Molecular Reproduction & Development 78:9–21 (2011)
Integral Role of GDF-9 and BMP-15 in Ovarian Function
122FUMIO OTSUKA,* KIRSTEN J. McTAVISH,AND SHUNICHI SHIMASAKI
1 Endocrine Center of Okayama University Hospital, Okayama, Japan
2 Department of Reproductive Medicine, University of California San Diego, School of
Medicine, La Jolla, California
The oocyte plays an important role in regulating and promoting follicle growth, and thereby its own development, by the production of oocyte growth factors that BMP-15 and GDF-9 play a role predominantly act on supporting granulosa cells via paracrine signaling. Genetic both in promoting early follicle studies in mice demonstrated critical roles of two key oocyte-derived growth factors growth as well as restraining belonging to the transforming growth factor-b (TGF-b) superfamily, growth and dominant/pre-ovulatory follicle differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in development in mono-ovulatory ovarian function. The identi？cation of Bmp15 and Gdf9 gene mutations as the causal mammals. mechanism underlying the highly proli？c or infertile nature of several sheep strains in a dosage-sensitive manner also highlighted the crucial role these two genes play in ovarian function. Similarly, large numbers of mutations in the GDF9 and BMP15 * Corresponding author: genes have been identi？ed in women with premature ovarian failure and in mothers of Endocrine Center of Okayama dizygotic twins. The purpose of this article is to review the genetic studies of GDF-9 University Hospital and BMP-15 mutations identi？ed in women and sheep, as well as describing the Okayama 700-8558, Japan. various knockout and overexpressing mouse models, and to summarize the molecu- E-mail: firstname.lastname@example.org- u.ac.jp lar and biological functions that underlie the crucial role of these two oocyte factors in Grant sponsor: USDA National Institute of female fertility. Food and Agriculture; Grant number: 2008-35203-19024; Grant sponsor: NIH; Grant numbers: RO1 HD414940-08, Mol. Reprod. Dev.78: 9–21, 2011. ß 2010 Wiley-Liss, Inc. HD414940-08S1, R21-HD41494-02, R21- HD41494-02S1; U54 HD012303; Grant sponsors: Takeda Science Foundation; WESCO Scienti？c Promotion Foundation
Published online 10 December 2010 in Wiley Online Library (wileyonlinelibrary.com). Received 8 October 2010; Accepted 16 November 2010 DOI 10.1002/mrd.21265
There is a large amount of evidence supporting the
concept that the oocyte plays a central role in regulating Abbreviations: ALK, activin receptor-like kinase; BMP, bone morphogenetic follicle growth and development (Matzuk et al., 2002; Gilchr- protein; BMP-RII, BMP type II receptor; PTGS2, cyclooxygenase 2; EGF, ist et al., 2008; Su et al., 2009). Pioneer work by Falck and epidermal growth factor; ERK, extracellular signal-regulated kinase; FGF, ？bro- blast growth factor; FSHR, follicle-stimulating hormone receptor; GDF, growth colleagues demonstrated in 1959 that intact rabbit preovu- and differentiation factor; HAS, hyaluronan synthase; 3bHSD, 3b-hydroxyster- latory follicles do not luteinize when transplanted into the oid dehydrogenase; IGF, insulin-like growth factor; KL, kit ligand; LHCGR, anterior chamber of the eye, in contrast to oocyte-free luteinizing hormone/choriogonadotropin/receptor; MAPK, mitogen-activated protein kinase; PCOS/PCO, polycystic ovary syndrome/polycystic ovary; PI3K, explants of either the follicle wall or granulosa cells that do phosphatidylinositol 3-kinase; POF, premature ovarian failure; Cyp19a1, P450 undergo morphological luteinization (Falck, 1959). There- aromatase; Cyp11a1, P450 steroid side-chain cleavage enzyme; StAR, ste- fore, the oocyte acted to inhibit luteinization of the other roidogenic acute regulatory protein; TG, transgenic; TGF, transforming growth follicular cells. Subsequently, Nalbandov and colleagues factor; ZP, zona pellucida.
ß 2010 WILEY-LISS, INC.
Molecular Reproduction & Development TSUKA ET AL. O
con？rmed this work using rabbit dominant follicles in situ, of granulosa cell mitotic ability at the end of the primary showing that removal of the oocyte caused spontaneous stage.
luteinization of granulosa and theca cells and elevated Differentiation of all follicle compartments, including the secretion of progesterone to levels produced by corpora oocyte, granulosa, and theca cells, is influenced by GDF-9 lutea (El-Fouly et al., 1970; Nalbandov, 1970). These stud- (Dong et al., 1996; Carabatsos et al., 1998; Elvin et al., ies were integral in establishing that the oocyte is involved in 1999b). GDF-9-de？cient oocytes grow more rapidly than governing follicle luteinization, possibly by secretion of the control oocytes and attain a larger size despite the halt in putative luteinizing inhibitor(s) that has been of persistent follicle growth at the primary stage (Carabatsos et al., interest in the ？eld. 1998). While GDF-9-de？cient oocytes displayed relatively Decades later, it has been established that ovary- normal meiotic competence for early stage follicles, there
were some ultrastructural meiotic defects, including an derived transforming growth factor-b (TGF-b) superfamily
members play an integral role in inhibiting granulosa cell absence of cortical granules and clustering of organelles progesterone production. Initial work showed that two theca around the germinal vesicle. This suggests that late secre- cell-derived bone morphogenetic proteins (e.g., BMP-4 and tory events in oogenesis are affected by the absence of
GDF-9. In the immature primary follicles with degenerated BMP-7) inhibit FSH-dependent progesterone production
oocytes, granulosa cells showed characteristics associated while stimulating FSH-dependent estradiol production in
rat granulosa cells in vitro (Shimasaki et al., 1999). This with dominant preovulatory follicles (Elvin et al., 1999b). Of ？nding is consistent with in vivo steroidogenesis during note, Cyp19a1 expression that is normally upregulated in the follicular phase of the estrus/menstrual cycle. Subse- preovulatory follicles was 50% of wild-type levels in Gdf9 quent studies have demonstrated that other members null ovaries, despite the lack of follicles past the primary
stage (Dong et al., 1996), suggesting that GDF-9 may of the TGF-b superfamily (e.g., oocyte-derived BMP-15
normally act to inhibit aromatase expression. In addition, and BMP-6) also inhibit FSH-stimulated progesterone pro-
duction without changing estradiol production in rat gran- theca cell recruitment was impaired in Gdf9 null ovaries, as ulosa cells (Otsuka et al., 2001b, 2000). Therefore, it determined by the lack of CYP17, LH receptor (LHCGR), appears that the long-sought oocyte-derived luteinization and c-kit expression in theca cells (Elvin et al., 1999b). inhibitors are most likely BMP family members (Shimasaki Therefore, the oocyte-derived GDF-9 appears to be essen- et al., 2004). tial for normal oocyte and granulosa cell function as well as Following identi？cation of the BMP system in the ovary, theca cell formation.
remarkable progress has been made towards a molecular Further evidence for the important paracrine role of GDF- understanding of how oocyte factors regulate the function of 9 comes from the ？nding that in GDF-9-de？cient mice,
granulosa cells during follicle growth and ovulation (Matzuk granulosa cell expression of kit ligand (KL), and inhibin-a et al., 2002; Gilchrist et al., 2008; Su et al., 2009). Among is upregulated (Elvin et al., 1999b). Generation of double- oocyte-derived BMP family members, the biological and mutant mice lacking both GDF-9 and inhibin-a revealed that physiological activities of growth and differentiation factor-9 the block at the primary follicle stage in Gdf9 null ovaries (GDF-9) and BMP-15 have been most extensively re- was overcome by co-deletion of the Inha gene. Thus, searched as genetic studies revealed essential roles for despite the absence of GDF-9, follicle growth proceeds both factors in female fertility in several mammalian species normally until the type 5b (late preantral) follicle stage in (Moore et al., 2004; Juengel et al., 2004a; McNatty et al., Inha/Gdf9 double-null ovary. Therefore, in mice it appears 2005d; Gilchrist et al., 2008; Su et al., 2009). In this review, that one of the critical functions of GDF-9 that promotes we focus on the role of the two most homologous oocyte- early follicle growth is downregulation of inhibin-a. factors, GDF-9 and BMP-15, in ovarian function.
