Oocyte-derived Growth Differentiation Factor Research Articles

Oocyte-derived growth differentiation factor 9 suppresses the expression of CYP17A1 and androgen production in human theca cells

ObjectiveTo investigate the direct effect of growth differentiation factor 9 (GDF9) on androgen production in human theca cells. DesignExperimental study SettingTertiary hospital-based research laboratory Patient(s)Women underwent IVF/intracytoplasmic sperm injection (ICSI) at our clinic were included in this study. Intervention(s)Primary cultured human theca cells from women undergoing IVF/ICSI treatment were treated with GDF9, Activin receptor-like kinase 5 (ALK5) inhibitor, and SMAD4 agonist. Main Outcome Measure(s)The expression of androgen synthesis-related gene StAR, CYP17A1, and LHCGR, levels of androstenedione and testosterone, phosphorylation of SMAD2/3 and the interaction between bone morphogenetic protein receptor II (BMPRII) and ALK5 were evaluated by RT-qPCR, Western blot, enzyme-linked immunosorbent assays, and co-immunoprecipitation assays respectively. ResultsGDF9 decreased StAR, CYP17A1, and LHCGR expression levels in human theca cells, which was prevented by the treatment with the ALK5 inhibitor, and suppressed production of androgen in human theca cells. GDF9 increased SMAD2/3 phosphorylation, and the ALK5 inhibitor also suppressed this effect. BMPRII and ALK5 bound to each other following GDF9 stimulation. The SMAD4 agonist kartogenin also decreased mRNA levels of StAR and CYP17A1, and protein levels of StAR in human theca cells. ConclusionGDF9 can activate the BMPRII-ALK5-SMAD2/3 signaling pathway, suppress CYP17A1 expression and androgen production in human theca cells.

Growth differentiation factor 9 promotes follicle-stimulating hormone-induced progesterone production in chicken follicular granulosa cells

The function of oocyte-derived growth differentiation factor 9 (GDF9) in ovarian follicles has thus far been poorly defined in avian species compared with the defined function in mammals. Our aim here is to investigate the effects of GDF9 on steroidogenesis and on chicken ovarian granulosa cell (GC) mitosis. Primary GCs from both prehierarchical (6–8 mm in diameter, phGCs) and preovulatory follicles (F1–F5, poGCs) were cultured in the presence or absence of the GDF9 protein. The progesterone (P4) levels in the culture medium were then measured by radioimmunoassay (RIA), and the expression levels of steroidogenesis genes were detected by quantitative PCR. We found that GDF9 alone showed no significant effect on the P4 levels by regulating the expression of steroidogenesis genes, such as STAR, CYP11A1 and HSD3B. Further experiments indicated that GDF9 promoted follicle-stimulating hormone (FSH)-induced P4 production and STAR expression. GDF9 also rescued the FSH-induced decrease of FSH receptor (FSHR) expression but had no effect on the forskolin-induced P4, STAR and forskolin-inhibited FSHR expression levels, suggesting that GDF9 might achieve its regulatory role of P4 by enhancing FSHR and STAR expression. In addition, GDF9 also promoted GC cell cycle progression, regulated the gene transcription of related genes, potentiated DNA replication and inhibited apoptosis. Interestingly, these effects differed between the phGCs and the poGCs. To our knowledge, this is the first report that illustrates the function of GDF9 on chicken GCs and the effects on ovarian steroidogenesis. Our findings highlight the regulation of central oocytes on the surrounding granulosa cells and emphasize the interaction between paracrine signals and endocrine hormones on ovarian progesterone production; these findings contribute to the understanding of the development of avian ovarian follicles.

Loss of oocyte Rps26 in mice arrests oocyte growth and causes premature ovarian failure

