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Martin M Matzuk - One of the best experts on this subject based on the ideXlab platform.

  • Growth Differentiation factor 9 bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Jia Peng, John J. Eppig, Karen Wigglesworth, Adithya Rangarajan, Chandramohan Kattamuri, Randall T Peterson, Thomas B Thompson, Martin M Matzuk
    Abstract:

    The TGF-β superfamily is the largest family of secreted proteins in mammals, and members of the TGF-β family are involved in most developmental and physiological processes. Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-β superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.

  • transforming Growth factor β receptor type 1 is essential for female reproductive tract integrity and function
    PLOS Genetics, 2011
    Co-Authors: Julio E Agno, Mark A Edson, Ankur K Nagaraja, Takashi Nagashima, Martin M Matzuk
    Abstract:

    The transforming Growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this Growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Mullerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in Growth Differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell Differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.

  • Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells.
    Development, 2007
    Co-Authors: Koji Sugiura, Stephanie A Pangas, Martin M Matzuk, Karen Wigglesworth, Marilyn J. O'brien, Jason P. Affourtit, John J. Eppig
    Abstract:

    Oocyte-derived bone morphogenetic protein 15 (BMP15) and Growth Differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15 -/- and Bmp15 -/- Gdf9 +/- double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production.

  • oocyte derived bmp15 and fgfs cooperate to promote glycolysis in cumulus cells
    Development, 2007
    Co-Authors: Koji Sugiura, Stephanie A Pangas, Martin M Matzuk, Karen Wigglesworth, Francisco Javier Diaz, Marilyn J Obrien, Shweta Sharma, Shunichi Shimasaki, John J. Eppig
    Abstract:

    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.

  • knockout of pentraxin 3 a downstream target of Growth Differentiation factor 9 causes female subfertility
    Molecular Endocrinology, 2002
    Co-Authors: Simona Varani, Janet L Demayo, Heidi F. Horton, Michael C. Byrne, Francesco J Demayo, Julia A Elvin, Martin M Matzuk
    Abstract:

    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 TGFβ 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 L...

Robert B Gilchrist - One of the best experts on this subject based on the ideXlab platform.

  • 134 epidermal Growth factor receptor mapk3 1 pathway cross talk enables Growth Differentiation factor 9 to signal through smad2 3 in mouse granulosa cells
    Reproduction Fertility and Development, 2009
    Co-Authors: Maxime Sasseville, Lesley J Ritter, Darryl L Russell, David G Mottershead, Thao Nguyen, Robert B Gilchrist
    Abstract:

    Oocyte-secreted Growth Differentiation factor 9 (GDF9) plays a critical role throughout folliculogenesis. It has been shown to control many functions of granulosa cells, including gene expression, steroidogenesis and proliferation. This study investigates the cellular requirements that allow GDF9 to act on granulosa cells. Our results showed that GDF9 (20 ng/ml)-stimulated mouse granulosa cells 3H-thymidine incorporation was inhibited by a type 1 receptor Alk4/5/7 inhibitor (SB431542, 5 μM), by an epidermal Growth factor (EGF) receptor inhibitor (AG1478, 5μM) and a MEK1 inhibitor (U0126, 10 μM). Interestingly, activin A- and TGFβ-stimulated 3H-thymidine incorporation shared similar inhibitor sensitivity. Moreover, when denuded oocytes were used as the mitogenic agent, SB431542, AG1478 and U0126 all prevented the increase in 3H-thymidine incorporation. Oocyte-stimulated 3H-thymidine incorporation in secondary follicles and cumulus-oocyte complexes were also sensitive to Alk4/5/7, EGF receptor and MEK1 inhibition. Basal and EGF-stimulated levels of phopho-MAPK3/1 were inhibited by using the EGF receptor inhibitor, but were not affected by inhibition of Alk4/5/7 or by adding GDF9 in granulosa cells. Using granulosa cells transfected with a SMAD3-luciferase reporter construct, GDF9-stimulated SMAD3 response could be inhibited by Alk4/5/7, EGFR and MEK1 inhibitors. Genes involved in cumulus cells expansion (Ptx3 and Has2) were upregulated in granulosa cells by co-culturing with denuded oocytes and that upregulation was inhibited by Alk4/5/7 as well as by EGF receptor inhibition. These results suggest that TGFβ superfamily members signalling through Smad2/3 share a common requirement of EGF receptor-dependant phospho-MAPK3/1 throughout folliculogenesis. These results strongly suggest that, apart from its role in the transmission of the ovulatory LH signal within the ovarian follicle, EGF receptor pathway might serve as modulators of GDF9 action on granulosa cells. Hence the interaction between endocrine and oocyte signalling may be mediated at the level of MAPK and Smad2/3 cross-talk in granulosa cells.

