compartment of the follicle are essential to produce a growth and differentiation of the follicle.3,8,18 Upon
healthy egg. entry into the growth phase, the squamous granulosa
Several characteristics of postnatal oocyte cells that characterize the primordial follicle become
development within the follicle make it especially cuboidal in shape and begin to proliferate mitotically
attractive for experimental study. First, the follicle so that they continue to fully cover the expanding
presents a relatively simple anatomy, consisting of surface of the growing oocyte.19,20 These follicles,
three principal cell types, each occupying a well- now termed primary, are delimited by a basement
defined spatial position. Second, cohorts of primor- membrane that lies apposed to the basal side of the
dial follicles regularly enter and complete the growth granulosa cells. As the granulosa cells continue to
phase, so the growth and differentiation process can proliferate, they generate multiple layers around the
be studied throughout most of the postnatal life of a oocyte; such follicles are termed secondary. Steroido-
female. Third, culture systems have been developed genic cells known as the theca are recruited to folli-
that recapitulate much of postnatal oocyte and follic- cles containing two layers of granulosa cells external
ular development. As a result, much has been learned to the basement membrane.21 Near the time that the
about the signaling mechanisms that control the oocyte completes its growth, a fluid-filled cavity
development of the female germ cell. Here, I review termed the antrum appears. The antrum separates
pathways of communication between the oocyte and the granulosa cells into two populations—the mural
the somatic compartment of the ovarian follicle, granulosa, which line the inner wall of the follicle
focusing on work carried out using the mouse as a and the cumulus granulosa, which surround the
model system. oocyte. Under the opposing influences of signals from
the oocyte and from extra-follicular sources—nota-
bly, follicle-stimulating hormone (FSH) secreted by
POSTNATAL OOCYTE the pituitary gland, the mural and cumulus granulosa
DEVELOPMENT: GROWTH AND cells express different genes and fulfill different func-
MEIOTIC MATURATION tions.22,23 Follicles containing a large antrum are
Postnatal oocyte development comprises two termed Graafian and it is these that typically ovulate
phases—a prolonged period of growth within the fol- in response to luteinizing hormone (LH).
licle followed by a much briefer period known as
meiotic maturation that occurs coincident with ovu-
Meiotic Maturation
lation (Figure 1). Current evidence indicates that no
new functional oocytes are created after birth under The final phase of oocyte development is known as
physiological conditions.4–7 Instead, the population meiotic maturation, and is triggered by the ovulatory
of oocytes present at birth represents the lifetime release of LH from the pituitary gland.24–27 The
endowment of the female. oocyte cell cycle, which has been arrested at late dip-
lotene of prophase I since fetal life, resumes and the
oocytes enter M-phase. The chromosomes assemble
Oocyte and Follicular Growth on a spindle, which is translocated by an actin-
During growth, which requires 3–4 months in humans dependent process to the cortex where the first mei-
and about 3 weeks in mice, the volume of the oocyte otic division occurs, segregating one homologue of
increases more than 100-fold. This increase in size each pair into the small first polar body.28,29 The
reflects the accumulation of messenger (m) RNAs and chromosomes remaining in the oocyte then align on
proteins, as well as organelles such as mitochondria, a spindle in preparation for the second meiotic divi-
that are essential for embryonic development after sion, which is triggered by fertilization. These nuclear
fertilization.8–10 For example, mRNA synthesis falls to events are accompanied by cytoplasmic maturation,
an undetectable level in a fully grown oocytes and is notably the translational activation of a subset of
not fully reactivated until a species-specific stage after previously silent mRNAs and the silencing and in
fertilization,9,11 and the embryo does not resume mito- some cases degradation of previously active
chondrial replication until after it has implanted into mRNAs.30–32
the uterus. Oocyte growth is also marked by important All aspects of postnatal oocyte development
epigenetic modifications, including methylation of spe- depend on signaling interactions with the granulosa
cific DNA sequences that influences their subsequent cells of the ovarian follicle. I now turn to a discussion
transcriptional activity,12–15 and by ultrastructural of the different mechanisms of signaling and their
changes that are necessary for embryogenesis.16,17 specific roles during oocyte development. First,
2 of 22 © 2017 Wiley Periodicals, Inc. Volume 7, January/February 2018

WIREs Developmental Biology Regulation of female germ cell development by intercellular signaling
(a)
Oocyte and follicular growth
Translated mRNA
Stored mRNA
Mitochondria
Mural
Zona Antrum
pellucida
Granulosa Cumulus
Oocyte Theca
Primordial Primary Secondary Antral Pre-ovulatory

(Graafian)
(b)
Meiotic maturation
Immature Germinal vesicle Spindle Metaphase I Metaphase II

(germinal-vesicle) breakdown assembly
FI GU RE 1 | Postnatal oocyte and follicular development. (a) The arrangement of the principal cell types of the follicle at different stages of
oocyte and follicular growth is shown. Each primordial follicle contains one oocyte enclosed by a small number of squamous granulosa cells. The
first morphological sign that a follicle and its oocyte have entered the growth phase is a transition of the granulosa from a squamous to cuboidal
morphology. As the oocyte grows, the cuboidal granulosa cells proliferate so that they continue to cover the surface of the oocyte. Continued
proliferation of the granulosa cells generates a second layer, defining the follicle as secondary. Thecal cells are recruited around the exterior of the
follicle and are separated from the granulosa cells by a basement membrane. As the follicle continues to grow, a fluid-filled cavity termed the
antrum appears. This divides the granulosa into mural and cumulus subpopulations, which express different genes and follow different fates. Even
though the follicle will continue to increase in size, growth of the oocyte slows or stops at the antral stage. This transition is accompanied by
transcriptional arrest and a change in the degree of condensation and spatial arrangement of the DNA in the oocyte nucleus, from a
nonsurrounded nucleolus (NSN) to a surrounded nucleolus (SN) configuration, as shown in the oocyte in the preovulatory follicle. The fully grown
oocyte contains, in addition to actively translated mRNAs, products including stored translationally inactive mRNAs and mitochondria that are
required during meiotic maturation or early embryogenesis. (b) The nuclear events of meiotic maturation are highlighted. The membrane
surrounding the nucleus (germinal vesicle) breaks down and the chromosomes condense and align on the spindle, which becomes translocated to
the periphery of the oocytes. The first meiotic division segregates homologous chromosomes and one set is discarded in the first polar body. The
chromosomes remaining in the oocyte then align on a second spindle. Meiosis becomes arrested at metaphase II until fertilization activates the
egg. During maturation, the cumulus granulosa cells secrete a matrix that separates them from each other and from the oocyte and physical
contact between the two cell types is terminated. (Modified with permission from Ref 3. Copyright 2016 Springer International)
however, it is important to highlight a potential express mainly N-cadherin, and other components of
impediment to contact and communicate between the junctional complexes such as nectins are also detecta-
oocyte and granulosa cells and how this is bypassed. ble.33 During its growth, however, the oocyte pro-
duces an extracellular coat termed the zona
pellucida.34–36 Composed of three or four glycopro-
TRANSZONAL PROJECTIONS— teins depending on the species, the zona pellucida is
BRIDGES FOR INTERECELLULAR initially assembled in small aggregates or chunks.
These subsequently become knit together to form a
SIGNALING continuous envelope around the oocyte that separates
Within a primordial follicle, the oocyte and granulosa it from the bodies of the adjacent granulosa cells. The
cells are directly apposed to each other, and adherens zona pellucida thickens as the oocyte grows, reaching
and gap junctions link the two cell types. Oocytes a final size of about 7 μm in mice and 15 μm in
express mainly E-cadherin whereas granulosa cells humans. In view of the formidable physical barrier

