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Regulators of Ovarian Preantral Follicle
Development
Elizabeth A. McGee, MD1 Renju S. Raj, MD1
1Department of Obstetrics, Gynecology and Reproductive Sciences,
the University of Vermont College of Medicine, Burlington, Vermont
Semin Reprod Med 2015;33:179–184
Address for correspondence Elizabeth A. McGee, MD, Department of
Ob/Gyn, 111 Colchester Avenue, Smith Building, Burlington, VT
05401-1473 (e-mail: eamcgee@uvm.edu).
The progressive development of ovarian follicles from the
primordial or resting stage through the ovulatory stage is
essential for mammalian fertility.1 During this development,
follicles not only get bigger but the components of the follicle
undergo qualitative changes. Granulosa cells transform from
fibroblast-like cells to cuboidal classic epithelial cellswith gap
junctions and zona occludens. A basement membrane is
produced and progressively remodeled.2 The granulosa cells
divide and form layers that predestine cells to the locations
(mural, periantral, or cumulus) that will define their future
differentiation and functionality. The basement membrane
both defines and shapes the cells around it, but is also
modified by them. The granulosa/basementmembrane/theca
relationship is a key component of early folliculogenesis.2 The
oocyte is not a passive passenger in this process. It increases in
size, undergoes cytoplasmic changes, and develops a zona
pellucida that will inform development of the putative cu-
mulus cells. Notably, this all occurs before the development of
the antrum.1
Preantral follicles are small, but essential and dynamic
participants in the reproductive process. The term preantral
has caused some confusion over the years because different
authors have used the term to mean different things. Some
have used it to mean multilaminar follicles just prior to
coalescence of the antral fluid in a central cavity, whereas
others have meant any time or stage prior to antrum forma-
tion. In this article, we use the broader second definition and
also specify the stage of preantral to which we refer when
appropriate. Whenwe compare follicles within a species, it is
often useful to classify preantral follicles by layers of gran-
ulosa cells present rather than by a specific term.3
Preantral Follicles Are Gonadotropin
Responsive
Follicle-stimulating hormone (FSH) was once thought to only
play a role in development of larger follicles beyond the antral
stage.1 Though follicles beyond the antral stage are clearly
gonadotropin dependent, the smaller preantral follicles are
gonadotropin responsive.4,5 FSH is not a survival factor for
preantral follicles as it is for preovulatory and antral follicles,4
but it promotes follicle growth, granulosa cell division, and
differentiation in follicles from the primary stage onward.4,5
FSH treatment can increase multilaminar preantral follicle
growth in vivo.5 When juvenile rats in which the ovaries only
contain primary follicles were treated with exogenous FSH,
Keywords
► preantral follicle
► ovary
► Smad
► FSH
► TGF-β
Abstract Preantral follicle development has become an increasingly recognized area of study in
the last two decades. Factors that regulate the growth survival and differentiation of
these small, yet complex structures have been identified. The field of fertility preserva-
tion and a need for increased numbers of mature oocytes for stem cell research revealed
how little we knew of how follicles got from the primordial stage to the antral stage with
a healthy and competent oocyte inside. This work discusses the role of gonadotropins in
regulating preantral follicles and also the role of the TGF-β family members and their
associated Smad signaling molecules in preantral follicle development. Preantral follicle
development is a necessary step to fertility in females and further understanding of this
process is essential for progress in both infertility care and the enlarging field of in vitro
folliculogenesis.
Issue Theme Innovations in
Reproductive Endocrinology: A Tribute to
Bruce Carr, MD; Guest Editors, Serdar E.
Bulun, MD, and Richard S. Legro, MD
Copyright © 2015 by Thieme Medical
Publishers, Inc., 333 Seventh Avenue,
New York, NY 10001, USA.
Tel: +1(212) 584-4662.
DOI http://dx.doi.org/
10.1055/s-0035-1552584.
ISSN 1526-8004.
179
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mailto:eamcgee@uvm.edu
http://dx.doi.org/10.1055/s-0035-1552584
http://dx.doi.org/10.1055/s-0035-1552584
the follicles readily underwent growth and differentiation.