Global transcriptional activity increases as oocytes grow and is silenced in fully grown oocytes. Thus, the chromatin configuration varies during oocyte growth, but the molecular mechanisms regulating these changes remain to be clarified. Here, we studied a susceptibility gene of polycystic ovary syndrome (PCOS), RPS26, which is a ribosomal protein-encoding gene that is highly expressed in the ovary, but the functions of which remain unknown. Specific knockout of Rps26 in mouse oocytes resulted in retarded follicle development from pre-antral follicles to antral follicles, while the chromatin configurations of the oocytes were arrested at the transition from the non-surrounded nucleolus (NSN) to surrounded nucleolus (SN)-type. As a consequence, all oocytes died by postnatal day 84 resulting in premature ovarian failure (POF). Loss of Rps26 in oocytes led to decreased mRNA transcription and low levels of histone trimethylation on H3K4/H3K9 and DNA methylation at 5-cytosine, high levels of which are required for oocytes to transform from NSN to SN-type. Low protein levels of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15, and the oocyte-granulosa cell gap junction protein connexin 37 inhibited oocyte growth and retarded follicle development. The disruption of the phosphoinositide 3-kinase/protein kinase B/Forkhead box O-3a pathway contributed to oocyte death and follicle atresia. These results provide genetic clues for the clinical diagnosis of POF, especially in PCOS patients without treatment.

Oocyte-derived BMP15 but not GDF9 down-regulates connexin43 expression and decreases gap junction intercellular communication activity in immortalized human granulosa cells

In the ovary, connexin-coupled gap junctions in granulosa cells play crucial roles in follicular and oocyte development as well as in corpus luteum formation. Our previous work has shown that theca cell-derived bone morphogenetic protein (BMP)4 and BMP7 decrease gap junction intercellular communication (GJIC) activity via the down-regulation of connexin43 (Cx43) expression in immortalized human granulosa cells. However, the effects of oocyte-derived growth factors on Cx43 expression remain to be elucidated. The present study was designed to investigate the effects of oocyte-derived growth differentiation factor (GDF)9 and BMP15 on the expression of Cx43 in a human granulosa cell line, SVOG. We also examined the effect relative to GJIC activity and investigated the potential mechanisms of action. In SVOG cells, treatment with BMP15 but not GDF9 significantly decreased Cx43 mRNA and protein levels and GJIC activity. These suppressive effects, along with the induction of Smad1/5/8 phosphorylation, were attenuated by co-treatment with a BMP type I receptor inhibitor, dorsomorphin. Furthermore, knockdown of the central component of the transforming growth factor-β superfamily signaling pathway, Smad4, using small interfering RNA reversed the suppressive effects of BMP15 on Cx43 expression and GJIC activity. The suppressive effects of BMP15 on Cx43 expression were further confirmed in primary human granulosa-lutein cells obtained from infertile patients undergoing an in vitro fertilization procedure. These findings suggest that oocyte-derived BMP15 decreases GJIC activity between human granulosa cells by down-regulating Cx43 expression, most likely via a Smad-dependent signaling pathway.

Embryotropic actions of follistatin: paracrine and autocrine mediators of oocyte competence and embryo developmental progression

Despite several decades since the birth of the first test tube baby and the first calf derived from an in vitro-fertilised embryo, the efficiency of assisted reproductive technologies remains less than ideal. Poor oocyte competence is a major factor limiting the efficiency of in vitro embryo production. Developmental competence obtained during oocyte growth and maturation establishes the foundation for successful fertilisation and preimplantation embryonic development. Regulation of molecular and cellular events during fertilisation and embryo development is mediated, in part, by oocyte-derived factors acquired during oocyte growth and maturation and programmed by factors of follicular somatic cell origin. The available evidence supports an important intrinsic role for oocyte-derived follistatin and JY-1 proteins in mediating embryo developmental progression after fertilisation, and suggests that the paracrine and autocrine actions of oocyte-derived growth differentiation factor 9, bone morphogenetic protein 15 and follicular somatic cell-derived members of the fibroblast growth factor family impact oocyte competence and subsequent embryo developmental progression after fertilisation. An increased understanding of the molecular mechanisms mediating oocyte competence and stage-specific developmental events during early embryogenesis is crucial for further improvements in assisted reproductive technologies.

BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

SMAD7 antagonizes key TGFβ superfamily signaling in mouse granulosa cells in vitro

Transforming growth factor β (TGFβ) superfamily signaling is essential for female reproduction. Dysregulation of the TGFβ signaling pathway can cause reproductive diseases. SMA and MAD (mothers against decapentaplegic) (SMAD) proteins are downstream signaling transducers of the TGFβ superfamily. SMAD7 is an inhibitory SMAD that regulates TGFβ signaling in vitro. However, the function of SMAD7 in the ovary remains poorly defined. To determine the signaling preference and potential role of SMAD7 in the ovary, we herein examined the expression, regulation, and function of SMAD7 in mouse granulosa cells. We showed that SMAD7 was expressed in granulosa cells and subject to regulation by intraovarian growth factors from the TGFβ superfamily. TGFB1 (TGFβ1), bone morphogenetic protein 4, and oocyte-derived growth differentiation factor 9 (GDF9) were capable of inducing Smad7 expression, suggesting a modulatory role of SMAD7 in a negative feedback loop. Using a small interfering RNA approach, we further demonstrated that SMAD7 was a negative regulator of TGFB1. Moreover, we revealed a link between SMAD7 and GDF9-mediated oocyte paracrine signaling, an essential component of oocyte-granulosa cell communication and folliculogenesis. Collectively, our results suggest that SMAD7 may function during follicular development via preferentially antagonizing and/or fine-tuning essential TGFβ superfamily signaling, which is involved in the regulation of oocyte-somatic cell interaction and granulosa cell function.

DAN (NBL1) Specifically Antagonizes BMP2 and BMP4 and Modulates the Actions of GDF9, BMP2, and BMP4 in the Rat Ovary1

Although differential screening-selected gene aberrative in neuroblastoma (DAN, official symbol NBL1) is the founding member of the DAN subfamily of bone morphogenetic protein (BMP) antagonists, its antagonizing targets, gene regulation, and physiological functions remain unclear. Using diverse cell expression systems, we found that the generation of bioactive DAN is likely to be cell type specific. Unlike other phylogenetically close members, which are covalently linked homodimers, DAN forms a noncovalently linked homodimer during folding. Purified recombinant DAN specifically blocked signaling of BMP2 and BMP4 but not that of other ovarian-expressed transforming growth factor-beta members. Although widely distributed in many organs, DAN transcript level was periodically regulated by gonadotropins. Ovarian microdissection indicated that NBL1 (DAN) mRNA is mainly expressed in granulosa cells, where its transcript level is up-regulated by the gonadotropin-driven cAMP cascade. We further investigated the local regulation and ovarian functions of DAN. NBL1 (DAN) mRNA expression in granulosa cells was up-regulated by oocyte-derived growth differentiation factor 9 (GDF9), whereas treatment with DAN significantly reversed the inhibitory effect of BMP4 on follicle-stimulating hormone-induced progesterone production in cultured granulosa cells. Our findings suggest the DAN gradient in granulosa cells, established by oocyte-derived GDF9, may serve as an antagonist barrier that modulates the actions of theca-derived BMP4 and granulosa/theca-derived BMP2 during folliculogenesis both spatially and temporally.

Estrogen Promotes the Development of Mouse Cumulus Cells in Coordination with Oocyte-Derived GDF9 and BMP15

The differentiation and function of cumulus cells depend upon oocyte-derived paracrine factors, but studies on the estrogen receptor knockout mice suggested that estrogen also participates in these processes. This study investigates the possible coordination of estrogen and oocytes in the development and function of cumulus cells using cumulus expansion and the expression of transcripts required for expansion as functional endpoints. Preantral granulosa cell-oocyte complexes developed in vitro with 17β-estradiol (E2) exhibited increased levels of cumulus expansion and Has2 transcripts, encoding hyaluronan synthase 2, compared with those developed without E2. Moreover, cumulus cell-oocyte complexes (COCs) isolated from antral follicles and maintained in culture without E2 exhibited reduced cumulus expansion and Has2 mRNA levels compared with freshly isolated COCs. Exogenous E2, provided during the maintenance culture, alleviated these deficiencies. However, when oocytes were removed from COCs, E2 supplementation did not maintain competence to undergo expansion; the presence in culture of either fully grown oocytes or recombinant growth differentiation factor 9 (GDF9) was required. Recombinant bone morphogenetic protein 15, but not fibroblast growth factor 8, augmented the GDF9 effect. Oocytes or GDF9 suppressed cumulus cell levels of Nrip1 transcripts encoding nuclear receptor-interacting protein 1, a potential inhibitor of estrogen receptor signals. Therefore, E2 and oocyte-derived paracrine factors GDF9 and bone morphogenetic protein 15 coordinate to promote the development of cumulus cells and maintain their competence to undergo expansion. Furthermore, suppression of Nrip1 expression in cumulus cells by oocyte may be one mechanism mediating cross talk between oocyte and E2 signals that promotes follicular development.