Ewes With Gdf9 Mutations GENETIC STUDIES OF MUTATIONS IN THE Gdf9 Sheep have proven to be a valuable model to elucidate GENE the importance of GDF-9 in female reproductive function. Gdf9 Knockout Mice Normal ewes ovulate one or two oocytes per cycle
(Montgomery et al., 1992). The Cambridge and Belclare Generation of a Gdf9 null mouse model by the Matzuk lab sheep breeds were known to have highly proli？c natures, provided a new avenue for studying the role of GDF-9 in
although there was extreme variation in the fertility of both ovarian function (Dong et al., 1996). Homozygous Gdf9 null mice were grossly indistinguishable from heterozygous or breeds with some ewes ovulating up to 3–6 oocytes per wild-type mice. A complete loss of GDF-9 in homozygous cycle, some ovulating a normal number of oocytes and males showed that GDF-9 is not required for male fertility. other ewes were infertile. Analysis of the inheritance pat- This is in contrast to homozygous null females that were terns of the ovarian hypoplasia present in the infertile ewes
in these strains suggested an autosomal gene was likely to infertile, while heterozygous Gdf9 null female mice were be involved. Although ewes from a different breed (called phenotypically normal (Dong et al., 1996). Ovaries from
adult female homozygous mice were signi？cantly smaller Inverdale) that have a similar phenotype were previously than wild-type ovaries, and exhibited a complete absence identi？ed to carry a mutation in the Bmp15 gene (addressed of normal follicles beyond the primary stage. The block in the Ewes with Bmp15 Mutations section), histological in follicle growth appeared to be attributed to the loss analysis of the hypoplastic ovaries from Cambridge and
10 Mol Reprod Dev 78:9–21 (2011)
ROLE OF GDF-9 AND BMP-15 IN OVARIAN FUNCTION
TABLE 1. Naturally Occurring Mutations in Ovine Gdf9 and Bmp15 Genes Sheep breeds Gene allele Nucleotide changes Amino acid changes Mature protein Refs.
HBelclare/Cambrige FecG GDF-9S77F Hanrahan et al. (2004) c.1184 (230) C>T Ser395Phe Icelandic Thoka FecTT S109R Nicol et al. (2009) GDF-9c.1279 (326) A>C Ser427Arg IInverdale FecX BMP-15V31D Galloway et al. (2000) c.896 (92) T>A Val299Asp HHanna FecX BMP-15Q23term Galloway et al. (2000) Lc.871 (67) C>T Gln291Term Lacaune FecX BMP-15C53Y Bodin et al. (2007) RRasa Aragonesa c.962 (160) G>A Cys321Tyr FecX BMP-1517 bp-del Martinez-Royo et al. (2008) c.525–541 del (17 bp) 208Term and Monteagudo et al. (2009) Belclare/Cambridge HHanrahan et al. (2004) (double heterozygotes) FecG S77F GDF-9c.1184 (230) C>T Ser395Phe G FecX BMP-I5Q-29term c.718 C>T Gln239Term Belclare/Cambridge HGDF-9S77F Hanrahan et al. (2004) c.1184 (230) C>T Ser395Phe (double heterozygotes) FecG BBMP-15S99I FecX c.1100 (296) G>T Ser367lle 2wWoodlands Unknown Unknown Feary et al. (2007) FecXUnknown
Belclare ewes revealed different abnormalities to those Women With GDF9 Mutations
associated with the Inverdale ewes, such as the presence of follicles with an antrum and oocytes with thickened zona The identi？cation of variant GDF-9 sequences in non- pellucidae (ZP) surrounded by abnormally dispersed layers symptomatic, premature ovarian failure (POF) patients of cells (Hanrahan et al., 2004). (Dixit et al., 2006; Laissue et al., 2006; Kovanci et al., Therefore, since GDF-9 is most homologous to BMP-15 2007; Zhao et al., 2007) suggests that altered GDF-9 in primary structure as well as spatio-temporal expression in function may also be involved in ovarian dysfunction in the ovary, a potential involvement of this gene in the peculiar women. Further, GDF-9 missense and nonsense mutations phenotypes observed in Cambridge and Belclare ewes was have also been detected in mothers of dizygotic twins, investigated. Consequently, genetic studies uncovered suggesting that some variants may also be linked to a eight single nucleotide polymorphisms across the coding polyovulatory phenotype (Montgomery et al., 2004; Palmer
et al., 2006). Three missense mutations, GDF-9P103S region of the Gdf9 gene (Galloway et al., 2000; Hanrahan ,GDF-9P374L, and GDF-9R454C, occur in conserved amino et al., 2004); of these, one mutation, GDF-9S77F, was
directly associated with the sterility phenotype in both the acid positions and are signi？cantly related to an increased Cambridge and Belclare breeds (Table 1). This mutation ovulation rate in women (Palmer et al., 2006). Intriguingly, from Ser to Phe replaces an uncharged polar group with a the GDF-9P103S mutation is detected both in mothers of non-polar group, and is considered to affect the binding to dizygotic twins and in women with POF. Therefore, GDF-9 BMP type I receptors. Interestingly, ewes homozygous for is likely to alter the ovulation rate in women as well as in the GDF-9S77F mutation are infertile, while heterozygous ewes.
Interestingly, many of these mutations were found in the carriers have an increased ovulation rate associated with a
proregion of the GDF-9 proprotein. In this regard, one of the larger litter size than normal. In addition to the Gdf9 poly-
important features in the posttranslational processing of the morphisms, four Bmp15 polymorphisms were also identi- ？ed in Cambridge and Belclare sheep, two of which were TGF-b superfamily members is that the proregion is nec- linked to the infertility phenotype in homozygotes or double essary for dimerization of the mature protein (Shimasaki heterozygotes. Double heterozygotes (GDF-9S77F/BMP- et al., 2004). As such, proregion mutations may influence 15S99I or GDF-9S77F/BMP-15Q-29Ter) exhibit an additive dimerization of the proprotein, and thereby negatively im- effect on ovulation rate, thus the biological effects of pact the production of functional mature protein dimers.
