Knockout of pentraxin 3, a downstream target of growth differentiation factor-9, causes female subfertility

The ovulatory process is tightly regulated by endocrine as well as paracrine factors. In the periovulatory period, extensive remodeling of the follicle wall occurs to allow the extrusion of the oocyte and accompanying cumulus granulosa cells. Growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) are secreted members of the TGFbeta superfamily that are expressed beginning in the oocyte of small primary follicles and through ovulation. Besides its critical role as a growth and differentiation factor during early folliculogenesis, GDF-9 also acts as a paracrine factor to regulate several key events in preovulatory follicles. By analyzing GDF-9-regulated expression profiles using gene chip technology, we identified TNF-induced protein 6 (Tnfip6) and pentraxin 3 (Ptx3 or PTX3) as novel factors induced by GDF-9 in granulosa cells of preovulatory follicles. Whereas Tnfip6 is induced in all granulosa cells by the LH surge, Ptx3 expression in the ovary is specifically observed after the LH surge in the cumulus granulosa cells adjacent to the oocyte. PTX3 is a member of the pentraxin family of secreted proteins, induced in several tissues by inflammatory signals. To define PTX3 function during ovulation, we generated knockout mice lacking the Ptx3 gene. Homozygous null (Ptx3(-/-)) mice develop normally and do not show any gross abnormalities. Whereas Ptx3(-/-) males are fertile, Ptx3(-/-) females are subfertile due to defects in the integrity of the cumulus cell-oocyte complex that are reminiscent of Bmp15(-/-)Gdf9(+/-) double mutant and BMP type IB receptor mutant mice. These studies demonstrate that PTX3 plays important roles in cumulus cell-oocyte interaction in the periovulatory period as a downstream protein in the GDF-9 signal transduction cascade.     

Integral role of GDF-9 and BMP-15 in ovarian function

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 predominantly act on supporting granulosa cells via paracrine signaling. Genetic studies in mice demonstrated critical roles of two key oocyte-derived growth factors belonging to the transforming growth factor-β (TGF-β) superfamily, growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in ovarian function. The identification of Bmp15 and Gdf9 gene mutations as the causal mechanism underlying the highly prolific 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 genes have been identified in women with premature ovarian failure and in mothers of dizygotic twins. The purpose of this article is to review the genetic studies of GDF-9 and BMP-15 mutations identified in women and sheep, as well as describing the various knockout and overexpressing mouse models, and to summarize the molecular and biological functions that underlie the crucial role of these two oocyte factors in female fertility.     

GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.     

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are members of the transforming growth factor-beta superfamily. Both molecules are closely related in their primary structures and share a nearly identical spatiotemporal expression pattern in the oocyte during folliculogenesis in mammals. Here we have established a series of cell lines, which express recombinant BMP-15, GDF-9, or both, and investigated whether they form homodimers and/or heterodimers. We demonstrate the first evidence that both BMP-15 and GDF-9 can form non-covalent homodimers when expressed individually, while when both are co-expressed BMP-15/GDF-9 heterodimers are produced. Interestingly, when GDF-9 and BMP-15 are co-expressed the processing of both proproteins are significantly impaired as compared with that of the singly expressed proproteins, suggesting that the proprotein heterodimer is less susceptible to proteolytic cleavage than the individual homodimers. Since BMP-15 mutant sheep, called Inverdale, exhibit severe defects in ovarian function we have also established stable transformants expressing the mutant BMP-15 (InvBMP-15) alone or together with GDF-9. Although InvBMP-15 was previously predicted to be unable to form homodimers, we show here that it does form non-covalent dimers; however, the processing efficiency of InvBMP-15 proprotein is significantly lower than wild-type BMP-15. Surprisingly, when GDF-9 is co-expressed, the processing and secretion of InvBMP-15 is abolished, and the processing of GDF-9 is also severely impaired, suggesting that the heterodimers of InvBMP-15/GDF-9 proproteins are not susceptible to proteolytic cleavage and thus degrade in the cells. Based on these findings we propose a novel hypothesis that a decrease in GDF-9 secretion may be involved in causing infertility in homozygous Inverdale ewes.     

Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells

Mammalian oocytes are deficient in their ability to carry out glycolysis. Therefore, the products of glycolysis that are necessary for oocyte development are provided to oocytes by companion cumulus cells. Mouse oocytes secrete paracrine factors that promote glycolysis in cumulus cells. The objective of this study was to identify paracrine factors secreted by oocytes that promote glycolysis and expression of mRNA encoding the glycolytic enzymes PFKP and LDHA. Candidates included growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and fibroblast growth factors (FGFs). Bmp15-/- and Gdf9+/- Bmp15-/- (double mutant, DM) cumulus cells exhibited reduced levels of both glycolysis and Pfkp and Ldha mRNA, and mutant oocytes were deficient in promoting glycolysis and expression of Pfkp and Ldha mRNA in cumulus cells of wild-type (WT) mice. Alone, neither recombinant BMP15, GDF9 nor FGF8 promoted glycolysis and expression of Pfkp and Ldha mRNA in WT cumulus cells. Co-treatment with BMP15 and FGF8 promoted glycolysis and increased expression of Pfkp and Ldha mRNA in WT cumulus cells to the same levels as WT oocytes; however, the combinations of BMP15/GDF9 or GDF9/FGF8 did not. Furthermore, SU5402, an FGF receptor-dependent protein kinase inhibitor, inhibited Pfkp and Ldha expression in cumulus cells promoted by paracrine oocyte factors. Therefore, oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells.     

Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles

The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.     

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.     

Functional and molecular characterization of naturally occurring mutations in the oocyte-secreted factors bone morphogenetic protein-15 and growth and differentiation factor-9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that are critical local regulators of ovarian physiology. Recent studies have identified a number of mutations in these genes that cause increased fertility and infertility in heterozygous or homozygous ewes carrying the mutations, respectively. Interestingly, heterozygous ewes with a mutation in both BMP-15 and GDF-9 exhibit higher fertility than those having mutation in only one of the genes. Here, we have produced recombinant human BMP-15 and GDF-9 that carry the mutations identified in those sheep, i.e. I31D and S99I in BMP-15 and S77F in GDF-9. We found that when individually expressed, both BMP-15 mutations had no effect on the processing, secretion, and dimerization of the mature proteins or on the biological activity of the molecules. However, when mutant BMP-15 was co-expressed with wild-type GDF-9, the secretion of BMP-15 and GDF-9 was significantly reduced, suggesting that the mechanisms by which the BMP-15 mutations affect sheep fertility occurs at the level of protein secretion rather than dimerization and biological activity. Moreover, when mutant GDF-9 was co-expressed with mutant BMP-15, the secretion levels of both proteins were significantly lower than those of cells co-expressing wildtype GDF-9 and mutant BMP-15, suggesting a possible mechanism for the extreme fertility observed in the compound heterozygous mutant sheep.     

Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion

Bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9 are oocyte-secreted growth factors that are critical local regulators of ovarian function and may be involved in preovulatory cumulus expansion. As cumulus expansion occurs in response to the ovulatory surge, the present study was designed: 1) to investigate whether GDF9 and BMP15 are regulated by gonadotropins in the mouse ovary; and 2) to visualize changes in both GDF9 and BMP15 immunostaining in response to gonadotropins. Immature 21-day-old mice were sequentially treated with recombinant human FSH (r-hFSH), 5 IU daily, at Days 21, 22, and 23 of life, then injected with 5 IU hCG at Day 24 of life. In response to r-hFSH, steady-state Bmp15 mRNA expression levels increased in both total ovaries and cumulus-oocyte complexes, whereas Gdf 9 mRNA levels did not. In addition, BMP15 protein levels increased in total ovaries. The GDF9 immunostaining was exclusively seen in growing oocytes in both control and gonadotropin-treated mice, whereas that of BMP15, which was also primarily seen in growing oocytes, exhibited important changes in response to gonadotropins. Strong BMP15 immunostaining was observed in the follicular fluid of atretic antral follicles after FSH treatment and in expanded, but not in compact, cumulus cells after hCG. The present results show for the first time that BMP15 levels increase during gonadotropin-induced follicular development, in parallel with oocyte maturation, and that this local factor is likely involved in cumulus expansion as previously suggested by studies in Bmp15-null mice.     

Potential role of bone morphogenetic protein-15 in zebrafish follicle development and oocyte maturation

Bone morphogenetic protein (BMP)-15 is a member of the transforming growth factor beta (TGF-beta) superfamily and is closely related to growth and differentiation factor (GDF)-9, both structurally and functionally. In mammals, BMP-15 is predominantly produced by oocytes and exerts important regulatory functions within the ovary, such as promoting early folliculogenesis, preventing premature luteinization and enhancing cumulus cell expansion. The role of BMP-15 in mammalian ovary differs between monoovulatory and polyovulatory species. Recent studies in zebrafish have provided initial evidence that BMP-15 is also an important regulator of ovarian functions. BMP-15 is produced by the zebrafish ovary throughout follicle development and maturation. In vitro studies using zebrafish follicles have revealed that incubation with recombinant human BMP-15 or over-expression of BMP-15 in oocytes results in an inhibition of gonadotropin- and maturation inducing hormone (MIH)-induced oocyte maturation. Conversely, immnunoneutralization with BMP-15 antiserum or silencing of BMP-15 expression using morpholino antisense oligonueclotides enhances oocyte maturation. A key step in BMP-15 action is the sensitivity of follicles to MIH. In vivo injection of BMP-15 antiserum causes a significant decrease in maturation-incompetent (insensitive to MIH) small early growth phase follicles and a concomitant increase in mature follicles. These findings support a role in BMP-15 in preventing precocious oocyte maturation in zebrafish. We propose that the suppression of premature oocyte maturation by BMP-15 may be important to maintain oocyte quality and subsequent ovulation and fertilization.     

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.     

Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary.

The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-beta superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P(4)) but not estradiol (E(2)) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P(4) and E(2) production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P(4) production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action.