Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrop... more Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.
The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone m... more 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 3H-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 3H-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 alpha-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.
Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member w... more Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.
The Journal of Clinical Endocrinology & Metabolism, 1999
Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that i... more Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.
Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator o... more Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.
The development of ovarian follicles and subsequent corpus luteum formation is accompanied by ver... more The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.
Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrop... more Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.
The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone m... more 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 3H-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 3H-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 alpha-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.
Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member w... more Growth differentiation factor-9 (GDF-9) is a transforming growth factor-b (TGF-b) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131-136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R. , Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531-535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-b superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-b family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.
The Journal of Clinical Endocrinology & Metabolism, 1999
Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that i... more Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.
Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator o... more Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.
The development of ovarian follicles and subsequent corpus luteum formation is accompanied by ver... more The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.
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Papers by Mika Laitinen