GDF-9 proregion mutations identi？ed in POF women and BMP-15 and GDF-9 are likely to be distinct (Table 1).
mothers of dizygotic twins may, therefore, cause impaired More recently, another point mutation in the Gdf9 gene processing of the proproteins by forming mis-folded pro- resulting in a non-conservative amino acid change (GDF-
9S109R) in the C-terminus of the mature GDF-9 protein was protein dimers.
identi？ed in Icelandic Thoka sheep (Nicol et al., 2009). It is notable that ovaries from 25- to 31-week-old Similar to the GDF-9S77F mutation, this mutation was female Gdf9 null mice contained either single unilateral or also associated with increased fecundity in heterozygous bilateral ovarian follicular cysts lined by several layers of ewes and infertility in homozygotes. There is a complete flattened granulosa cells (Dong et al., 1996). This cystic absence of follicle development in GDF-9S109R homo- ovarian phenotype bears some resemblance to the histo- zygotes, despite normal activation of oocytes and ex- logical characteristics seen in polycystic ovary syndrome pression of a number of oocyte-speci？c genes involved (PCOS) in women. PCOS is one of the most common in ZP formation (Nicol et al., 2009). These studies provided causes of anovulation, infertility, and menstrual irregulari- evidence that GDF-9 is a critical player in ovine follicle ties in women, affecting between 5% and 10% of women of growth. reproductive age (Knochenhauer et al., 1998). Analysis of
11 Mol Reprod Dev 78:9–21 (2011)
Molecular Reproduction & Development TSUKA ET AL. O
human ovary tissues from women with PCOS revealed that GDF-9 also plays an important role during the ？nal
GDF-9 mRNA expression is substantially delayed and stages of follicle growth prior to ovulation. Prior to the LH reduced during the growth and differentiation phase surge, cumulus cells require GDF-9 to support the meta- (Teixeira Filho et al., 2002). In contrast, PCOS/PCO had bolic cascades such as glycolysis and sterol biosynthesis no detectable effect on the expression pattern of BMP-15 (Sugiura et al., 2005). Evidence for this comes from ？ndings
(Teixeira Filho et al., 2002). The disparity between altera- of reduced cholesterol synthesis from acetate in cumulus/ tions in these two critical oocyte factors, with changes in oocyte complexes of Bmp15 null mice, and to a greater /þGDF-9 but not BMP-15 expression, may be due to the extent, of Bmp15Gdf9/ double mutant mice. As speci？c role of GDF-9 in maintaining follicle structure, mouse oocytes are de？cient in cholesterol synthesis, they although there are controversial data showing no signi？cant depend on cumulus cells to provide cholesterol. Important- change in GDF-9 or BMP-15 protein levels associated with ly, oocyte-derived GDF-9 seems to act to promote choles- PCOS (Zhao et al., 2010). Further studies are needed to terol biosynthesis in cumulus cells, thereby ensuring the determine if changes in BMP-15/GDF-9 can directly trigger oocyte receives an adequate supply of essential metabolic follicular arrest in PCOS/PCO women or if altered expres- precursors (Su et al., 2008).
sion is secondary to ovarian dysfunction. In addition to regulation of cumulus cell metabolism,
GDF-9 controls diverse processes and gene expression
during the preovulatory stage. In mouse preovulatory folli-
cles, GDF-9 inhibits FSH-induced steroidogenesis while BIOLOGICAL FUNCTIONS OF GDF-9 IN promoting cumulus cell progesterone production by stimu- REGULATING FOLLICULOGENESIS lating the expression of an intrinsic prostaglandin-E2/EP2 In vitro studies using recombinant GDF-9 protein have receptor signaling pathway (Elvin et al., 2000). GDF-9 also clari？ed the biological roles and importance of GDF-9 ac- enhances cumulus cell expansion in the presence of FSH tions in follicle growth and development at all stages of (Elvin et al., 1999a), but not without FSH (Dragovic et al., folliculogenesis. There is compelling evidence in several 2005), which may relate to the GDF-9 enhancement of mammalian species that GDF-9 is essential for early stages hyaluronan synthase 2 (HAS2) and cyclooxygenase 2
(PTGS2) mRNAs (Elvin et al., 1999a). Addition of the of follicle development. Gdf9 null mice (Dong et al., 1996)
and ewes either homozygous for naturally occurring muta- extracellular domain of the type II BMP receptor (BMP-
RII) to granulosa cell cultures suppressed GDF-9-induced tions in the Gdf9 gene (Hanrahan et al., 2004) or immunized
cumulus cell expansion, but only partially neutralized oo- against GDF-9 (Juengel et al., 2002) all exhibit a block in
cyte-induced expansion (Dragovic et al., 2005). This sug- follicle growth at the primary stage. Further, GDF-9 also
elicited a reduction in primordial follicle number in vivo (Vitt gests that GDF-9 is not the sole oocyte-derived cumulus et al., 2000b), and in vitro exposure of rodent (Hayashi et al., expansion-enabling factor, and that other factors are also 1999; Nilsson and Skinner, 2002) or human (Hreinsson involved in cumulus expansion. Thus, GDF-9 plays an et al., 2002) ovarian tissue to GDF-9 promotes primary important role in regulating several aspects of granulosa
cell function during the preovulatory stage of follicle follicle progression.
At the preantral stage, GDF-9 has been shown to be development.
effective in stimulating the growth of in vitro cultured rat Oocyte-secreted factors possess anti-apoptotic actions. preantral follicles (Hayashi et al., 1999). GDF-9 also pro- In a study using bovine cumulus–oocyte complexes
(Hussein et al., 2005), oocyte removal signi？cantly in- motes early preantral follicle growth in human ovaries
(Hreinsson et al., 2002). In the transition to the antral creased cumulus cell apoptosis compared with intact stage, it appears that GDF-9 promotes follicular survival cumulus–oocyte complexes. Increased apoptosis in oocy- by suppressing granulosa cell apoptosis and follicular tectomized cumulus cell complexes could be reversed in a atresia (Orisaka et al., 2006). This may be achieved, in dose-dependent manner by co-culture with denuded oo- part, by GDF-9 stimulation of follicular FSH receptor cytes. In the study, GDF-9 had no signi？cant effect on
(FSHR) expression as adequate FSHR levels in granu- cumulus cell apoptosis whereas two other oocyte-secreted
factors (i.e., BMP-15 and BMP-6) reduced cumulus cell losa cells is essential for FSH-dependent antral follicle
growth. apoptosis (Hussein et al., 2005). In a study using rat pre- GDF-9 also promotes granulosa cell proliferation while antral follicles, however, GDF-9 was shown to exert anti- inhibiting FSH-induced steroidogenesis and LHCGR ex- apoptotic effects in preantral follicles and protects granu- pression in rat granulosa cells collected from early antral losa cells from undergoing apoptosis via activation of the and preovulatory follicles (Vitt et al., 2000a). The GDF-9 phosphatidylinositol 3-kinase/Akt pathway (Orisaka et al., inhibition of FSH action was postulated to occur via modu- 2006). Differences between the anti-apoptotic actions of lation of FSHR levels or by FSHR coupling levels to Gs GDF-9 in these two models suggest that there are differ-
ences in GDF-9 anti-apoptotic activity between species or protein (Vitt et al., 2000a). However, ？ndings in rats that
GDF-9 stimulates FSHR expression in preantral follicles that GDF-9 has anti-apoptotic actions during early, but not (Orisaka et al., 2006) and in cultured granulosa cells from late stages of follicle development.
early antral follicles (our unpublished ？ndings) suggest that As primary human granulosa cells are dif？cult to obtain in
large quantities, most studies have utilized luteinized hu- GDF-9 inhibition of FSH-actions acts via modi？cation of
man granulosa cells to examine GDF-9 actions in the postreceptor signaling.