(a) (b) (c) (d) (e)
Granulosa
Granulosa
Oocyte
TZP TZP
zp
Cumulus Oocyte zp
granulosa
Oocyte
Adherens junction Gap junction

Growth factor
(f) Growth factors
Receptors
Stretching
1 2 3 4 Actin filaments
Newborn
Granulosa granulosa
cells
Zona
pellucida
Oocyte
Pushing
1 2 3 4
F I G U R E 2 | Transzonal projections (TZPs). (a) A dense forest of TZPs, indicated by the arrow, projects from the cumulus granulosa cells to the
oocyte. Phalloidin (green) stains their actin-rich cytoskeleton as well as the cortex of the oocyte. Nuclei are stained using DAPI (blue).
Bar = 25 μm. (b) Higher-power magnification shows that several TZPs often appear to emerge from a single locus (arrows). (c) Electron-
micrograph showing an oocyte at an early stage of growth, as indicated by the thin zona pellucida (zp), and an incipient TZP (arrow) projecting
from a granulosa cell. Bar = 500 nm. (d) Electron-micrograph showing an oocyte at a later stage of growth and several narrow and elongated
TZPs (one marked by arrow). Bar = 500 nm. (e) Cartoon depicting interactions between TZP tips and oocyte plasma membrane. The TZPs are
anchored by adherens and possibly other types of junctions. Gap junctions permit the passage of molecules up to 1 kDa. The TZP–oocyte interface
is presumably also the site of contact between membrane-associated growth factors and their membrane-associated receptors. (f ) Two
nonexclusive models of how TZPs may form: Stretching model (upper panel). (1) Prior to deposition of the zona pellucida, the plasma membranes
of the oocyte (pink) and adjacent granulosa cells (light brown) are in physical contact at numerous sites (two shown here per granulosa for
simplicity). (2) Deposition of the zona pellucida (light blue) pushes the bodies of the granulosa cells away from the oocyte, but the cells remain
connected at the original points of contact. (3) As the oocyte continues to grow and the zona pellucida thickens, the cytoplasmic strands of the
granulosa cell elongate to produce TZPs. New granulosa cells (dark brown) are born to enable a continuous layer to be maintained around the
expanding oocyte surface. (4) Because granulosa cells born after the deposition of the zona pellucida have never been in direct contact with the
oocyte, they do not generate TZPs. Hence, the number of TZPs does not increase as oocytes grow. Pushing model (lower panel). (1, 2) Deposition
of the zona pellucida prevents physical contact of the oocyte and surrounding granulosa cells. (3) Granulosa cells elaborate filopodia-like structures
that extend towards the oocyte. New granulosa cells (dark brown) are generated as the oocyte surface expands. (4) The newborn granulosa cells
elaborate new TZPs. Hence, the number of TZPs increases as oocytes grow.
that the zona pellucida imposes between the oocyte diameter and morphologically resemble filopodia,
and granulosa cells, how do the two cell types main- originate from the granulosa cells and extend to the
tain contact and communication? oocyte where they contact the plasma membrane
Contact between the granulosa cells and oocyte (Figure 2(c) and (d)).29,37 TZPs that project from the
is mediated by structures termed transzonal projec- cumulus granulosa cells in the layer immediately
tions (TZPs) (Figure 2(a) and (b)). These thin cyto- adjacent to the zona pellucida are easiest to trace
plasmic filaments, which may be up to about 1 μm in morphologically or using markers29,38,39 and likely

constitute the bulk of the population. However, there parallel filaments along the length of the TZPs, sug-
is no a priori reason to suppose that TZPs do not gesting that cytoskeletal elements provide a structural
project from more distal granulosa cells also.40 Elec- backbone.38 Most TZPs appear to possess an actin-
tron micrographs reveal that the foot of the TZP is rich cytoskeleton; thus phalloidin, which binds to
frequently enlarged and cradled by exvaginations of and stabilizes polymerized actin, is routinely used to
the oocyte plasma membrane.39,41 TZPs have also mark the TZPs that project to an oocyte.29,41,46,51,52
been observed to penetrate deeply into invaginations The actin cytoskeleton further reinforces the similar-
of the oocyte membrane.42 These structural features ity between TZPs and filopodia.53 TZPs containing a
increase the area of membrane contact between the tubulin backbone have also been detected. These
two cell types and potentially facilitate increased appear to be less abundant than the actin-rich TZPs;
communication. The density of the TZPs surround- however, the relative proportions of the two have
ing a fully grown oocyte (Figure 2(a)) suggests that not been reported. Nor is it known whether both
many can emanate from a single granulosa cell,42 actin- and tubulin-rich TZPs may project from an
and it appears that several TZPs may emerge from a individual granulosa cell. It has been proposed that
single point of origin or node (Figure 2(b)). TZPs the tubulin-rich TZPs mediate cell adhesion whereas
were first described more than a century ago43 and the actin-rich TZPs mediate cell communication.29
physically couple the granulosa cells to the oocyte in There is not yet direct evidence to support this attrac-
all mammals studied including humans.38,39,44–47 tive model, and the impaired gap junctional commu-
Analogous structures have also been identified in a nication in oocyte-specific knockout of Ptk251
wide range of nonmammalian species including frog, implies that adherens-type junctions are required to
chicken, and starfish.48–50 Thus, the principle that stably attach TZPs containing gap junctions to the
filopodia-like structures mediate contact between the oocyte.
somatic compartment of the follicle and the oocyte The origin of the TZPs remains mysterious. At
appears to be highly evolutionarily conserved. least two nonexclusive mechanisms may be envisioned
As the sole vehicle of contact between the gran- (Figure 2(f )). TZPs may represent sites of attachment
ulosa cells and the oocyte, TZPs serve two key func- that exist between the oocyte and adjacent granulosa
tions (Figure 2(e)). First, they enable contact- cells before the zona pellucida becomes assembled
dependent communication between the cells. The tips around the oocyte.36,43 According to the ‘stretching’
of the TZPs (though not necessarily all TZPs) harbor model, as the zona pellucida is deposited and pushes
gap junctions and possibly also membrane-associated the bodies of the granulosa cells away from the
growth factors.29,37 As discussed below, oocyte oocyte, the two cells remain attached at the original
development depends crucially on communication points of contact (see Figure 2(c)). The subsequent
mediated through these pathways. Second, they are thickening of the zona pellucida elongates this cyto-
essential to maintain adhesion between the two cell plasmic finger, thereby generating the TZPs. Alterna-
types, which is probably necessary to keep the tively, TZPs may arise after the zona pellucida has
granulosa–oocyte complex (GOC) intact. This func- been deposited. According to the ‘pushing’ model,
tion is likely mediated at least in part by adherens TZPs are elaborated by the granulosa cells and grow
junctions that have been identified at the points of towards the oocyte. This model is consistent with the
membrane contact.33 Experimental support for the idea that TZPs are specialized filopodia that are gener-
role of adherens junctions was recently obtained. ated by the granulosa cells, possibly in response to sig-
Proline-rich tyrosine kinase (PTK) 2 regulates the nals sent by the oocyte.
assembly of adherens junctions and has been immu- No direct proof yet supports either model. Sev-
nologically localized in foci at the plasma membrane eral lines of evidence nonetheless indicate that as
of oocytes.51 Although it is not known whether these oocytes grow, new TZPs are generated by the granu-
foci are located where the TZP tips meet the oocyte losa cells. First, gap junctional communication
plasma membrane, oocyte-specific deletion of Ptk2 between the oocyte and adjacent granulosa cells
reduces the number of TZPs by about one-third. increases during oocyte growth.54,55 A simple expla-
Moreover, this decrease in the number of TZPs in nation for this observation is that as oocytes grow,
the absence of PTK2 in the oocyte is accompanied by the number of TZPs increases in parallel with the
a decrease in gap junctional coupling.51 Thus, the increase in the number of granulosa cells adjacent to
cell-communication and cell-adhesion functions of the oocyte. Alternatively, the gap junctional plaques
the TZPs are tightly coupled. on existing TZPs could become larger thereby
Two types of TZPs have been identified. Early enabling increased coupling. Second, the number of
studies using electron microscopy revealed multiple TZPs was reported to increase in parallel with oocyte