  • Growth Differentiation factor 9 is a germ cell regulator of sertoli cell function
    Endocrinology, 2009
    Co-Authors: Robert B Gilchrist, Craig A Harrison, Peter G Stanton, Peter K Nicholls, Paul G Farnworth
    Abstract:

    Oocyte-secreted Growth Differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFbeta ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

  • exogenous Growth Differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice
    Human Reproduction, 2007
    Co-Authors: Robert B Gilchrist, Jeremy G Thompson, Michelle Lane
    Abstract:

    BACKGROUND: Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as Growth Differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. METHODS: Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal Growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. RESULTS: Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. CONCLUSIONS: Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.

  • exogenous Growth Differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice
    Human Reproduction, 2007
    Co-Authors: Robert B Gilchrist, Jeremy G Thompson, Michelle Lane
    Abstract:

    BACKGROUND: Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as Growth Differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. METHODS: Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal Growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. RESULTS: Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. CONCLUSIONS: Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.

  • oocyte secreted factor activation of smad 2 3 signaling enables initiation of mouse cumulus cell expansion
    Biology of Reproduction, 2007
    Co-Authors: Rebecca Dragovic, Lesley J Ritter, S J Schulz, D T Armstrong, Jeremy G Thompson, F Amato, Robert B Gilchrist
    Abstract:

    Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming Growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that Growth Differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal Growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

Kenneth P Mcnatty - One of the best experts on this subject based on the ideXlab platform.

  • Growth Differentiation factor 9 bone morphogenetic protein 15 gdf9 bmp15 synergism and protein heterodimerization
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: David G Mottershead, Craig A Harrison, Thomas D Mueller, Peter G Stanton, Kenneth P Mcnatty
    Abstract:

    Although the title of the paper by Peng et al. (1) gave the impression that the authors were working with a purified Growth Differentiation factor 9:bone morphogenetic protein 15 (GDF9:BMP15) heterodimer preparation, at best, what was being tested was a mixture of BMP15 homodimers (75%) and GDF9:BMP15 heterodimers (25%), a point made by the authors early on in the results section. However, this important point was not reiterated, and the hG9:B15 preparation was misleadingly referred to as a GDF9/BMP15 heterodimer. In this context, there is a lack of data on characterization of the hG9:B15 preparation; e.g., no silver-stained SDS/PAGE gels are presented to indicate purity, and no chemical crosslinking data are presented in support of heterodimer formation. Given that GDF9 and BMP15 dimers interact noncovalently, they may be capable of forming monomers; hence, an equilibrium may exist between monomers, heterodimers, and homodimers (Fig. 1). The authors should not have assumed that heterodimerization of GDF9 and BMP15 would take place simply because they were coexpressed in the same cells, and copurification of GDF9 along with BMP15 via affinity chromatography was not sufficient evidence to establish heterodimer formation. For example, a dimer of homodimers (as shown in Fig.1, part 4) would similarly copurify and may well be biologically extremely potent.

  • effects of active immunization against Growth Differentiation factor 9 and or bone morphogenetic protein 15 on ovarian function in cattle
    Reproduction, 2009
    Co-Authors: Jennifer L Juengel, Kenneth P Mcnatty, N L Hudson, Lynda Whiting, M C Berg, Keith Hamel, Peter Smith, S B Lawrence
    Abstract:

    Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular Growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived Growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15 in vivo on ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone or together, altered follicular development and ovulation rate in cattle. Thus, as has been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal follicular development and ovulation rate in cattle.

  • bone morphogenetic protein 15 and Growth Differentiation factor 9 co operate to regulate granulosa cell function
    Reproduction, 2005
    Co-Authors: Kenneth P Mcnatty, David G Mottershead, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve Lawrence, Andrea Western, Mohamed F Meerasahib, Nigel P Groome
    Abstract:

    The oocyte-secreted polypeptide Growth factors, Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular Growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3 H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3 H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any Growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these Growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive α-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either Growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the Growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted Growth factors co-operate to regulate proliferation and gonadotropin-induced Differentiation of granulosa cells in mammals.

  • bone morphogenetic protein 15 and Growth Differentiation factor 9 co operate to regulate granulosa cell function in ruminants
    Reproduction, 2005
    Co-Authors: Kenneth P Mcnatty, David G Mottershead, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve Lawrence, Andrea Western, Mohamed F Meerasahib, Nigel P Groome
    Abstract:

    The oocyte-secreted polypeptide Growth factors, Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these Growth factors have used the rodent model. However, the evidence suggests that these Growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating 3 H-thymidine incorporation by ovine granulosa cells compared with each Growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two Growth factors in stimulating 3 H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these Growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the Growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these Growth factors alone.