12 Mol Reprod Dev 78:9–21 (2011)
OLE OF GDF-9 AND BMP-15 IN OVARIAN FUNCTION R
granulosa cell growth. Direct effects of androgens are human ovary. In luteinized human granulosa cells, GDF-9
has no direct effects on basal or FSH-induced progesterone mediated by the androgen receptor that is expressed in production, which is inconsistent with the effect in rodent granulosa cells. Granulosa cells also have signaling path- granulosa cells. Rather, GDF-9 reverses activin A suppres- ways for FSH and IGF-1, which are functionally linked to sion of FSH-induced progesterone secretion (Shi et al., each other and also appear to be modulated by androgens. 2010), while enhancing activin A-induced inhibin B secre- In porcine granulosa cells, GDF-9 potently enhances tion (Shi et al., 2009b). GDF-9 has been detected in lutei- IGF-1-stimulated proliferation and inhibits FSH-stimulated nized human granulosa cells in addition to oocytes, and progesterone secretion (Hickey et al., 2005). Androgens appears to be involved in an autocrine-enhancement of dose-dependently enhance the mitotic activity of oocytes or activin A-induced inhibin B secretion from the luteinized GDF-9, and this enhancement is opposed by co-treatment granulosa cells (Shi et al., 2009a). Thus, in humans, it with anti-androgen drugs. Therefore, in the pig, androgens appears that increasing GDF-9 expression during folliculo- interact with GDF-9 to amplify granulosa proliferation at the genesis enhances granulosa cell response to activin A, early antral stage.
leading to an upregulation of inhibin B levels. As GDF-9 and BMP-15 are both present in follicles GDF-9 also plays a role during in vitro maturation of throughout most stages of follicular growth, it is important oocytes and subsequent fetal viability (Yeo et al., 2008). to consider whether these growth factors are synergistic or The addition of exogenous GDF-9 to mouse cumu- redundant in processes where they have similar activity
alone. GDF-9 can act co-operatively with BMP-15, with lus–oocyte complexes cultured with FSH prior to in vitro some studies in granulosa cells showing the effects of a fertilization and transfer to recipient females had no effect
combined GDF-9/BMP-15 treatment that are different from on implantation rate or on fetal and placental weights, but
either growth factor alone (McNatty et al., 2005b). In addi- increased the number of viable fetuses. This suggests that
GDF-9 supports embryo development and fetal viability. tion, GDF-9 and BMP-15 have species-dependent effects. GDF-9 has been reported to regulate granulosa cell In ovine granulosa cells, ovine BMP-15 given together with mitosis through both Smad-dependent and -independent mouse GDF-9 or ovine GDF-9 was more potent in stimu- signaling pathways (Huang et al., 2009). In human lutei- lating ovine granulosa cells mitosis compared with each nized granulosa cells, GDF-9 upregulated cyclin D1 and E growth factor independently, whereas in bovine granulosa mRNA, and protein expression and phosphorylation of cells there is little or no co-operative action between ovine cyclin D1 and Rb protein via ERK1/2 activation, and sup- BMP-15 and ovine GDF-9 in terms of mitotic activity pressed the negative cell-cycle regulators p15INK4B (McNatty et al., 2005c). The species of origin of GDF-9 andp16INK4A via Smad3 activation. Consistent with the general also affected the progesterone response as well as inhibin consensus that GDF-9 signals through the type I receptor production by ovine granulosa cells. Therefore, the effects ALK-5, SB431542 (an ALK-4/5/7 inhibitor) was able to block of GDF-9 and BMP-15, when combined, can be cooperative the mitotic activity of GDF-9 (Huang et al., 2009). compared with each growth factor alone; however, the GDF-9 also plays a key role in controlling theca cell response of granulosa cells from different species to
GDF-9 from a particular species can vary, as does the functions. GDF-9 effects on theca cells were initially uncov-
response of granulosa cells to different sources of GDF-9. ered by an in vivo study in which intra-peritoneal injections
The use of recombinant GDF-9 in in vitro assays is of GDF-9 into immature rats promoted the progression of
primordial and primary follicles to small preantral follicles critical for determining the biological actions of GDF-9 in and caused an increase in the expression of the theca cell- the ovary. However, it appears that different preparations of speci？c marker CYP17 (Vitt et al., 2000b). GDF-9 stimu- recombinant GDF-9 protein may have different activities. lates testosterone production in cultured rat preantral folli- Speci？cally, the inclusion of a C-terminal, af？nity-puri？ca-
cles. Taken together with the ？nding that the androgen tion tag may affect GDF-9 bioactivity (Mottershead et al.,
2008), although in vitro GDF-9 studies to date have mostly receptor antagonist flutamide blocks GDF-9-induced pre-
utilized N-terminal tag or non-tag preparations. Moreover, antral growth, it is suggested that GDF-9 actions in theca
cells as well as granulosa cells are important for promoting whether or not FSH is required for the activity of GDF-9 in preantral follicle growth to the early antral stage (Orisaka cumulus cell expansion remains to be determined, as dis- et al., 2009). GDF-9 actions in theca cells were also re- cussed previously (Pangas and Matzuk, 2005). Future ported in bovine follicles (Spicer et al., 2008). GDF-9 sti- studies should ideally adopt the use of a standard method
of production of recombinant GDF-9. mulates the proliferation of theca cells derived from small
follicles, inhibiting theca cell IGF-1- and LH-induced pro-
gesterone and androstenedione production. Hence, GDF-9
may play a role in increasing proliferation but suppressing GENETIC STUDIES OF MUTATIONS IN THE differentiation of theca cells from small follicles in bovine Bmp15 GENE ovary.