Growth Differentiation Factor-9 Mediates Follicle-Stimulating Hormone-Thyroid Hormone Interaction in the Regulation of Rat Preantral Follicular Development

FSH regulates follicular growth in a stage-development fashion. Although preantral follicle stage is gonadotropin responsive, FSH is not required for preantral follicular growth. With the antrum, the follicles continue growing under the influence of FSH and become gonadotropin dependent. Although thyroid hormone is important for normal female reproductive function, its role and interaction with FSH in the regulation of preantral ovarian follicular growth is yet to be defined. In the present study, we have examined the action and interaction of FSH and T(3) in the regulation of the growth of preantral follicles, especially in their transition from preantral to early antral stage, using an established follicle culture system and evaluated the involvement of growth differentiation factor-9 (GDF-9) in this process in vitro. We have demonstrated that although T(3) alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth. Although FSH alone significantly down-regulated FSH receptor (FSHR) mRNA abundance in the preantral follicles and T(3) alone was ineffective, expression of the message was significantly increased in the presence of both hormones. In addition, intra-oocyte injection of GDF-9 antisense oligonucleotides (GDF-9 morpholino) induced follicular cell apoptosis and suppressed follicular growth induced by FSH and T(3). These responses were attenuated by exogenous GDF-9. Our findings support the concept that thyroid hormone regulates ovarian follicular development through its direct action on the ovary and that promotes FSH-induced preantral follicular growth through up-regulation of FSHR, a mechanism dependent on the expression and action of oocyte-derived GDF-9.

Growth Differentiation Factor 9 Promotes Rat Preantral Follicle Growth by Up-Regulating Follicular Androgen Biosynthesis

The transition from preantral to early antral stage is the penultimate stage of ovarian follicular development in terms of gonadotropin dependence and follicle destiny. Although oocyte-somatic cell communication is important in early follicular development, our knowledge of the precise role of the oocyte-derived growth differentiation factor (GDF)-9 during preantral follicle growth is incomplete. We examined whether and by what means oocyte-derived GDF-9 controls follicular development and steroidogenesis during the preantral to early antral transition, by a combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Intraoocyte injection of GDF-9 antisense suppressed rat preantral follicle growth in vitro, whereas GDF-9 enhanced follicular development. GDF-9 augmented testosterone production in preantral follicles. GDF-9 antisense suppressed androgen production and CYP17A1 mRNA expression in cultured follicles, a response attenuated by exogenous GDF-9. The nonaromatizable androgen 5alpha-dihydrotestosterone rescued the follicular growth arrest caused by GDF-9 down-regulation. The specific androgen receptor antagonist flutamide suppressed GDF-9-induced preantral follicle growth in vitro. The data suggest that GDF-9 plays an important role in promoting preantral follicle growth by up-regulating follicular androgen biosynthesis. GDF-9 is essential for CYP17A1 expression during follicular development from the preantral to the early antral stage.

Oocyte-granulosa-theca cell interactions during preantral follicular development

The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions.

Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF91

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3-6 mm) and large (8-22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.

Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.

Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.

Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth

Ovarian folliculogenesis is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of oocyte growth and somatic cell proliferation and differentiation. Within the follicle, paracrine interactions between the oocyte and surrounding granulosa cells are critical for normal cell development and function. This review focuses on the role of paracrine interactions during early oocyte and follicular development that ensure proper coordination of oocyte and somatic cell function. Particular emphasis is given to granulosa cell-derived Kit Ligand (KitL), whose functional importance for oocyte growth has been demonstrated by a wide range of in vivo and in vitro studies. Reported interactions between KitL and oocyte-derived growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) suggest the molecular basis of oocyte-granulosa cell interactions, but also hint at the complexity of these communications. These paracrine interactions and the structure of the oocyte-granulosa cell interface are follicle stage-specific and regulated by FSH. Elucidation of the molecular mechanisms that promote the development of healthy oocytes with good developmental competence has potential applications for improving fertility and for in vitro growth systems for oocytes from domestic animals and humans.

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

Members of the transforming growth factor-β (TGF-β) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-β 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-β superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-β, 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.

Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells

Ovarian inhibin production is stimulated by FSH and several TGFbeta family ligands including activins and bone morphogenetic proteins. Growth differentiation factor-9 (GDF-9) derived by the oocyte is a member of the TGFbeta/activin family, and we have previously shown that GDF-9 treatment stimulates ovarian inhibin-alpha content in explants of neonatal ovaries. However, little is known about GDF-9 regulation of inhibin production in granulosa cells and downstream signaling proteins activated by GDF-9. Here, we used cultured rat granulosa cells to examine the influence of GDF-9 on basal and FSH-stimulated inhibin production, expression of inhibin subunit transcripts, and the GDF-9 activation of Smad phosphorylation. Granulosa cells from small antral follicles of diethylstilbestrol-primed immature rats were cultured with FSH in the presence or absence of increasing concentrations of GDF-9. Secreted dimeric inhibin A and inhibin B were quantified using specific ELISAs, whereas inhibin subunit RNAs were analyzed by Northern blotting using (32)P-labeled inhibin subunit cDNA probes. Similar to FSH, treatment with GDF-9 stimulated dose- and time-dependent increases of both inhibin A and inhibin B production. Furthermore, coincubation of cells with GDF-9 and FSH led to a synergistic stimulation of both inhibin A and inhibin B production. GDF-9 treatment also increased mRNA expression for inhibin-alpha and inhibin-beta subunits. To investigate Smad activation, granulosa cell lysates were analyzed in immunoblots using antiphosphoSmad1 and antiphosphoSmad2 antibodies. GDF-9 treatment increased Smad2, but not Smad1, phosphorylation with increasing doses of GDF-9 leading to a dose-dependent increase in phosphoSmad2 levels. To further investigate inhibin-alpha gene promoter activation by GDF-9, granulosa cells were transiently transfected with an inhibin-alpha promoter-luciferase reporter construct and cultured with different hormones before assaying for luciferase activity. Treatment with FSH or GDF-9 resulted in increased inhibin-alpha gene promoter activity, and combined treatment with both led to synergistic increases. The present data demonstrate that oocyte-derived GDF-9, alone or together with pituitary-derived FSH, stimulates inhibin production, inhibin subunit mRNA expression, and inhibin-alpha promoter activity by rat granulosa cells. The synergistic stimulation of inhibin secretion by the paracrine hormone GDF-9 and the endocrine hormone FSH could play an important role in the feedback regulation of FSH release, thus leading to the modulation of follicle maturation and ovulation.

Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17alpha-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated.

Expression of mRNA encoding growth differentiation factor 9 and bone morphogenetic protein 15 during follicular formation and growth in a marsupial, the brushtail possum ( Trichosurus vulpecula)

The oocyte derived growth differentiation factor (GDF) 9 and bone morphogenetic protein 15 (BMP15; also known as GDF9b) are essential for normal follicular growth. However, little is known about expression of these factors during ovarian development. Therefore, we determined the ontogeny of expression of GDF9 and BMP15 mRNA in the developing ovary of the brushtail possum. Ovaries were collected from pouch young ( n=3–5 per group) around times of key developmental events namely: (1) morphological sexual differentiation (i.e. days 1–5 following birth), (2) after sexual differentiation (i.e. days 10–15), (3) before and during initiation of germ-cell meiosis (i.e. days 22–45), (4) shortly after initiation of follicular growth (i.e. days 78–85), (5) during preantral follicular growth (i.e. days 96–113) and (6) during antral follicular growth (i.e. days 155–190). Ovaries were also collected from three juvenile and four adult animals and gene expression was determined by in situ hybridization. The mRNAs encoding GDF9 and BMP15 were first observed in oocytes of newly-formed primordial follicles (i.e. days 78–85). Expression of both mRNAs was restricted to the oocyte and was present in follicles irrespective of whether they were non-growing primordial follicles or undergoing preantral or antral development. Thus, since the mRNAs encoding GDF9 and BMP15 were not observed until follicular formation, it is unlikely that these proteins have any role in early germ cell development. Nevertheless, the findings that the mRNAs encoding both proteins were observed in oocytes from the primordial stage of follicular formation suggest a possible role for these proteins in the maintenance of primordial follicles as well as a key role during follicular development. These results highlight important species differences in the ontogeny of expression of GDF9 and BMP15 between possums and other species such as the human, sheep or rat.