growth in an electron-microscopic study of human the expanding surface of the growing oocyte. It also
follicular development.42 Third, when cryopreserved suggests that the granulosa cells may be the source of
follicles were subsequently grown in culture, the the signal that initiates oocyte growth.
number of TZPs increased, as assessed by an increase Indeed, multiple lines of evidence suggest that
in the birefringence of the zona pellucida.52 These granulosa cells trigger oocyte growth by activating
results favor the model that the granulosa cells sur- phosphatidylinositide 3-kinase (PI3-kinase) signaling
rounding growing oocytes generate new TZPs. Pre- in the germ cell (Figure 3). When Pten, whose activ-
cise quantification of the number of TZPs associated ity inhibits the pathway, is deleted from oocytes
with oocytes at different stages of growth and analy- within primordial follicles, most begin to grow
sis of TZP dynamics during growth in vitro should shortly after birth.58,59 This is accompanied by an
clarify the mechanism by which they are generated. increase in phosphorylation of protein kinase B (also
If the granulosa cells surrounding growing known as AKT), suggesting that PI3-kinase signaling
oocytes generate new TZPs, many questions about has been activated. Similarly, deletion of Tsc1 or
this process remain to be answered. What signals Tsc2, which also inhibit PI3-kinase signaling, in
induce their formation and why do they grow oocytes of primordial follicles also causes most
towards the oocyte? Intriguingly, in mice lacking oocytes to begin to grow.60,61 Tsc2 deletion was
growth-differentiation factor (GDF) 9, a growth fac- associated with an increase in phosphorylation in
tor produced by the oocyte, the number of TZPs is ribosomal protein S6, as predicted; curiously, how-
reduced and they frequently lie parallel rather than ever, this was not observed when Tsc1 was deleted.
perpendicular to the oocyte surface.56 These results Importantly, in all three cases, the granulosa cells
hint at a role for the oocyte in promoting TZP for- often did not assume the cuboidal morphology char-
mation. What role is played by gonadotropins, which acteristic of primary and later-stage follicles, but
regulate the differentiation and function of the granu- instead retained the squamous morphology of pri-
losa cells? A larger number of TZPs are associated mordial follicles. This is strong evidence that activat-
with the oocytes of mice lacking Fshb, encoding the ing PI3-kinase signaling within the oocyte is
essential β-subunit of FSH, than with wild-type sufficient to initiate its growth, even when the gran-
oocytes.57 Moreover, when either wild-type or ulosa cells of the follicle remain in the primordial
Fshb−/− mice were ‘primed’ by injection of an FSH state. Two inferences may be drawn from these
analog prior to collection of cumulus cell–oocyte observations. First, PI3-kinase signaling promotes
complexes, the density of TZPs was reduced. These oocyte growth directly, rather than indirectly such
results suggest that FSH antagonizes the formation of as by causing the oocyte to send a signal to the
TZPs. Identifying the molecular pathways that link granulosa cells that induces them to send a growth-
FSH to TZPs and how the reduced number of TZPs promoting signal to the oocyte. Second, oocyte
affects the developmental progression of the oocyte growth per se does not trigger granulosa cell prolif-
are important issues for future work to address. eration, although factors produced by growing
oocytes might play a role.
These experiments convincingly show that acti-
CELL SIGNALING AT THE INITIATION vating PI3-kinase signaling in the oocyte can cause it
to start growing, but is it the physiological mechan-
OF OOCYTE GROWTH
ism? And if so, how does it become activated under
Shortly after birth in the mouse, a cohort of primor- normal conditions? Kit ligand (KITL) is constitutively
dial follicles and their enclosed oocytes enter the expressed by the granulosa cells62,63 and interacts
growth phase while the remaining follicles remain at with the Kit receptor (KIT) expressed by oocytes.64,65
the primordial follicle stage. Although the oocytes of This ligand–receptor pair has long been suspected to
this cohort are not subsequently ovulated, presuma- play a key role in multiple aspects of germ cell devel-
bly because the female is not yet sexually mature, opment. Because KIT signaling is required for prena-
they are an invaluable resource to dissect the signal- tal development of female germ cells,66 mutants
ing pathways that control entry into the growth typically possess very few oocytes at birth, making it
phase. The first morphological sign that an oocyte challenging to study potential postnatal functions.
has entered the growth phase is a morphological The advent of the Cre-lox strategy, enabling genes to
remodeling of the granulosa cells from a squamous be modified in specific cells at specific stages of differ-
to cuboidal shape. This may reflect (re-)entry of the entiation, has been an especially valuable boon to
granulosa cells into the mitotic cell cycle, as they untangling the multiple roles of KIT signaling during
must proliferate in order to continue to fully cover oocyte development.

(a)
PTEN
Growth PDK1 AKT1

factor PIP2 PIP3
PI3
Receptor
kinase
tyrosine TSC1/2
kinase
MTOR
RPS6KB1 EIF4EBP1
Protein synthesis
(b) ? ?
MTOR MTOR
Protein Protein
synthesis synthesis
KITL
P
PI3K AKT PI3K AKT PI3K AKT
TSC1/2 TSC1/2 TSC1/2
MTOR MTOR MTOR
Protein Protein Protein