Bmp15 Knockout and Bmp15 Transgenic Mice GDF-9 actions in theca cells to regulate androgen pro-
duction subsequently affect granulosa cells at a number of Knocking-out the Bmp15 gene unexpectedly revealed levels, with androgen acting as a substrate to facilitate minimal alterations in follicle development and fertility in granulosa cell estrogen production from androgen aroma- mice (Yan et al., 2001). Heterozygous and null males and tization and also via direct effects of androgens on females were all viable and developed normally. Bmp15 null
13 Mol Reprod Dev 78:9–21 (2011)
Molecular Reproduction & Development TSUKA ET AL. O
2008). Interestingly, however, a chimeric protein consisting males were fertile with normal testis size, whereas Bmp15
of the human proregion, human cleavage site, and mouse homozygous mutant females were subfertile, with reduced
litter size compared with heterozygous and wild-type mature region (termed hhmBMP-15) could be processed,
leading to the secretion of mature protein (Hashimoto et al., females. Bmp15 null ovaries typically contained follicles at all stages of development and multiple corpora lutea. How- 2005). On this basis, the role of BMP-15 in folliculogenesis
was investigated by generation of transgenic (TG) mice ever, on occasion Bmp15 null ovaries contained very few overexpressing hhmBMP-15 exclusively in oocytes follicles and increased ZP remnants, indicating increased
follicle atresia. Further, in response to ovulation stimuli, (McMahon et al., 2008a). BMP-15 TG mice carrying a Bmp15 null females ovulated fewer oocytes, with some flox-stopped hhmBmp15 transgene were generated using oocytes remaining trapped in follicles. Also these mice have a Cre/loxP system. Female transgenic hhmBMP-15-flox defects in early embryonic development, resulting in sub- mice were mated with males expressing Cre recombinase fertility. Overall in mice, folliculogenesis is largely unaffect- under control of the oocyte-speci？c ZP3-promoter. BMP-15
ed by the loss of BMP-15 and the defects are con？ned to the mature protein levels were ？vefold higher in BMP-15 TG
ovulation process and subsequent fertilization. than wild ovaries, with BMP-15 protein detected exclusively
in oocytes from the primary follicle stage throughout folli- GDF-9 expression was not altered in Bmp15 null ovaries, culogenesis (McMahon et al., 2008a). therefore compensation by GDF-9 most likely does not
explain the less severe phenotype of the Bmp15 knockout Granulosa cells from immature BMP-15 TG mice dis-
played an increased mitotic potential and reduced FSHR mice compared with Gdf9 null mice. Generation of double /þmRNA expression. Primordial follicle formation and the Gdf9 and Bmp15 mutant mice (Bmp15/Gdf9/ ) facili- primordial to primary transition occurred normally in tated analysis of biological interactions between the two molecules (Yan et al., 2001). Normal folliculogenesis and BMP-15 TG mice. However, a signi？cant decrease in pri- /mary follicles with a concomitant increase in secondary corpora lutea were occasionally observed in Bmp15/ þfollicles was observed, which may reflect elevated granu- Gdf9/ ovaries, similar to Bmp15 null ovaries. However, losa cell mitosis in primary follicles. Although secondary several abnormalities were observed, including reduced numbers of late-stage follicles, increased oocyte loss and follicles are not dependent on FSH, they are sensitive to an accumulation of ZP remnants, and an absence of cor- FSH, thus increased secondary follicle atresia in BMP-15 pora lutea. While early phases of folliculogenesis were TG mice may be the result of suppressed FSHR signaling. /þAlthough comparable numbers of dominant follicles reach relatively normal and Bmp15/Gdf9/ mice ovulated a the preovulatory stage initially, the ovarian reserve is ex- high number of oocytes in response to exogenous hor-
mones, the ability of the oocytes to be fertilized in vivo was hausted earlier in BMP-15 TG mice due to accelerated dramatically reduced. This was also observed, though less follicle development and increased atresia, resulting in a //premature onset of acyclicity. Therefore, BMP-15 TG pronounced, in Bmp15mice. Similar to Bmp15mice,
cumulus cells failed to stably adhere to eggs isolated from mice con？rmed important in vivo BMP-15 functions, /þincluding the ability to stimulate granulosa cell mitosis the oviducts of Bmp15/Gdf9/ mutant mice. Analysis of /(Otsuka et al., 2000) and to inhibit FSHR expression /þBmp15and Bmp15/Gdf9/ ovaries from mice stim- (Otsuka et al., 2001c). Further, the early onset of acyclicity ulated with PMSG/hCG revealed a frequent absence of in BMP-15 TG mice shows suppression of BMP-15 actions cumulus expansion and the existence of larger oocytes,
during early folliculogenesis is important for restraining suggesting that BMP-15 is functionally involved in the
process of cumulus expansion and in the functional inter- follicle development to prevent premature loss of the follicle action between cumulus cells and oocytes. Ovaries of the reserve. //Collectively these mouse models show that BMP-15 Bmp15/Gdf9mice at early time-points resembled /plays a key role during late stages of follicle development Gdf9ovaries with a block at the type 3B primary follicle /and following ovulation. Importantly, the biological impor- stage, thus the block in follicle development in Gdf9mice tance of BMP-15 versus GDF-9 during folliculogenesis may is not due to unopposed BMP-15 actions (Yan et al., 2001). differ between mice and other species, and this may reflect At later ages, additional unique abnormalities were ob- //inherent differences between mono- and poly-ovulatory served in Bmp15/Gdf9mouse ovaries compared with /mammals (Yan et al., 2001; Galloway et al., 2002). Gdf9ovaries, including enhanced oocyte loss, in- creased ZP remnants, and interstitial cell proliferation.
Thus, BMP-15 appears to play synergistic, but independent
roles in oocyte survival with GDF-9. Ewes With Bmp15 Mutations
Variation in ovulation rate in sheep due to genetic differ- A major defect detected in female mice lacking BMP-15
is during the ovulation process, which supports ？ndings that ences is well known. Inverdale and Hanna sheep, naturally functional mature BMP-15 protein level in mouse oocytes is occurring strains, provided a major breakthrough in the
reproductive biology of BMP-15 (Galloway et al., 2000). very low until after the LH surge in vivo (Gueripel et al., 2006;
The gene responsible for the ovulation variability in these Yoshino et al., 2006). In addition, the recombinant mouse
BMP-15 proprotein was not readily processed into the sheep was known to be X-linked, then in 2000 the two functional mature protein in transfected HEK293 cells causal independent point mutations were localized to the (Hashimoto et al., 2005), although in a different experimen- Bmp15 gene, namely BMP-15V31D and BMP-15E23Term fortal setting the mature protein was detected (McIntosh et al., Inverdale and Hanna strains, respectively (Galloway et al.,
14 Mol Reprod Dev 78:9–21 (2011)
OLE OF GDF-9 AND BMP-15 IN OVARIAN FUNCTION R
In vitro studies provided an interesting interpretation 2000). Homozygous carriers of the Inverdale and Hanna
Bmp15 mutations are infertile with streak ovaries showing regarding the role of endogenous GDF-9 in the Bmp15 folliculogenesis arrested at the primary stage, whereas mutant sheep. When recombinant human BMP-15 with a heterozygous careers exhibit higher ovulation rates and mutation at the 31st amino acid from Ile to Asp (BMP- larger litter size than controls (Galloway et al., 2000; 15I31D), to mimic ovine BMP-15V31D identi？ed in Inverdale
Shimasaki et al., 2003). sheep (Galloway et al., 2000), was expressed in HEK293 The importance of BMP-15 in sheep fertility was con- cells, the BMP-15I31D proprotein was processed and the
mature dimeric BMP-15I31D was normally secreted. How- ？rmed with the identi？cation of two additional Bmp15 point ever, when BMP-15I31D was co-expressed with GDF-9, the mutations, BMP-15Q-29Term and BMP-15S99I, in Cambridge processing of proprotein and thus secretion of the mature and Belclare sheep that resulted in the same phenotype as
the Inverdale and Hanna ewes (Hanrahan et al., 2004) protein were concomitantly disrupted (Liao et al., 2003). (Table 1). Subsequently, two more, new BMP-15 mutants Aberrant processing and secretion are suggested to occur (BMP-1517bp-del and BMP-15C53Y) have been identi？ed in by formation of BMP-15I31D/GDF-9 heterodimers that are
Rasa Aragonesa and Lacaune sheep, respectively (Bodin not susceptible to normal proteolytic cleavage leading to et al., 2007; Martinez-Royo et al., 2008; Monteagudo et al., degradation within cells expressing these molecules (Liao 2009). Both Rasa Aragonesa and Lacaune sheep display a et al., 2003). Given the negative impact of the BMP-15I31D
similar phenotype to other mutant ewes with increased mutation on secretion of normal GDF-9, it is reasonable to ovulations in the heterozygotes and sterility in the homo- propose that decreased secretion of GDF-9 may be, at least zygotes (Table 1). In vitro studies showed that the BMP- in part, functionally involved in the aberrant phenotype of 15the homozygous Inverdale ewes. C53Y mutation impairs the maturation process of BMP-15
protein, resulting in defective secretion of both the mature
and proprotein (Bodin et al., 2007). In addition, another
sheep strain with increased ovulation rate, Woodlands Women With BMP15 Mutations
sheep, was reported to bear a putative genetic mutation Critical roles of BMP-15 in female fertility have also been W(FecX2) that is different from known Bmp15 and Gdf9 demonstrated in women. The discovery of BMP15 muta- mutations (Feary et al., 2007). Ovaries of Woodlands ewes tions in female patients with reproductive defects provided express reduced levels of BMP-15 in oocytes and ALK-6 in the opportunity to translate basic BMP-15 knowledge to the both oocytes and granulosa cells, while the expression clinic. The ？rst BMP-15 mutation in women that was asso- patterns of GDF-9 and BMP-RII were not altered. There- ciated with hypergonadotropic ovarian failure, due to ovari- fore, functional defects in BMP-15 may be involved in the an dysgenesis, was an A-G transition at position 704 of Wincreased ovulation rate in FecX2mutant ewes (Feary BMP15 gene (Di Pasquale et al., 2004). This mutation et al., 2007). The prevalence of BMP-15 mutations identi- results in a non-conserved substitution of Y235C in the ？ed in sheep breeds with altered ovulation rates convinc- proregion of BMP-15 proprotein (BMP-15Y235C), which acts
ingly shows the important role this oocyte protein plays in in a dominant negative fashion by altering the processing of reproductive function in sheep. the wild-type BMP-15 proprotein (Di Pasquale et al., 2004).