This is also seen in thehypogonadal (hPG)mouse inwhich the
growth of initial wave of folliculogenesis is suppressed as
early as day 7, the first time point evaluated. The number of
follicles beyond the primary stage was greatly reduced at all
ages, but robust follicle growth resumedwhen the mice were
treated with FSH.6
FSH is also a growth-promoting factor for preantral fol-
licles in culture. Suppression of apoptosis of granulosa cells is
essential for maintaining a follicle pool and for follicle devel-
opment to occur. There are many different hormones and
growth factors that play a role in suppressing or promoting
apoptosis of granulosa cells.7,8 For preantral follicles, activa-
tion of the cyclic guanosinemonophosphate (cGMP) pathway
is a potent promoter of preantral follicle survival.9 When
apoptosis is suppressed in cultured follicles, FSH is able to
induce follicle growth and differentiation.4 The growth pro-
moting effects of FSH can be difficult to demonstrate when
cell death is not prevented, because the cell loss masks the
increase in cell division. FSH has been demonstrated to be
necessary for preantral follicle growth and development in
vitro in several species, including primates10
The FSH receptor (FSHR) is expressed in very early follicles
from the one layer primary stage of follicle on,11 which is
consistent with a role for FSH in early follicle growth. Trans-
genicmice that have had the FSHR gene deleted (FORKO) have
altered early folliculogenesis.12 These animals have a slightly
different phenotype from the hPG mice, but their ovaries do
not have follicleswithmore than four layers of granulosa cells.
These transgenic mice are also interesting because they also
have a distinct, but less severe subfertility phenotype in the
heterozygotes.
Though FSH is not absolutely necessary for antral follicles
to develop in the ovaries of mammals, the above evidence
together demonstrate that FSH can promote optimal follicle
growth and progression through the preantral stages of
follicle development.
Transforming Growth Factor-β and Activin
Signaling in Follicle Development
The related growth factors transforming growth factor-β
(TGF-β) and activin exert their effects at least partly through
a canonical Smad signaling pathway (►Fig. 1). The growth
factor binds the type 2 receptor, a serine/threonine receptor
kinase, which then activates the type 1 receptor. The type 1
receptor activates a receptor-activated Smad, phosphorylat-
ing it on themad homology-2 (MH2) domain. Though there is
some variation and promiscuity among the receptors and
signalingmolecules, in general activin binds the activin type 2
receptor (ACVR2A), which then activates the activin type 1
receptor (ACVR1B), also known as ALK4. The TGF-β type 2
receptor is typically TGFβRII that activates TGFβRI (also
known as ALK5).
These receptors activate both Smad2 and Smad3. These
two Smads are very similar in structure and function, but not
identical. Smad3 is able to bind DNA directly, but Smad 2
cannot because it has an L2 loop that structurally blocks this
domain. Both Smads interact with the co-Smad, Smad 4, and
translocate to the nucleus where they have various effects.
Smads work with a multitude of nuclear factors that create
binding specificity based on the combinations of factors that
are present and available for interaction in the specific cell
type or stage of maturation. This creates a wide variety of
actions that thesetwo growth factors can elicit in a stage and
development specific manner.
The expression of Smad2 and Smad3 in the ovary is stage
specific.13 Smad2 is more ubiquitous and present in primary
through antral follicles, reduced in preovulatory follicles, but
returns in corpora lutea. In contrast, Smad3 is present largely
in small follicles. Its expression is diminished in large antral
follicles and does not reappear in corpora lutea. Thus the
Smad3 effects are mostly limited to preantral follicles
(►Fig. 2).
In granulosa cells, both Smad2 and Smad3 translocate to
the nucleus in response to treatment with either TGF-β or
activin (►Fig. 3a). However, the relative sensitivity to stimu-
lation is not the same. Smad3 is activated by TGF-β at lower
doses than Smad2. Smad2 is more sensitive to activin stimu-
lation than TGF-β.13 Thus each factor will activate both
Smads, but the relative activation may differ based on the
concentration of factor available. Lower levels of TGF-β will
favor Smad3 activation, whereas lower activin would favor
Smad2 activation.
Smad3 and Follicle Development
In mice that do not express the Smad3 protein, fertility is
compromised. Ovaries begin with the normal number of
primordial follicles, so initial endowment of the follicle pool
is not disturbed.14However, there is a delay in the appearance
of multilaminar follicles in the infantile and juvenile animal.