synthesis synthesis synthesis
FI GU RE 3 | Initiation of oocyte and follicular growth. (a) Schematic representation of the canonical phosphatidylinositide 3-kinase (PI3-kinase)
signaling pathway. (b) Possible signaling pathway that activates oocyte growth. Oocytes in primordial follicles are enclosed by squamous
granulosa cells. Unknown signals trigger an increase in protein synthesis in the granulosa cells and a transition to a cuboidal morphology. This
may reflect entry into the mitotic cell cycle. The granulosa cells may increase production of Kit ligand (KITL); another possibility is that synthesis of
the more bioactive membrane-bound form becomes favored. The consequent activation of KIT signaling within the oocyte increases protein
synthesis, thus driving an increase in cell size.
A constitutively active form of KIT (D318V), to grow. This strongly suggest that the granulosa
corresponding to the most common activating muta- cells generated the growth-promoting signal, but the
tion in human germ cell tumors, was engineered and KIT-deficient oocytes were unable to respond.
targeted to oocytes using a Vasa-driven Cre.67 Strik- Complementary experiments targeting the gran-
ingly, almost all oocytes of primordial follicles began ulosa cells support this model. When Tsc1 was deleted
to grow shortly after birth. Phosphorylated AKT from the granulosa cells, thereby activating PI3-kinase
could be detected in these oocytes, confirming that signaling in these cells, KITL expression in the granu-
PI3-kinase signaling had been activated. The sur- losa cells was increased and most oocytes began to
rounding granulosa cells in most cases remained grow.68 As would be expected given that they are the
squamous, however, indicating that the somatic com- source of the growth-promoting signal, the granulosa
partment of the follicle remained in a primordial cells of the growing follicles became cuboidal. Con-
state. Perhaps more crucial are experiments in which versely, deletion of Rptor in the granulosa cells, which
KIT signaling in oocytes was ablated, by generating a would decrease protein synthesis, prevented the
mutant that lacks the exon encoding the kinase squamous-cuboidal transition in these cells as well as
domain.67 Even though granulosa cells of primordial the initiation of oocyte growth.68 Taken together,
follicles became cuboidal, the enclosed oocytes failed these experiments suggest that KITL produced by the

granulosa cells in the primordial follicle triggers Gap Junctions

growth of the enclosed oocyte (Figure 3). The best understood function of the granulosa cells is
These findings also raise many questions for fur- to transfer essential nutrients and signals to the oocyte
ther study. Differential splicing of its mRNA generates via the gap junctions that link the two cell types.65,76
two isoforms of KITL—a soluble form and a Gap junctions are intercellular channels composed of
membrane-associated form that has a higher bioactiv- transmembrane proteins termed connexins that per-
ity. Although increased production of KITL may be the mit the exchange of molecules up to about 1 kDa
trigger that initiates growth, it is also plausible that a between the coupled cells.77,78 Oligomers of six con-
switch from soluble to membrane-associated forms nexins form hemi-channels and the docking of hemi-
plays a role. The source and the nature of the signal channels of adjacent cells generates the gap junction.
that increases protein synthesis in the granulosa cells of Many individual gap junctions may cluster in one
one primordial follicle but not those of its neighbor region of the plasma membrane, generating a struc-
remains unknown. A clue may lie in observations that, ture termed a plaque. Mammalian connexins are
when ovary-like structures have been generated by encoded by approximately 20 genes, and the
aggregating germ cells and granulosa cells, many of the subtype(s) of connexin present in a gap junction can
follicles in these reconstituted ovaries rapidly initiate affect its properties and thus the type or efficiency of
growth.69,70 This strongly suggests that the regulated intercellular communication that it supports.
entry of primordial follicles into the growth pool Although many gap junctions contain only a single
depends on cellular interactions in the intact ovary that connexin subtype, there also exist heterotypic junc-
are not recapitulated in the de novo-generated struc- tions where the two hemi-channels contain different
tures. Finally, the transcription factor, FOXO3, translo- connexins and heteromeric hemi-channels where the
cates from the nucleus to the cytoplasm at an early hexamer contains different connexins. Not all con-
stage of growth and genetic deletion of Foxo3 cause nexins can interact to form heteromers or heterotypic
most oocytes in primordial follicles to begin to junctions, but the potential combination of different
grow.59,71 Similarly, deletion of the oocyte-specific tran- connexins within one gap junction may permit addi-
scription factor Sohlh2 or oocyte-specific deletion of tional diversity in their signaling properties.
transcription factor Lhx8 also trigger initiation of Both oocytes and granulosa cells express numer-
oocyte growth, apparently independently of signals ous connexin genes; however, GJA4 (connexin-37) is
from the granulosa cells.72–74 These results indicate that the principal essential connexin identified in oocytes,
PI3-kinase signaling in the oocyte somehow decreases whereas GJA1 (connexin-43) predominates in the
the activity these transcription factors, but the nature of granulosa cells.79–82 Deletion of either gene severely
the link remains to be defined. Alternatively, it may be compromises oocyte development. In mice lacking
that oocyte growth can be triggered through independ- Gja4, no gap junctional coupling is detectable
ent pathways. between the oocyte and granulosa cells.82 The oocytes
grow to only about half the volume of wild-type
oocytes and they fail to acquire a property termed
CELL SIGNALING DURING OOCYTE meiotic competence, defined as the ability to undergo
GROWTH meiotic maturation when removed from the follicle
and incubated in vitro.83,84 When wild-type oocytes
Signals from the granulosa cells play an essential role are enclosed by Gja4−/− granulosa cells, however, they
not only in initiating oocyte growth but also in sustain- grow apparently normally.84 Thus, GJA4 is essential
ing it. This was clearly demonstrated when GOCs were in oocytes but not in granulosa cells for gap junctional
isolated from ovarian follicles and cultured intact or fol- coupling between the two cell types and for normal
lowing physical separation of the two cell types.75 oocyte development. Intriguingly, the granulosa cells
Oocytes within intact GOCs continued to grow, of Gja4−/− mice become luteinized and begin to secrete
whereas oocytes not in direct contact with the granu- progesterone,82 a process that normally occurs only
losa cells grew little or not at all, even when the granu- after ovulation. This suggests that oocyte–granulosa
losa cells were provided in co-culture. Subsequent cell coupling is also required to maintain normal
studies showed that the rate of growth of an oocyte granulosa cell physiology.
was proportional to the number of granulosa cells that In mice lacking Gja1, granulosa–granulosa cell
were present in the GOC containing that oocyte.54 coupling is severely reduced and the granulosa cells
These results highlight the indispensable role played by are compromised as shown by their failure to gener-
the granulosa cells as well as the crucial importance of ate the normal multilaminar structure around the
physical contact between the two cell types. growing oocyte.81 Even though coupling between the