  • effects of immunization against bone morphogenetic protein 15 and Growth Differentiation factor 9 on ovulation rate fertilization and pregnancy in ewes
    Biology of Reproduction, 2004
    Co-Authors: Jennifer L Juengel, N L Hudson, Lynda Whiting, Kenneth P Mcnatty
    Abstract:

    Abstract Immunization of ewes against Growth Differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3–4 days following mating. Subsequen...

Aaron J W Hsueh - One of the best experts on this subject based on the ideXlab platform.

  • Growth Differentiation factor 9 gdf9 stimulates proliferation and inhibits steroidogenesis by bovine theca cells influence of follicle size on responses to gdf9
    Biology of Reproduction, 2008
    Co-Authors: L J Spicer, D T Allen, Sabine Mazerbourg, Anita H Payne, Aaron J W Hsueh
    Abstract:

    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 smallfollicle 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 ,o rCYP17A1 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, oocyteand granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature Differentiation of the theca interna during early follicle development.

  • adenoviral gene transfer allows smad responsive gene promoter analyses and delineation of type i receptor usage of transforming Growth factor β family ligands in cultured human granulosa luteal cells
    The Journal of Clinical Endocrinology and Metabolism, 2005
    Co-Authors: Noora Kaivooja, Robert B Gilchrist, Aaron J W Hsueh, Sabine Mazerbourg, David G Mottershead, Samu Myllymaa, Nigel P Groome, Sebastien Duprat, Olli Ritvos
    Abstract:

    In the human ovary, cell Growth and Differentiation are regulated by members of the TGF-β superfamily, including Growth Differentiation Factor-9 (GDF9), TGF-β, and activin. TGF-β and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-β, and activin responses. In rodent granulosa cells, TGF-β and GDF9 signal through the TGF-β type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase rep...

  • Growth Differentiation factor 9 signaling is mediated by the type i receptor activin receptor like kinase 5
    Molecular Endocrinology, 2004
    Co-Authors: Sabine Mazerbourg, Cynthia Klein, Olli Ritvos, David G Mottershead, Noora Kaivooja, Olexander Korchynskyi, Aaron J W Hsueh
    Abstract:

    Growth Differentiation Factor-9 (GDF-9) is an oocyte-derived Growth factor and a member of the TGF-beta superfamily that includes TGF-beta, activin, and bone morphogenetic proteins (BMPs). GDF-9 is indispensable for the development of ovarian follicles from the primary stage, and treatment with GDF-9 enhances the progression of early follicles into small preantral follicles. Similar to other TGF-beta family ligands, GDF-9 likely initiates signaling mediated by type I and type II receptors with serine/threonine kinase activity, followed by the phosphorylation of intracellular transcription factors named Smads. We have shown previously that GDF-9 interacts with the BMP type II receptor (BMPRII) in granulosa cells, but the type I receptor involved is unknown. Using P19 cells, we now report that GDF-9 treatment stimulated the CAGA-luciferase reporter known to be responsive to TGF-beta mediated by the type I receptor, activin receptor-like kinase (ALK)5. In contrast, GDF-9 did not stimulate BMP-responsive reporters. In addition, treatment with GDF-9 induced the phosphorylation of Smad2 and Smad3 in P19 cells, and the stimulatory effect of GDF-9 on the CAGA-luciferase reporter was blocked by the inhibitory Smad7, but not Smad6. We further reconstructed the GDF-9 signaling pathway using Cos7 cells that are not responsive to GDF-9. After overexpression of ALK5, with or without exogenous Smad3, the Cos7 cells gained GDF-9 responsiveness based on the CAGA-luciferase reporter assay. The roles of ALK5 and downstream pathway genes in mediating GDF-9 actions were further tested in ovarian cells. In cultured rat granulosa cells from early antral follicles, treatment with GDF-9 stimulated the CAGA-luciferase reporter activity and induced the phosphorylation of Smad3. Furthermore, transfection with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 actions. In conclusion, although GDF-9 binds to the BMP-activated type II receptor, its downstream actions are mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins. Because ALK5 is a known receptor for TGF-beta, diverse members of the TGF-beta family of ligands appear to interact with a limited number of receptors in a combinatorial manner to activate two downstream Smad pathways.