The carriers of this mutation were two sisters who inherited The infertile and superfertile phenotypes of the Bmp15 mutant ewes were both reproducible by immunizing wild- the mutation from their father. Interestingly, they were type ewes against synthetic peptides derived from sheep heterozygous carriers, yet their streak ovaries resembled BMP-15 mature protein (Juengel et al., 2002, 2004b). the ovarian phenotype of homozygous mutant BMP-15 Speci？cally, the use of a short-term immunization protocol ewes. The exaggerated phenotype may indicate this muta- achieved an increase in ovulation rates (Juengel et al., tion has dominant negative activity.
2002, 2004b), while a longer-term and higher-dose protocol A number of other mutations in the BMP15 gene have typically resulted in an anovulatory phenotype (Juengel been identi？ed in women with POF (Di Pasquale et al., et al., 2002, 2004b). Further studies showed that antibodies 2004, 2006; Dixit et al., 2005, 2006; Laissue et al., 2006). In generated against the N-terminal region of BMP-15 or GDF- addition, rare deletions and missense mutations in the 9 were most effective at inhibiting the paracrine actions of coding region of BMP-15 have also been identi？ed in
these oocyte-secreted factors in vivo and in vitro, inducing mothers of dizygotic twins (Montgomery et al., 2004; Palm- an anovulatory phenotype (McNatty et al., 2007). Similarly er et al., 2006). Therefore, these phenotypes associated in cattle, immunization against BMP-15 and/or GDF-9 al- with BMP15 gene mutations are similar to GDF9 mutations. tered follicle development (Juengel et al., 2009). In terms of However, in contrast to the ？nding that GDF-9P103S ,fertility, however, the outcome varied, with some cattle GDF-9P374L, and GDF-9R454C mutations signi？cantly in-
immunized to BMP-15 peptide alone becoming anovulatory creased ovulation rate in women (Palmer et al., 2006), no whereas others immunized with BMP-15 or BMP-15 with signi？cant relationship was detected between 35 BMP15 GDF-9 peptides had increased ovulation rates (Juengel gene SNPs and dizygotic twinning (Zhao et al., 2008). et al., 2009). Overall, BMP-15 appears to be important for To explore the biological signi？cance of the BMP15 and promoting early stages of follicle growth while restraining GDF9 gene mutations found in women with POF, two the number of dominant preovulatory follicles, and is a key representative BMP-15 (BMP-15R76C and BMP-15R206H )determination of ovulation quota and litter size in sheep and and GDF-9 (GDF-9K67E and GDF-9P103S) mutants that cattle (Shimasaki et al., 2004). occur with a high incidence in POF patients but have not
15 Mol Reprod Dev 78:9–21 (2011)
Molecular Reproduction & Development TSUKA ET AL. O
been identi？ed in normal cases (Dixit et al., 2005, 2006; 2001a). Follistatin is strongly expressed in dominant folli- Kovanci et al., 2007) were selected for study (Inagaki and cles, with very low or undetectable levels in atretic follicles. Shimasaki, 2010). All these mutations are located in the As BMP-15 inhibits FSHR expression, follistatin regulation proregion of the proprotein, thus the mature protein should of BMP-15 actions is likely important for maintaining gran- be indistinguishable from wild-type if the posttranslational ulosa cell responsiveness to FSH.
proprotein processing occurs normally. Interestingly, these Studies to explore BMP-15 bioactivity were initiated mutations impaired proprotein posttranslational proces- using recombinant human BMP-15 tagged with a Flag sing, leading to reduced production of mature BMP-15 and epitope at the C-terminus (Otsuka et al., 2000), and mount- GDF-9 proteins (Inagaki and Shimasaki, 2010). As a con- ing biological data were reported (Otsuka et al., 2000, sequence, these BMP-15 and GDF-9 mutations may be 2001a,c; Otsuka and Shimasaki, 2002a,b; Moore et al., associated with a short early period of enhanced fertility, 2003). Recently, N-terminally tagged recombinant human leading to the increased likelihood of dizygotic twins and/or BMP-15 protein was also produced and showed activity in rapid exhaustion of the ovarian reserve leading to stimulating the transcription of genes including Pentraxin3 POF (Inagaki and Shimasaki, 2010). Further phenoty- and HAS2, inhibitory Smad6/7, BMP antagonists, and in- pe–genotype analysis is necessary to conclude the biologi- hibin/activin bA and bB subunits (Li et al., 2009). Addition- cal impact of human BMP-15 mutations on the occurrence ally, recombinant ovine BMP-15 and recombinant ovine or of POF and dizygotic twinning. mouse GDF-9 were shown to exhibit a co-operative regu- Overall, parallels between phenotypes in women and lation on proliferation and FSH-induced differentiation of sheep with BMP-15 mutations suggest that reduced BMP- ovine, bovine, and rat granulosa cells (McNatty et al., 15 and/or GDF-9 levels are linked to increased ovulation 2005b,c).
rate and litter size. Further, proper posttranslational proces- BMP-15 is also involved in the regulation of cumulus cell sing of the BMP-15 proprotein is a critical aspect of female apoptosis. In bovine ovaries, removal of the oocyte from fertility in sheep as well as in humans. cumulus–oocyte complexes triggers cumulus cell apopto- sis. The induction of cumulus cell apoptosis can be pre-
vented by treatment with BMP-15, but not with GDF-9
(Hussein et al., 2005). Thus, BMP-15 may contribute to BIOLOGICAL FUNCTIONS OF BMP-15 IN decreasing the incidence of apoptosis within cumu- REGULATING FOLLICULOGENESIS lus–oocyte complexes until the point of ovulation. BMP- BMP-15 is exclusively expressed in the oocyte within the 15 is also involved in stimulating cumulus expansion ovary, with expression increasing in relation to follicle (Yoshino et al., 2006) by enhancing cumulus cell expres- growth and development (Otsuka et al., 2000). Consistent sion of epidermal growth factor (EGF)-like growth factors. with responsiveness to BMP ligands throughout fol- Oocytes promote the expression of EGF receptors on liculogenesis, expression of the type I (ALK-6) and type II cumulus cells, which is crucial for cumulus cells to be (BMP-RII) receptors utilized by BMP-15 has been con- capable of responding to LH-induced mural granulosa cell ？rmed in granulosa cells from the primordial to primary signals. Oocyte removal reduces EGF receptor expression stages onwards (Moore et al., 2003; McNatty et al., in mouse cumulus cells, and treatments with either GDF-9 2005a). In vitro studies using recombinant human BMP- or GDF-9 plus BMP-15 can restore EGF receptor levels, 15 showed that BMP-15 stimulates proliferation of undiffer- suggesting that both GDF-9 and BMP-15 are crucial for the entiated rat granulosa cells in an FSH-independent manner process of cumulus cell expansion through induction of the (Otsuka et al., 2000). However, BMP-15 can also inhibit EGF receptor (Su et al., 2010).