This delay in follicle growth is also displayed by follicles in
culture that do not express Smad3.15 The follicles do not grow
in response to FSH stimulation, though follicles from their
Fig. 1 Smad activation by the transforming growth factor-β family
transmembrane serine threonine kinase complex allows translocation
of the R-Smad/Smad4 complex to the nucleus. MH1, mad homology-1;
MH2, mad homology-2.
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Regulators of Ovarian Preantral Follicle Development McGee, Raj180
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wild-type siblings do (►Fig. 3b). Occasional estrous cycles
and even ovulation can occur very rarely in these animals, but
egg quality is poor and no pregnancies resulted.15
Lack of Follicle-Stimulating Hormone Receptor
Responsiveness
An analysis of whole ovaries, isolated granulosa cells, and
cultured granulosa cells was performed in Smad3-deficient
animals and their wild-type siblings. In all of the Smad3-
deficient tissues, the three messenger RNAs (mRNAs) for
FSHR, aromatase, and cyclin D2 were expressed at lower
levels. Cultured granulosa cells of follicles deficient in
Smad3 have lower baseline expression of FSHR and aroma-
tase than wild-type granulosa cells and they do not increase
expression in response to FSH stimulation.15 When Smad3
expression is returned to granulosa cells via adenovirus, the
ability of FSH to upregulate its receptor returns (►Fig. 3c).
Follicle-Stimulating Hormone Receptor Promoter
To understand the mechanism of delayed follicle growth and
reduced FSHR expression in follicles of Smad3-deficient mice,
the FSHR promoter was evaluated. A Smad-binding element
is present in the FSHR promoter region at�440 bp. When the
FSHR promoter is expressed in granulosa cells, its activity can
be upregulated by treatment of the cells with TGF-β. This
stimulation can be abolished by mutating or deleting the
Smad-binding site (►Fig. 3d). The specificity of this site was
confirmed with chromatin immunoprecipitation assays.
The aforementioned studies provide strong evidence that
Smad3 functions to regulate the expression of FSHR in
preantral follicles and promote early folliculogenesis.
Inhibitory Smads Modulate Smad Pathway
Signaling
In addition to the receptor-activated Smads, there are also
inhibitory Smads that function to regulate the activity of the
Smad pathway. Both Smad6 and Smad7 are inhibitory Smads.
Smad6 function is more specific to the bone morphogenetic
protein (BMP) pathway of growth factors, but Smad7 can
inhibit both the Smad1, 5, 8 and the Smad2, 3 pathways.
Smad7may also have Smad pathway–independent functions.
The roles of Smad6 and Smad7 are likely very different in
the ovary. This is accentuated by the very different expression
patterns of the two molecules.16 Smad6 expression is limited
largely to the oocytes, though expression is seen in granulosa
cells in culture. However, Smad7 is expressed in granulosa,
oocytes, and theca cells, and it is highly expressed in blood
vessels in the ovary.
The two factors also differ in their regulation by TGF-β.
TGF-β can upregulate the expression of Smad7, but not of
Smad6 in granulosa cells (►Fig. 4a). Though TGF-β is a potent
regulator of Smad7, activin treatment does not increase the
expression of Smad7 in granulosa cells (►Fig. 4b). FSH
treatment does not alter the TGF-β stimulation of Smad7
expression, nor does activation of the cAMP pathway with
other agents.16
There are several Smad responsive elements in the pro-
moter region of Smad7. Deletional andmutational analysis of
the promoter region of Smad7 revealed a canonical SBE site at
�141 bp that was critical for TGF-β to activate the promoter
construct.16 Both Smad2 and Smad3 are able to independent-
ly activate this promoter. When either one is ablated
Fig. 2 Immunohistochemical analysis of Smad2 and Smad3 in the ovary. The micrographs (upper row) are of Smad2 staining patterns in small
follicles (upper left panel) and in antral follicles (upper right panel). Serial sections were also stained for Smad3 in the lower row. (Modified from
Xu et al.13)
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Regulators of Ovarian Preantral Follicle Development McGee, Raj 181
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chemically or genetically, the promoter is still activated and
the mRNA is still transcribed. However, when they are both
blocked by chemical inhibitors or receptor dysfunction, the
TGF-β stimulation is also blocked, demonstrating that the
canonical Smad signaling pathway is required for TGF-β
stimulation of Smad7 expression.16
To further evaluate the role of Smad7 in granulosa cells, an
expression vector was used to overexpress Smad7 protein in
granulosa cells. When Smad7 is overexpressed in granulosa
cells, a marked increase in apoptosis is noted in granulosa
cells in culture (►Fig. 4c). In contrast, when the expression of
Smad7 is reduced by small interfering RNA (siRNA), the
ability of TGF-β treatment to induce apoptosis of granulosa
cells is markedly reduced (►Fig. 4d). Thus, Smad7 is a
mediator of TGF-β–induced apoptosis in granulosa cells.