  • Growth Differentiation factor 9 promotes the Growth development and survival of human ovarian follicles in organ culture
    The Journal of Clinical Endocrinology and Metabolism, 2002
    Co-Authors: Julius Hreinsson, Aaron J W Hsueh, Jennifer E Scott, Carsten Rasmussen, Marja Liisa Swahn, Outi Hovatta
    Abstract:

    Growth Differentiation Factor-9 (GDF-9) is a Growth factor and a member of the TGFbeta superfamily that is secreted by oocytes in growing ovarian follicles. In the current study we cultured human ovarian follicles within slices of ovarian cortical tissue in the presence and absence of recombinant rat GDF-9. Ovarian tissue was obtained by biopsy during cesarean section (31 women) or gynecological laparoscopy (3 women). The mean age (+/-SD) of the women who donated tissue was 32.8 +/- 5.0 yr (range, 19-41 yr). In these biopsies a total of 900 follicles were analyzed. A significantly higher proportion of cultured primordial follicles showed Growth initiation and reached the secondary stage of development in the presence of GDF-9. In serum-free cultures after 7 d, 53% of the follicles had reached the secondary stage with GDF-9 vs. 31% in the control group (P < 0.01). Follicle viability was also improved in the presence of GDF-9 after 7 d in culture (74% GDF-9 vs. 48% control), which resulted in a smaller reduction in follicle numbers due to atresia. Treatment with GDF-9 in vitro promoted the survival and progression of human follicular development to the secondary stage. This oocyte-derived factor may be essential for the development of somatic cells in early human follicles and useful in designing culture conditions for maturation of follicles and oocytes in vitro.

  • Growth Differentiation factor 9 stimulates rat theca interstitial cell androgen biosynthesis
    Biology of Reproduction, 2000
    Co-Authors: Elena V Solovyeva, Abraham Amsterdam, Aaron J W Hsueh, Masaru Hayashi, Karen Margi, Claudine Barkats, Cynthia Klein, A Tsafriri
    Abstract:

    Growth Differentiation Factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived Growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and Differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and Differentiation.

Nigel P Groome - One of the best experts on this subject based on the ideXlab platform.

  • bone morphogenetic protein 15 and Growth Differentiation factor 9 co operate to regulate granulosa cell function
    Reproduction, 2005
    Co-Authors: Kenneth P Mcnatty, David G Mottershead, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve Lawrence, Andrea Western, Mohamed F Meerasahib, Nigel P Groome
    Abstract:

    The oocyte-secreted polypeptide Growth factors, Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular Growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3 H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3 H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any Growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these Growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive α-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either Growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the Growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted Growth factors co-operate to regulate proliferation and gonadotropin-induced Differentiation of granulosa cells in mammals.

  • bone morphogenetic protein 15 and Growth Differentiation factor 9 co operate to regulate granulosa cell function in ruminants
    Reproduction, 2005
    Co-Authors: Kenneth P Mcnatty, David G Mottershead, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve Lawrence, Andrea Western, Mohamed F Meerasahib, Nigel P Groome
    Abstract:

    The oocyte-secreted polypeptide Growth factors, Growth Differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these Growth factors have used the rodent model. However, the evidence suggests that these Growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating 3 H-thymidine incorporation by ovine granulosa cells compared with each Growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two Growth factors in stimulating 3 H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these Growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the Growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these Growth factors alone.

  • adenoviral gene transfer allows smad responsive gene promoter analyses and delineation of type i receptor usage of transforming Growth factor β family ligands in cultured human granulosa luteal cells
    The Journal of Clinical Endocrinology and Metabolism, 2005
    Co-Authors: Noora Kaivooja, Robert B Gilchrist, Aaron J W Hsueh, Sabine Mazerbourg, David G Mottershead, Samu Myllymaa, Nigel P Groome, Sebastien Duprat, Olli Ritvos
    Abstract:

    In the human ovary, cell Growth and Differentiation are regulated by members of the TGF-β superfamily, including Growth Differentiation Factor-9 (GDF9), TGF-β, and activin. TGF-β and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-β, and activin responses. In rodent granulosa cells, TGF-β and GDF9 signal through the TGF-β type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase rep...

  • Growth Differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep
    Biology of Reproduction, 2002
    Co-Authors: Jennifer L Juengel, N L Hudson, Karen L Reader, Steve Lawrence, Mark Cranfield, Peter Smith, Mika Laitinen, D A Heath, Anne R Oconnell, Nigel P Groome
    Abstract:

    Abstract The aim of this study was to test the hypothesis that both Growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9–10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that th...

  • Growth Differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep
    Biology of Reproduction, 2002
    Co-Authors: Jennifer L Juengel, N L Hudson, Karen L Reader, Steve Lawrence, Mark Cranfield, Peter Smith, Mika Laitinen, D A Heath, Anne R Oconnell, Nigel P Groome
    Abstract:

    The aim of this study was to test the hypothesis that both Growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these Growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of Growth.