FSH actions by suppressing rat granulosa cell FSHR ex- A link between BMP and ？broblast growth factor (FGF)
pression (Otsuka et al., 2001c). BMP-15 inhibits FSH-in- signaling has been established in a number of tissues. duced granulosa cell expression of steroidogenic acute FGFs play an important role during embryonic development
and in adult tissues for the regulation of the nervous system, regulatory protein (StAR), Cyp11a1, 3b-hydroxysteroid de-
hydrogenase (HSD), LH receptor, and inhibin/activin sub- tissue repair, and tumor angiogenesis. In the ovary, FGF-8 units (Otsuka et al., 2001c). Moreover, BMP-15 stimulates is expressed speci？cally in oocytes, and FGF receptors in KL expression in granulosa cells (Otsuka and Shimasaki, granulosa cells (Berisha et al., 2004; Buratini et al., 2005; 2002a). Interestingly, KL inhibits BMP-15 expression in the Miyoshi et al., 2010). Since mammalian oocytes are unable oocytes. Thus, BMP-15 and KL form a negative feedback to initiate glycolysis, companion cumulus cells must trans-
mit to the oocyte glycolysis products necessary for oocyte loop between the oocyte and surrounding granulosa cells;
when the oocyte produces BMP-15, this stimulates granu- development. Expression of cholesterol biosynthesis en- /þlosa cells to produce KL that, in turn, signals back to the zymes is reduced in both Bmp15/Gdf9/ double mutant oocyte via c-kit to further inhibit oocyte BMP-15 expression. cumulus cells and in wild-type cumulus cells after removal The combination of increased KL expression and subse- of oocytes (Su et al., 2008). Therefore, it appears that BMP- quent reduction in BMP-15 inhibition of FSH actions allows 15 and GDF-9 regulate cumulus cell cholesterol biosynthe- effective induction of granulosa cell proliferation (Otsuka sis as a compensation for the de？ciency in oocytes (Su
and Shimasaki, 2002a). et al., 2008), thus BMP-15 and FGF-8 cooperate to promote BMP-15 actions are regulated by follistatin, a protein that glycolysis in cumulus cells (Sugiura et al., 2007). The can bind BMP-15 and negate its activity (Otsuka et al., interaction between the FGF and BMP systems is also
16 Mol Reprod Dev 78:9–21 (2011)
OLE OF GDF-9 AND BMP-15 IN OVARIAN FUNCTION R
the involvement of BMP-15/GDF-9 in ovarian physiology important for the maintenance of FGF receptor signaling in
granulosa cells. FGF-induced SPRY2 expression negative- and pathophysiology in women.
ly affects FGF receptor signaling; however, BMP-15 and There are clinical implications for BMP-15 and GDF-9, GDF-9 suppress FGF-stimulated SPRY2 expression allow- speci？cally as a potential tool to help overcome female ing FGF signals to continue to act in cumulus cells (Sugiura infertility. POF is a common cause of infertility in women, et al., 2009). which can manifest as primary amenorrhea or secondary A mass-spectrometry analysis of the mature protein of amenorrhea after pubertal development. POF occurs either recombinant human BMP-15 revealed that the Ser residue through follicle dysfunction or follicle depletion, where pa- at the 6th position is phosphorylated (Saito et al., 2008). tients with follicle dysfunction are infertile despite follicles Further studies demonstrated that Golgi-apparatus casein remaining in the ovary while follicle depletion reflects pre- 6thkinase phosphorylates the Serof recombinant human mature loss of the primordial pool due to accelerated re- BMP-15 as well as GDF-9 (Saito et al., 2008). Importantly, cruitment or increased atresia. As BMP-15 and GDF-9 play the phosphorylation of these proteins is essential for their a role both in promoting early follicle growth as well as biological activities (McMahon et al., 2008b). Moreover, restraining dominant/preovulatory follicle development in de-phosphorylated BMP-15 and GDF-9 exhibited antago- mono-ovulatory mammals, there is potential for BMP-15 nistic activity toward not only their phosphorylated counter- and/or GDF-9 to help both overcome follicle dysfunction parts but also toward each other, as well as BMP-7, which and to slow further follicle depletion if identi？ed early enough.
shares the same type II receptor, BMP-RII. Thus, phos- The immunization studies in sheep and cattle showed phorylation may be a means of regulating the bioactivity of that the anovulatory phenotype of homozygous mutant
sheep could be reproduced by multiple, high-dose injec- BMP-15 and GDF-9, as well as other members of the TGF-b
superfamily. Identi？cation of the posttranslational modi？ca- tions of BMP-15 or GDF-9 peptides. Therefore, there is the tion of BMP-15 and GDF-9 is an important step towards potential to adopt this technique to use as a permanent, low understanding the molecular basis of their function. How- cost, non-surgical, and safe contraceptive option for women ever, it remains unknown whether their phosphorylation is or domesticated mammalian species. As this would arrest directly involved in the physiology of BMP-15 actions in vivo. follicle growth at the primary stage, this would disrupt Recently, Foxo3a, an oocyte transcription factor in- normal estrogen production, thus it may be considered to
be a too radical contraceptive option for normal women of a volved in the PI3K/Akt signaling pathway, has been shown
reproductive age. However, this could be a safe, non- to influence follicular development and female fertility.
Female transgenic mice with constitutively active Foxo3a surgical option for women who otherwise would undergo are infertile due to impaired oocyte growth and follicular surgical ovary removal for more serious and persistent
conditions that failed to be helped by traditional contra- development, leading to anovulation (Liu et al., 2007).
ceptives or pharmaceuticals, such as endometriosis or Constitutively active Foxo3a in oocytes causes a dramatic
reduction in the expression of BMP-15, connexin 37, and uterine ？broids, which are both estrogen responsive con- connexin 43, which are important molecules for the estab- ditions and a common reason for surgical ovary removal in lishment of paracrine and gap junction communications in women. However, before treatment with BMP-15 or GDF-9 follicles (Liu et al., 2007). This suggests that Foxo3a is a key could be used to assist with infertility, or immunization with signaling molecule that can reduce oocyte growth and GDF-9/BMP-15 peptides utilized as a contraceptive option, follicular development by inactivating BMP-15. translational studies evaluating the effects of treatment Overall, these studies have shown BMP-15 plays an doses and timing, as well as the effectiveness of exoge- important role in female fertility. While BMP-15 is not abso- nously administering mature protein versus designing a treat- lutely required for fertility of female rodents, a poly-ovulatory ment to effectively promote enhanced endogenous production species, it does contribute to the process of ovulation. In of the oocyte factors, would need to be performed.