The stimulation of Smad7 expression by TGF-β, but not by
activin, may be a means by which these two closely related
growth factors can exert different effects on growing follicles.
This may be the mechanism by which TGF-β induces pro-
nounced apoptosis in preantral rat follicles in serum-free
culture.17
Summary
In this article, we have summarized works that collectively
demonstrate that Smad3 is essential for FSH to be able to
upregulate its own receptor in small follicles. TGF-β, working
through the type 1 receptor, activates either Smad2 or Smad3
(or both) and increases the expression of Smad7, providing a
negative feedback loop in signaling and promoting granulosa
Fig. 3 Smad3 and follicle-stimulating hormone (FSH) effects on follicle growth and FSH receptor function. (a) Differential sensitivities of Smad2
and Smad3 to activin and transforming growth factor-β (TGF-β) stimulation. Nuclear staining was determined by immunohistochemistry (IHC) in
cells after treatment. Bars represent percent of cells with nuclear staining intensity higher than cytoplasmic staining intensity. (b) Growth of
isolated follicles in culture over 3 days of treatment with or without FSH. Follicles were dissected from ovaries of wild-type (WT) and Smad3-
deficient litter mates and grown in serum-free culture medium supplemented with 8-Br-cGMP.KO, knockout. (c) FSH receptor (FSHR) expression in
cultured granulosa cells. When Smad3 is added back to granulosa cells with an adenovirus system (stippled bars), both basal and FSH-stimulated
FSHR mRNA expression is similar to WT cells expression levels. (d) FSHR promoter construct activity with the Smad-binding element (SBE) deleted
or truncated eliminates the ability of TGF-β treatment to enhance promoter activity (black bars). The lower inset is of the gel bands from a
chromatin immunoprecipitation (chip) assay. Smad3 antibody bound specifically at the SBE site, but not at the AP1/E-box site. (Modified from
Gong and McGee.15)
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apoptosis. Understanding the complex interplay of TGF-β and
activin in defined tissue culture systems may be of use in
refining protocols for in vitro folliculogenesis. It also may aid
our understanding the interactions possible in vivo that can
promote specific follicles to successfully navigate the arduous
path to dominance and ovulation.
Fig. 4 Smad7 regulation and effects on apoptosis. (a) Smad6 (white bars) and Smad7 (black bars) mRNA expression in response to transforming
growth factor-β (TGF-β) treatment of granulosa cells. (b) TGF-β, activin, 8-BR-camp, and forskolin effects on the activity of the Smad7 promoter
transfected into spontaneously immortalized granulosa cells. The TGF-β–stimulated increase in activity is not altered by either of the cyclic
adenosine monophosphate (cAMP) pathway activators. Activin over a range of doses does not increase the activity of the Smad7 promoter. (c)
Transfection of primary granulosa cells with empty vector (white bars) or a full-length smad7 vector (black bars). Overexpression of Smad7 results
in increased apoptosis of cells measured as a percent of TUNEL-stained cells in culture at 6 or 24 hours. (d) Cells were treated with vehicle (white
bars) or TGF-β (black bars). Cells were not transfected (NT) or transfected with siRNA for Smad7 or a negative control. Knock-down of Smad7
expression eliminates the ability of TGF-β to increase apoptosis in cultured granulosa cells. (Modified from Quezada et al.16)
Seminars in Reproductive Medicine Vol. 33 No. 3/2015
Regulators of Ovarian Preantral Follicle Development McGee, Raj 183
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