oocyte and adjacent granulosa cell is retained, the the granulosa cell–oocyte gap junctions also remains
oocyte does not develop normally. They reach less unknown. However, wild-type oocytes develop
than half the volume of wild-type oocytes, generate apparently normally when reaggregated with granu-
only a thin zona pellucida, lack specific ultrastruc- losa cells that lack Gja4,84 as do oocytes in mice that
tural features including fibrous lattices and cortical express Gja1 under the control of the Gja4 pro-
granules, and do not acquire meiotic compe- moter.86 These results suggest that GJA4 does not
tence.81,84 When Gja1−/− oocytes are enclosed by confer an essential property to the gap junctions link-
wild-type granulosa cells, however, they grow appar- ing the granulosa cells to the oocyte.
ently normally.84 These results indicate that gap junc- What do the granulosa cells provide to the
tional communication mediated through GJA1 growing oocyte via the gap junctions (Figure 4)?
between the granulosa cells is required for normal Oocytes are unable to efficiently utilize glucose as an
oocyte development, with the caveat that such energy source,87 probably because several key
experiments cannot rule out an essential GJA1 func- enzymes in the glycolytic pathway are expressed at
tion not related to gap junctions. In any case, these relatively low levels.88 These enzymes are present at
results illustrate the general principle that, when the a much higher concentration, however, in cumulus
normal physiology of the granulosa cells is disrupted, granulosa cells (and presumably the granulosa cells
oocytes cannot develop normally. of preantral follicles). Thus, the granulosa cells are
A point that remains unresolved is the identity able to metabolize glucose to pyruvate, which could
of the granulosa cell connexin that establishes gap then be transmitted via gap junctions to the oocyte,
junctions with the oocyte. Immunofluorescence using where it serves as an energy substrate. Similarly,
subtype-specific antibodies reveal GJA1 at the inter- oocytes are unable to efficiently take up certain
face between granulosa cells as expected, but not amino acids—for example, they lack Slc38a3, encod-
between the granulosa cells and the oocyte,85 and ing an alanine transporter—and rely on the granu-
gap junctional communication with the oocyte is losa cells to provide them via gap junctions.89 The
retained when Gja1 is deleted from granulosa cells.81 granulosa cells of preantral follicles also provide a
These observations suggest that, whereas granulosa function via gap junctions that enables the oocyte to
cells use GJA1 to assemble gap junctions with other regulate its pH in response to environmental stress.90
granulosa cells, they use a different connexin, likely The granulosa cells may also provide cholesterol to
GJA4, to assemble those with the oocyte. This sug- the oocytes, which express the biosynthetic enzymes
gests that GJA1 and GJA4 are selectively trafficked at relatively low levels.91 Cholesterol presumably is
to different locations within the granulosa cells; alter- not transported through gap junctions but might be
natively, homotypic junctions may be formed or transferred at the sites where the plasma membranes
maintained more efficiently than heterotypic junc- of the TZPs and oocyte are sufficiently closely
tions. Whether GJA4 confers a particular function to apposed. Conceptually, the extensive gap junctional
(a) (b)
KIT
Pyruvate
nucleotides KITL
amino acids
GDF9
Cholesterol BMP15
FGF8B
??
FI GU RE 4 | Communication between the growing oocyte and adjacent granulosa cells. (a) Gap junctions enable the granulosa cells to transfer
pyruvate, nucleotides, and amino acids to the oocyte. The granulosa cells also provide the oocyte with cholesterol, which might be transferred
between cells where the plasma membranes lie in close apposition. It is unknown whether the oocyte supplies essential factors to the granulosa
cells via gap junctions. (b) Granulosa cells produce Kit ligand (KITL) whereas oocytes express the KIT membrane receptor. The membrane-
associated form of KITL promotes oocyte growth more efficiently than soluble KITL. Oocytes secrete the transforming growth factor (TGFβ) family
members, GDF9 and BMP15, as well as FGF8B, which activate receptors on the granulosa cell plasma membranes.

coupling between the oocyte and granulosa cells, as many years ago by the observation that when the
well as between the granulosa cells themselves, may oocyte was removed from the follicle, the granulosa
be considered to be a mechanism that, by generating cells rapidly underwent luteinization, which normally
an enormous syncytial-like structure, greatly occurs only after ovulation has expelled the oocyte
increases the effective plasma membrane surface area from the follicle.99 This probably reflects at least in
that the oocyte can exploit to obtain essential nutri- part the activity of the oocyte to suppress expression
ents from extracellular sources.92 in the granulosa cells of the Lhcgr gene encoding the
LH receptor.100 Specific signals sent by the oocyte to
the granulosa cells via gap junctions have not yet
Growth Factors been identified. Factors secreted by the oocyte do,
Oocytes that are physically separated from the granu- however, regulate the differentiation of the granulosa
losa cells grow very little or not at all, even when the cells and even the thecal calls.21,87,101
two cell types are co-cultured. In contrast, although GDF9 and bone morphogenetic protein (BMP)
oocytes lacking Gja1 lack detectable gap junctional 15 are closely related members of the transforming
communication with the granulosa cells, they reach a growth factor (TGF) β superfamily. Like other family
final diameter of ~50 μm, representing a more than members, GDF9 and BMP15 are secreted as dimeric
50-fold increase in volume.83,84 This suggests that not pro-peptides, and removal of the inhibitory N-
only gap junctional communication but also other cell terminal pro-domains by membrane-associated furin-
contact-dependent signals from the granulosa cells like proteases generates the mature biologically active
promote oocyte growth. These putative signals have form.102,103 The dimeric pro-peptides may also be
not been identified; however, several lines of evidence active.103 The receptor for each is a heteromeric com-
implicate KITL. The granulosa cells of preantral folli- plex comprising type I and type II serine–threonine
cle, which contain growing oocytes, express both kinases. Ligand binding to the BMPR2 type II recep-
Kitl1 and Kitl2.93–95 Addition of soluble KITL to cul- tor triggers recruitment and phosphorylation of a type
ture medium promoted an increase in the rate of I receptor, which in turn phosphorylates SMAD2/3
growth of granulosa cell-free oocytes.96,97 Addition- (GDF9) or SMAD1/5/8 (BMP15). The phosphory-
ally, when oocytes from which the zona pellucida had lated forms associate with the so-called common
been removed were incubated on monolayers of fibro- SMAD4, and the heterodimeric SMAD is translocated
blasts expressing the membrane-bound KITL2, their to the nucleus where it can regulate transcription of
growth was robustly stimulated and this effect was target genes. GDF9 and BMP15 can also form hetero-
suppressed by the addition of a function-blocking dimers whose signaling activity is much greater than
KIT antibody or inhibitors of PI3-kinase signaling.97 that of the homodimers.102,103 However, the relative
It is worth noting that only a small region of the proportions of homomeric and heterodimeric forms
spherical oocyte would have been in physical contact in vivo has not been reported.
with the KITL-expressing fibroblasts. Deletion of Kit In situ hybridization and immunohistochemical
from growing oocytes, which could be achieved using studies indicate that the oocyte is the major source of
the Zp3-Cre mouse, would directly test a role for KIT both GDF9 and BMP15.104–106 The mRNAs for both
signaling during oocyte growth. Finally, it is intri- factors increase when oocytes begin to grow and
guing that expression of both Kitl1 and Kitl2 are sub- GDF9 protein is detectable in oocytes of primary folli-
stantially decreased in the granulosa cells of antral cles and all subsequent stages of folliculogenesis. In
follicles, whose oocytes have reached full size, and the mouse, BMP15 may not be produced in large
fully grown but not growing oocytes are able to sup- amounts until near the time of ovulation, possibly due
press Kitl1 and Kitl2 expression.94,95,98 These results to KITL-mediated inhibition of production.107 Some
imply that there is an additional pathway by which studies have reported expression of Gdf9 and Bmp15
fully grown oocytes are able to suppress growth- in granulosa cells as well.108 Female mice lacking
promoting signals from the granulosa cells. Gdf9 are anovulatory and sterile.109 The granulosa
cells fail to proliferate normally and do not generate
more than a single layer around the growing oocyte.
Signaling From the Oocyte to the Granulosa This proliferative defect is due to increased produc-
Cells tion of inhibin in the Gdf9−/− females, because the
The developing oocyte not only receives and granulosa cells of Gdf9−/−; Inha−/− females proliferate
responds to signals from the surrounding cells, it also apparently normally.110,111
sends signals that regulate differentiation of the The follicles of Gdf9−/− females also fail to
somatic compartment of the follicle. This was hinted acquire a thecal layer.21,109,112 These cells arise from