In summary, the elucidation of species-dependent ge- contrast, in several mono-ovulatory species, including
netic differences in ovulation rate provided novel insight into sheep, cattle, and humans, BMP-15 is critical for female
fertility. potential target molecules for manipulating female repro-
duction. Utilizing the BMP-15/GDF-9 system could poten-
tially facilitate the development of a unique strategy that
may help overcome infertility in women with reproductive IMPLICATIONS FOR THE TRANSLATIONAL defects such as POF. STUDIES The clear association between mutations and deletions
in the Bmp15 and Gdf9 genes and altered reproductive ACKNOWLEDGMENTS function, in addition to characterization of the biological This work was supported by National Research Initiative functions of BMP-15 and GDF-9 in follicular cells, has
Competitive Grant no. 2008-35203-19024 from the USDA established the importance of these two molecules for
National Institute of Food and Agriculture (to S.S.) as well normal female fertility. In order to drive translation of the
as NIH Grants RO1 HD414940-08, HD414940-08S1, basic research on the role of BMP-15 and GDF-9 in ovarian
R21-HD41494-02, and R21-HD41494-02S1 (to S.S.). function to potential clinical therapeutic options, there is a
This work was also supported by NICHD/NIH through a requirement for translational research that will further de？ne
17 Mol Reprod Dev 78:9–21 (2011)
Molecular Reproduction & Development TSUKA ET AL. O
cooperative agreement (U54 HD012303) to S.S. F.O. was Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. 1999a. supported in part by Grants-in-Aid for Scienti？c Research, Paracrine actions of growth differentiation factor-9 in the mam- Takeda Science Foundation and WESCO Scienti？c Pro- malian ovary. Mol Endocrinol 13:1035–1048.
motion Foundation. Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM. 1999b. Molecular characterization of the follicle defects in the growth differentiation factor 9-de？cient ovary. Mol Endocrinol 13:1018– REFERENCES 1034.
Elvin JA, Yan C, Matzuk MM. 2000. Growth differentiation factor-9 Berisha B, Sinowatz F, Schams D. 2004. Expression and locali- stimulates progesterone synthesis in granulosa cells via a pros- zation of ？broblast growth factor (FGF) family members during taglandin E2/EP2 receptor pathway. Proc Natl Acad Sci USA the ？nal growth of bovine ovarian follicles. Mol Reprod Dev 97:10288–10293. 67:162–171.
Falck B. 1959. Site of production of oestrogen in rat ovary as Bodin L, Di Pasquale E, Fabre S, Bontoux M, Monget P, Persani L, studied in micro-transplants. Acta Physiol Scand 47:1–101. Mulsant P. 2007. A novel mutation in the bone morphogenetic
protein 15 gene causing defective protein secretion is associat- Feary ES, Juengel JL, Smith P, French MC, O’Connell AR, ed with both increased ovulation rate and sterility in Lacaune Lawrence SB, Galloway SM, Davis GH, McNatty KP. 2007. sheep. Endocrinology 148:393–400. Patterns of expression of messenger RNAs encoding GDF9,
BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular de- Buratini J, Jr., Glapinski VF, Giometti IC, Teixeira AB, Costa IB, velopment and characterization of ovarian follicular populations Avellar MC, Barros CM, Price CA. 2005. Expression of ？broblast in ewes carrying the Woodlands FecX2W mutation. Biol Reprod growth factor-8 and its cognate receptors, ？broblast growth 77:990–998. factor receptor (FGFR)-3c and-4, in fetal bovine preantral folli-
cles. Mol Reprod Dev 70:255–261. Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel
JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery Carabatsos MJ, Elvin J, Matzuk MM, Albertini DF. 1998. Charac- GW, Beattie AE, Davis GH, Ritvos O. 2000. Mutations in an terization of oocyte and follicle development in growth differen- oocyte-derived growth factor gene (BMP15) cause increased tiation factor-9-de？cient mice. Dev Biol 204:373–384. ovulation rate and infertility in a dosage-sensitive manner. Nat Di Pasquale E, Beck-Peccoz P, Persani L. 2004. Hypergonado- Genet 25:279–283. tropic ovarian failure associated with an inherited mutation of Galloway SM, Gregan SM, Wilson T, McNatty KP, Juengel JL, human bone morphogenetic protein-15 (BMP15) gene. Am J Ritvos O, Davis GH. 2002. Bmp15 mutations and ovarian Hum Genet 75:106–111. function. Mol Cell Endocrinol 191:15–18. Di Pasquale E, Rossetti R, Marozzi A, Bodega B, Borgato S, Gilchrist RB, Lane M, Thompson JG. 2008. Oocyte-secreted Cavallo L, Einaudi S, Radetti G, Russo G, Sacco M, Wasniews- factors: Regulators of cumulus cell function and oocyte quality. ka M, Cole T, Beck-Peccoz P, Nelson LM, Persani L. 2006. Hum Reprod Update 14:159–177. Identi？cation of new variants of human BMP15 gene in a large
cohort of women with premature ovarian failure. J Clin Endo- Gueripel X, Brun V, Gougeon A. 2006. Oocyte bone morphoge- crinol Metab 91:1976–1979. netic protein 15, but not growth differentiation factor 9, is in-
creased during gonadotropin-induced follicular development in Dixit H, Rao LK, Padmalatha V, Kanakavalli M, Deenadayal M,
the immature mouse and is associated with cumulus oophorus Gupta N, Chakravarty B, Singh L. 2005. Mutational screening of
expansion. Biol Reprod 75:836–843. the coding region of growth differentiation factor 9 gene in Indian
women with ovarian failure. Menopause 12:749–754. Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell Dixit H, Rao LK, Padmalatha VV, Kanakavalli M, Deenadayal M, R, Galloway SM. 2004. Mutations in the genes for oocyte-
derived growth factors GDF9 and BMP15 are associated with Gupta N, Chakrabarty B, Singh L. 2006. Missense mutations in
both increased ovulation rate and sterility in Cambridge and the BMP15 gene are associated with ovarian failure. Hum Genet
Belclare sheep (Ovis aries). Biol Reprod 70:900–909. 119:408–415.
Hashimoto O, Moore RK, Shimasaki S. 2005. Posttranslational Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM.
1996. Growth differentiation factor-9 is required during early processing of mouse and human BMP-15: Potential implication
in the determination of ovulation quota. Proc Natl Acad Sci USA ovarian folliculogenesis. Nature 383:531–535.
102:5426–5431. Dragovic RA, Ritter LJ, Schulz SJ, Amato F, Armstrong DT,
Hayashi M, McGee EA, Min G, Klein C, Rose UM, van Duin M, Gilchrist RB. 2005. Role of oocyte-secreted growth differentia-
tion factor 9 in the regulation of mouse cumulus expansion. Hsueh AJ. 1999. Recombinant growth differentiation factor-9
(GDF-9) enhances growth and differentiation of cultured early Endocrinology 146:2798–2806.
ovarian follicles. Endocrinology 140:1236–1244. El-Fouly MA, Cook B, Nekola M, Nalbandov AV. 1970. Role
Hickey TE, Marrocco DL, Amato F, Ritter LJ, Norman RJ, Gilchrist of the ovum in follicular luteinization. Endocrinology 87:288–
293. RB, Armstrong DT. 2005. Androgens augment the mitogenic
18 Mol Reprod Dev 78:9–21 (2011)