a progenitor cell population in the ovarian mesen- glucose to generate pyruvate that they transfer to the
chyme and can be identified by the expression of oocyte. Removing the oocyte from the cumulus-
Gli1, encoding a transcription factor.21 Ligands of oocyte complex (COC), in a procedure termed oocy-
the Hedgehog pathway secreted by the granulosa tectomy (OOX), leads to a decrease both in the
cells induce expression of Gli1 in the thecal cell pre- amount of mRNAs encoding key enzymes in the gly-
cursors and GDF9 promotes production of these colytic pathway and in the rate of glycolysis in the
ligands. Thus, GDF9 directs differentiation not only remaining cumulus-cell shell.88,116 Importantly, incu-
of the granulosa cells but also, albeit indirectly, of bating the OOX shells with fully grown oocytes
the thecal cells. It may be noted that this effect of restores both the quantities of the affected mRNAs
GDF9 is manifested in ovaries near the time of birth. and glycolytic activity. These mRNAs and glycolytic
As ovaries at this stage would not contain growing activity are also reduced in the cumulus granulosa
follicles, this result indicates that nongrowing oocytes cells of Bmp15−/−; Gdf9+/− mice, and incubating
also produce GDF9. OOX shells in the presence of BMP15 and fibroblas-
The oocytes of Gdf9−/− females are able to tic growth factor (FGF) 8B restores mRNA levels and
grow and, possibly due to a 30-fold increase in KITL glycolysis.116 Thus, ODPFs stimulate the granulosa
production by the GDF9-deprived granulosa cells to manufacture the pyruvate that they transfer
cells,111,112 reach a final size significantly larger than to the oocyte.
wild-type oocytes. Nonetheless, these oocytes are Similarly, in both Bmp15−/−; Gdf9+/− mice and
abnormal. In prepuberal mice, a large fraction of the OOX shells of wild-type mice, the quantities of
growing oocytes fail to achieve meiotic competence mRNAs encoding enzymes required for cholesterol
and oocytes of both prepuberal and adult mice show synthesis are reduced in the cumulus granulosa cells
specific ultrastructural abnormalities, including an and cholesterol synthesis is impaired, and these
apparently disorganized cytoskeleton.56,109 These effects can be rescued fully and partially, respectively,
abnormalities cannot be attributed to an autocrine by co-culture of the OOX shells with fully grown
effect of GDF9 on the oocyte, because deletion of oocytes.91 Similarly, expression of the amino acid
Smad4 within the oocyte has no phenotypic effect.113 transporter, Slc38a3, and uptake of alanine by the
Rather, it appears that the GDF9-deprived granulosa cumulus granulosa is reduced in OOX shells and
cells fail to interact appropriately with the oocyte. In these defects can be rescued by co-incubation of the
support of this, the TZPs of Gdf9−/− mice are reduced cumulus granulosa cells with the fully grown
in number and are frequently oriented parallel rather oocytes.89 Thus, the oocyte produces and secretes
than perpendicular to the oocyte surface. In view of factors that stimulate the neighboring granulosa cells
the rescue of granulosa cell proliferation defect in to produce molecular nutrients that they in turn pro-
Gdf9−/−; Inha−/− mice, it would be interesting to test vide to the oocyte.
whether the TZPs of these individuals recover their
normal orientation and whether their oocytes
develop normally. CELL SIGNALING DURING MEIOTIC
Bmp15−/− female mice show a mild impairment
of fertility, which is exacerbated in the Bmp15−/−;
MATURATION
Gdf9+/− females, suggesting that SMAD2/3- As discussed above, oocytes of primordial follicles
dependent signaling may be dominant in this spe- are arrested at prophase I of meiosis and they remain
cies.114,115 Sheep homozygous for inactivating muta- so until they have completed growth and the preovu-
tions of either gene are infertile. In contrast, latory LH surge triggers meiotic maturation. When
heterozygous females carrying one mutant allele of oocytes at mid-growth phase are removed from the
either gene show enhanced fecundity, which may be follicle and placed in culture, however, they will
linked to a change in the relative proportion of undergo maturation in the absence of LH. This result
homo- and heterodimeric forms.103 Mutations in indicates that some property of the follicular environ-
these genes have also been linked to infertility in ment actively prevents these meiotically competent
humans.108 Thus, the functions of GDF9 and oocytes from undergoing maturation until the appro-
BMP15 during follicular development are evolution- priate LH-mediated signal is received. This inhibitory
arily conserved. action requires contact between the cumulus and
Remarkably, the oocyte-derived paracrine fac- mural granulosa cells, because when the COC was
tors (ODPFs), through their effects on the granulosa microsurgically detached from the mural granulosa
cells, also promote development of the oocyte itself. cells within an intact antral follicle, the oocyte under-
As discussed above, the granulosa cells metabolize went maturation.117 Blocking gap junctional activity

F I G U R E 5 | Regulation of meiotic maturation. (a) Before the activation of the luteinizing hormone (LH) receptor (LHCGR), the mural granulosa
cells produce and release NPPC, which activates its receptor, NPR2, located on both mural and cumulus granulosa cells. Active NPR2 generates
cGMP, which diffuses through gap junctions and reaches a concentration in the oocyte that is sufficient to inhibit phosphodiesterase 3A (PDE3A).
This allows the concentration of cAMP, produced by the oocyte, to remain high thereby maintaining CDK1 in a hyper-phosphorylated inactive
form. (b) Binding of LH to LHCGR triggers the release of ligands from the mural granulosa cells that activate EGFR on the mural and cumulus
granulosa cells. cGMP levels within the granulosa cells fall due both to inhibition of its synthesis owing to dephosphorylation of NPR2 and (later)
reduced production of NPPC and to increased hydrolysis owing to activation by phosphorylation of PDE5A in the granulosa cells. The relative
contribution of LHCGR- and EGFR-mediated signaling to these events remains to be fully elucidated. As the concentration of cGMP within the
granulosa cells falls, it flows out of the oocyte to equalize its concentration throughout the granulosa cell–oocyte compartment. The decreased
cGMP in the oocyte permits PDE3A to become active and hydrolyze cAMP, enabling dephosphorylation and activation of CDK1. (Modified with
permission from Ref 3. Copyright 2016 Springer International)
pharmacologically or by injecting peptides or antibo- these experiments do not entirely rule out a role for
dies targeting either GJA1 or GJA4 into the antrum cAMP provided by the granulosa cells, it appears
also induces maturation of follicle-enclosed that the key inhibitory molecule provided by these
oocytes.118–120 These results imply that the cells is cyclic guanosine monophosphate (cGMP).
maturation-inhibiting signal requires the mural gran- This inhibits the cAMP-specific phosphodiesterase
ulosa cells and gap junctional communication to (PDE) 3A within the oocyte and thus maintains a
reach the oocyte. high concentration of cAMP.121
Maturation is initiated by a drop in cyclic aden- cGMP is synthesized by the membrane-
osine monophosphate (cAMP) within the oocyte, associated guanylyl cyclase natriuretic peptide recep-
which enables the dephosphorylation and activation tor (NPR2). NPR2 is not detectable in the oocyte,
of cyclin-dependent kinase (CDK) 1 that drives entry but is abundantly expressed in the cumulus granulosa
into M-phase25 (Figure 5). A plausible early hypothe- cells and the mural granulosa cells adjacent to the
sis was that the gap junctions allowed cAMP to pass antrum.123 Consistent with this pattern of expres-
from the granulosa cells to the oocyte. Subsequent sion, GDF9 alone or in combination with BMP15 sti-
studies showed that cAMP, which is generated by the mulates the expression of Npr2.123 Moreover, these
adenylyl cyclase activity of a GPR3-activated Gs-pro- ODPFs also increase expression in the cumulus gran-
tein, produced by the oocyte itself is required to ulosa cells of inosine monophosphate dehydrogenase,
maintain high intracellular cAMP.121,122 Although which is required to generate the substrate for cGMP

synthesis. Experiments using fluorescent cGMP sen- of the specific pathways involved, gap junctions play
sors have shown that the concentration of cGMP is a crucial role in the rapid decrease in cGMP. When
uniform throughout the oocyte–granulosa cell com- gap junctions are pharmacologically inhibited, addi-
partment in antral follicles.124 As the mural granu- tion of LH provokes a rapid fall in cGMP in the
losa cells are located far from the oocyte, this mural granulosa cells but a much slower decline in
highlights the efficiency with which gap junctions can the cumulus cells.124 This suggests that gap junctions
permit cGMP to be delivered from remote sites of permit a rapid flow of cGMP from the cumulus gran-
synthesis to the oocyte. ulosa cells and also from the oocyte outward to the
NPR2 is activated by the C-type natriuretic mural granulosa cells. Later during maturation, gap
peptide (NPPC or CNP). Nppc mRNA is detectable junctional communication is lost between the two
only in the mural granulosa cells in antral follicles.123 cell types. This may be due to phosphorylation-
It is released extracellularly and diffuses through the mediated changes in the properties of the gap junc-
follicular fluid to activate NPR2 receptors on both tions and due to displacement of the granulosa cells
mural and cumulus granulosa cells. The restriction of away from the oocyte during cumulus layer expan-
NPPC synthesis to the mural granulosa cells likely sion.131 Gap junctions thus mediate not only meiotic
explains why oocytes, even within COCs, undergo arrest but also meiotic resumption.
maturation when removed from the follicle. As the Recent work has uncovered an additional regu-
mural granulosa cells that supply the NPPC are no latory role for the cumulus granulosa cells during
longer present, synthesis of cGMP stops; when the maturation. It has long been known that a subset of
concentration of cGMP within the oocyte falls below mRNAs in the oocyte become translationally acti-
a threshold, PDE3A becomes activated. Consistent vated during maturation.30 Addition of amphiregulin
with this, addition of NPPC to the culture medium (or EGF) to cumulus-enclosed oocytes but not
inhibits maturation of cumulus-enclosed but not cumulus-free oocytes in vitro further boosts the
cumulus-free oocytes, and this inhibition requires gap translational activity of specific mRNAs.132 Moreo-
junctional communication.119,121,123,125 These results ver, oocytes of Areg−/− mice show a reduced ability
also indicate that, in vitro, the cumulus granulosa to develop as embryos, suggesting that this increased
cells can make sufficient cGMP to prevent oocyte translational activity is developmentally important.
maturation. As oocytes do not detectably express the EGF recep-
Although LH triggers meiotic maturation, its tor, these results indicate that its activation in the
receptors are not expressed by the oocyte or even by granulosa cells induces them to send a signal to the
the cumulus granulosa cells that surround it. Instead, oocyte that upregulates translation of specific
they are found on the mural granulosa cells (as well mRNAs. Although this signal remains to be identi-
as on the thecal cells). Thus, the signal must be fied, EGF-receptor activation in the cumulus granu-
relayed from the mural granulosa cells to the oocyte. losa cells triggers phosphorylation of AKT in the
The binding of LH to its mural granulosa cell recep- oocyte, and pharmacological inhibition of PI3-kinase
tors triggers the release of epidermal growth factor- signaling (which would affect signaling in both
related peptides—amphiregulin, epiregulin, and cumulus granulosa and the oocyte) blocks the trans-
β-cellulin—that diffuse through the antrum and bind lational activation. EGF receptor ligands also did not
to EGF receptors (EGFR) located on both the mural further increase translation of the reporter mRNAs in
and cumulus granulosa cells.26 Following application oocytes in which Pten, which antagonizes PI3-kinase
of LH to follicles in vitro, the cGMP concentration signaling, had been deleted—presumably because the
within the granulosa–oocyte compartment rapidly pathway had become constitutively activated. This
falls.124 This decrease is first seen in the mural granu- evidence suggests that the signal may be a growth
losa cells and is then detected in the cumulus granu- factor that is mechanistically coupled to PI3-kinase
losa cells and oocyte. The fall in cGMP reflects both signaling.
decreased synthesis, owing to dephosphorylation of Coincident with oocyte maturation, the cumu-
NPR2126,127 and reduced production of lus granulosa cells secrete an extracellular matrix that
NPPC,125,128,129 and to increased hydrolysis, owing separates the cumulus cells from each other and
to phosphorylation of PDE5.130 pushes them away from the oocyte. Termed expan-
It remains to be fully resolved whether the sion of the cumulus layer, this process terminates
decreased activity of NPR2 and decreased production contact between the granulosa cells and contributes
of Nppc are triggered by LH directly or indirectly via to the loss of contact between the oocyte and the
activation of EGFR, and recent work suggests that granulosa cells. Expansion is triggered by the EGFR
both pathways play key roles.26,121,126,128 Regardless ligands that are released by the mural granulosa cells

in response to LH. When the oocyte is microsurgi- Recent studies have identified EVs in ovarian
cally removed from COCs obtained from preovula- follicular fluid143–149 (Figure 6). Like those of other
tory follicles, however, the remaining shell of origins, follicular EVs contain mRNAs, miRNAs,
cumulus cells is unable to undergo expansion.133,134 and proteins, suggesting a potential role for these
Co-culture of the shells with oocytes or with oocyte- structures in intrafollicular communication.139,140
conditioned medium, however, restores the ability to Several strategies have been employed to test this
expand via a SMAD2/3-dependent pathway.133–135 idea. In one approach, EVs have been loaded with a
Thus, oocytes secrete an expansion-enabling factor. fluorescent marker and then either co-incubated with
Subsequent work has revealed that cumulus granulosa cells or injected directly into the follicle.
expansion is impaired in Bmp15−/− and in Bmp15−/−; The granulosa or follicular cells then have been
Gdf9+/− mice.114,115 Moreover, addition of GDF9 examined to see whether they contain fluorescent
and/or BMP15 to culture medium can induce cumulus particles within the cytoplasm.144,147,149 Both the
expansion as well as upregulation in the cumulus cells in vitro and the in vivo tests have confirmed that the
of genes implicated in this process.102–104,114 More EVs derived from follicular fluid can fuse with granu-
broadly, as exemplified by their activity to suppress losa cells, establishing their potential to carry infor-
expression of the LH receptor, the oocyte-derived fac- mation between cells.
tors promote differentiation towards the cumulus cell To address the function of EVs, it has been
phenotype and antagonize the mural granulosa cell tested whether EVs can alter physiology or behavior
phenotype.22,136,137 This is due in part to their activ- of the granulosa cells. As discussed above, the cumu-
ity to upregulate MTOR (mechanistic target of rapa- lus cell layer undergoes expansion at the time of ovu-
mycin) signaling in the cumulus cells.23 Recent work lation, owing to the secretion of a jelly-like matrix by
has identified additional growth factors that are the cumulus cells. Strikingly, follicular EVs were
secreted by oocytes during maturation, following found to induce cumulus expansion in vitro as well
translational activation of their encoding mRNAs. as increases in the amount of mRNAs that are upre-
Specifically, interleukin-7 (IL7) secreted by maturing gulated during expansion in vivo. However, the
oocytes enhances proliferation of the cumulus cells.138 effects were considerably attenuated compared to the
Identifying additional functions for IL7 and other fac- response to the EGFR ligands that are the physiologi-
tors secreted by maturing oocytes is an important goal cal trigger.147 Additionally, it has been shown that
of future research. mixing EVs with granulosa cells leads to an increase
in the granulosa cell content of miRNAs that are
abundant in the EVs143,149; conversely, the quantities
of putative mRNA targets of EV-enriched miRNAs
NEW HORIZONS—EXTRACELLULAR are decreased.143,149 This suggests that EVs could
VESICLES deliver miRNAs to target cells and thereby regulate
gene expression posttranscriptionally. Finally, it has
Recent studies have uncovered a new and unantici- been shown that the density of EVs in the follicular
pated means by which cells within the follicle may fluid decreases as the antrum increases in volume and
communicate. Analyses of fluids from a range of bio- that the content of the EVs differs between small and
logical sources have revealed the presence of small large follicles.144,146,149 These data suggest that the
membrane-bound structures known as extracellular generation and composition of EVs could be develop-
vesicles (EVs). Among these, two main types based mentally regulated.
on their size and origin have been identified so Unexpectedly, EVs may also allow macromo-
far.139–141 Microvesicles range in diameter from lecular transfer not just between somatic cells within
100 to 1000 nm in diameter and are budded off at the follicle but also between the cumulus cells and
the plasma membrane. Exosomes are smaller, ran- the oocyte (Figure 6). Electron microscopy has
ging from 50 to 200 nm in diameter. They arise from revealed structures that are morphologically similar
endocytotic invaginations within the late endosome to EVs at the tips of TZPs, adjacent to the oocyte
compartment, which generate multivesicular bodies plasma membrane.41,150 These results are especially
(MVBs) containing multiple exosomes. Fusion of intriguing in view of live-cell imaging that reveals
MVBs with the plasma membrane releases the exo- mRNAs traveling along the TZPs towards their tips.
somes into the extracellular space. They are often Strikingly, after incubation of wild-type oocytes on a
described together as EV, reflecting the difficulty in monolayer of EGFP-expressing cumulus granulosa
assigning the origin of a vesicle after it has been cells, Egfp mRNA has been detected in the oocytes.41
released from the cell. mRNAs could not pass through gap junctions, but

FI GU RE 6 | New mechanisms of intrafollicular communication: extracellular vesicles (EVs). EVs are present in follicular fluid and represent a
potential mechanism by which macromolecules including mRNA and miRNA could be transferred between cells. Structures resembling EV have also
been detected at the tips of transzonal projections (TZPs) and could permit similar transfer from cumulus granulosa cells to the oocyte. (Modified
with permission from Ref 142. Copyright 2017 Springer International)
could be packaged into EVs that are budded from specific miRNAs, mRNAs, or proteins could be pack-
the tip of the TZPs and by this mechanism delivered aged in the EVs has not been demonstrated. Most
to the oocyte. importantly, we do not yet know whether the bioma-
While these results provide tantalizing hints terials delivered by exosomes play an essential role in
that EVs might be a new player in the signaling net- the physiological function of their target cells.
work within the growing follicle, many important Because the growing follicle is a well-defined system
questions remain to be answered. If EVs carry spe- comprising a small number of spatially segregated
cific signaling molecules, it would be expected that and easily distinguished cell types, such studies
EVs from a nonfollicular source should be unable to should be feasible.
trigger the same biological effects. Indirect evidence
supporting this idea has been presented,147 but defin-
itive experiments remain to be done. It also remains
CONCLUSION
unclear, what fraction of the cells in the follicle can
be reached by the EVs—for example, are the inner The anatomically simple structure of the follicle and
layers of cumulus cells around the oocyte accessible? its accessibility for experimental studies have allowed
Furthermore, although there are differences in the us to gain considerable insight into the signaling
molecular content of EVs, from small and large interactions with the somatic compartment of the fol-
follicles,144,146 a mechanism by which, for example, licle that support and direct differentiation of the

oocyte. Not surprisingly, this knowledge has identi- extended and retracted, and it is not certain how
fied new black boxes and generated new questions to many types of TZP exist that might serve different
be pursued. As discussed earlier, we do not know functions. LH acting on the mural granulosa cells
why one primordial follicle begins to grow while its triggers meiotic maturation of the oocyte, but the rel-
neighbor does not. What is the nature of the signal ative roles of signaling directly through its receptor
that increases expression of KITL in the granulosa or indirectly through EGFR activity remain to be
cells of a primordial follicle, and do other signals clarified. Finally, the role of EVs in regulating differ-
contribute to triggering growth? Once the oocyte has entiation of the somatic follicular cells and the oocyte
begun to grow, we know that Kit signaling can pro- needs to be experimentally defined. Importantly, the
mote further growth, but not whether it plays this answers to these questions will likely benefit human
role under physiological conditions. If not KITL, reproductive health. While it has long been known
what is the contact-dependent, gap junction- that disease and aging can reduce oocyte quality, the
independent signal that granulosa cells provide that underlying mechanisms remain largely obscure. It is
sustains oocyte growth? Although TZPs are the only plausible that in some cases the root cause lies in
physical bridge that enables contact-dependent com- impaired signaling between the oocyte and its follicu-
munication between the growing oocyte and its fol- lar microenviroment. A more profound understand-
licular environment, we know almost nothing of ing of this complex and continuing interaction
their origin. Nor do we know whether they are should enable therapeutic strategies to be designed
dynamic structures, that might be continually and implemented to preserve fertility in women.
ACKNOWLEDGMENTS
Supported by grants to H.J.C. from the Eunice Kennedy Shriver National Institute of Child Health & Human
Development of the National Institutes of Health (R21HD086407), Canadian Institutes of Health Research
(CIHR), the Natural Sciences and Engineering Research Council of Canada, and the Research Institute of the
McGill University Health Centre (RI-MUHC). Research reported in this publication is solely the responsibility
of the author and does not necessarily represent the official views of the National Institutes of Health. We apol-
ogize to colleagues whose research could not be cited owing to space constraints.
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17597692, 2018, 1, Downloaded from https://wires.onlinelibrary.wiley.com/doi/10.1002/wdev.294 by Univ of Sao Paulo - Brazil, Wiley Online Library on [14/03/2023].

Regulation of germ cell

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation

INTRODUCTION cells in a structure termed a primordial follicle. Before

N ewborn mammalian females contain an enormous

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Primordial Primary Secondary Antral Pre-ovulatory

Immature Germinal vesicle Spindle Metaphase I Metaphase II

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(a) (b) (c) (d) (e)

Adherens junction Gap junction

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Growth PDK1 AKT1

MTOR MTOR MTOR

Protein Protein Protein

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granulosa cells in the primordial follicle triggers Gap Junctions

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