Bone Morphogenetic Protein 15 Protein Research Articles

Molecular Characterization of the Follicular Development of BMP15-edited Pigs.

Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta (TGF-β) superfamily, which is critical for facilitating ovarian folliculogenesis in mono-ovulatory mammalian species but is not essential in poly-ovulatory mice. Our previously established BMP15-edited pigs presented varied female reproductive phenotypes, suggesting the important role of BMP15 in ovarian folliculogenesis in poly-ovulatory pigs. To understand the regulatory mechanism underlying the effect of BMP15 on porcine ovarian follicular development, we molecularly characterized infertile biallelic-BMP15-edited gilts with ovarian hypoplasia. We found that an absence of BMP15 proteins in biallelic BMP15-edited gilts can lead to premature activation of primordial follicles, possibly through the up-regulation of KITLG-KIT-PI3K-AKT signaling pathways. However, this absence severely impaired SMAD (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling, causing severely reduced granulosa cell proliferation, leading to the arrest of follicular development during the preantral stage and ovarian hypoplasia, resulting in complete infertility. Our study expands the understanding of the molecular functions of BMP15 in non-rodent poly-ovulatory mammals.

Single-Nucleotide Polymorphisms Identified within Exon 2 of Fertility-Associated Bone Morphogenetic Protein (BMP15) Gene in Three Romanian Sheep Breeds

The improvement of the reproductive traits of animals is of great interest for livestock production. Due to its positive impact on the sheep industry’s profitability, prolificacy is one of the most economically significant biological traits, showing variation between and within breeds of domestic sheep (Ovis aries). Different mutations in BMPR-1B, BMP15 and GDF9 genes coding for the transforming growth factor-β (TGFβ) superfamily have been shown to influence the ovulation rate and litter size. Numerous single-nucleotide polymorphisms (SNPs) in the bone morphogenetic protein 15 (BMP15) gene have been linked to ewe fecundity. Using targeted PCR amplification and Sanger sequencing, we were able to identify heterozygous SNPs in exon 2 of BMP15 in three sheep breeds reared in Romania: Tsigai, Cluj Merino and Tsurcana. The sequence analysis revealed three previously documented mutations, namely the missense mutation c.755T>C (L252P), which is predicted to change the tertiary structure of the BMP15 protein, and two silent mutations, c.747T>C (P249P) and c.1047G>A (V349V). In addition, we also identified one novel silent mutation, c.825G>A (S275S). Based on our findings and publicly available data, we indicate four putative mutational hotspots within exon 2 of BMP15 that could be considered for improving the indigenous sheep breeds through targeted gene editing and SNP genotyping strategies.

Decreased GDF9 and BMP15 in follicle fluid and granulosa cells and outcomes of IVF-ET among young patients with low prognosis.

To analyze the level of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in follicle fluid (FF) and granulosa cells (GCs) derived from young patients with low prognosis for in vitro fertilization and embryo transfer (IVF-ET) treatment. A prospective cohort study was carried out by enrolling 52 young patients with low prognosis according to the POSEIDON classification group 3 (low prognosis group) and 51 young patients with normal ovarian reserve (control group). The concentration of the GDF9 and BMP15 proteins in FF was determined by enzyme-linked immunosorbent assay. The mRNA level of the GDF9 and BMP15 in the GCs was measured by quantitative real-time PCR. The concentration of GDF9 (1026.72 ± 159.12 pg/mL vs. 1298.06 ± 185.41 pg/mL) and BMP15 (685.23 ± 143.91 pg/mL vs. 794.37 ± 81.79 pg/mL) in FF and the mRNA level of GDF9 and BMP15 in the GCs and the live birth rate per treatment cycle started (30.77% vs. 50.98%) and oocytes retrieved (4.25 ± 1.91 vs.12.04 ± 4.24) were significantly lower, whereas the canceled cycle rate was significantly higher (9.62% vs. 0) in the low prognosis group compared with the control group (P < 0.05). The expression of GDF9 and BMP15 in the ovary was positively correlated with live birth (P < 0.05). The expression of GDF9 and BMP15 in the ovary was decreased in young patients with low prognosis accompanied by a poorer outcome of IVF-ET treatment. ChiCTR1800016107 (Chinese Clinical Trial Registry), May 11, 2018. ( http://www.chictr.org.cn/edit.aspx?pid=27216&htm=4 ).

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/μL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/μL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.

BMP15 Modulates the H19/miR-26b/SMAD1 Axis Influences Yak Granulosa Cell Proliferation, Autophagy, and Apoptosis.

Bone morphogenetic protein 15 (BMP15) regulates the growth and development of follicles. In particular, the long non-coding RNA H19 plays an important role in mammalian reproduction. However, the function and regulatory mechanism of the interaction of BMP15 with H19 in yak granulosa cell (GC) proliferation, autophagy, and apoptosis are poorly understood. In our study, quantitative reverse-transcription-polymerase chain reaction analysis showed that H19 were highly expressed in yak healthy follicles. H19 was induced by BMP15 protein in yak GCs. In addition, we confirmed that overexpression of H19 promoted yak GC proliferation and autophagy and inhibited apoptosis. Bioinformatic analysis and luciferase reporter assays demonstrated that H19 directly binds to miR-26b, and SMAD1 was identified as a target of miR-26b. miR-26b overexpression inhibited GC proliferation and autophagy and promoted apoptosis through decreased SMAD1 expression, which was attenuated by H19 overexpression. RNA immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase assays showed that miR-26b was sponged by H19 to preserve SMAD1 expression. Furthermore, SMAD1 mRNA expression was induced and miR-26b expression was reduced after yak GCs were treated with BMP15 protein. In conclusion, our results demonstrated that the H19/miR-26b/SMAD1 axis responds to BMP15 to regulate yack GC proliferation, autophagy, and apoptosis.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 μg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

The Identification of Mutation in BMP15 Gene Associated with Litter Size in Xinjiang Cele Black Sheep.

Simple SummaryThe gene for Bone Morphogenetic Protein 15 has multiple single-nucleotide polymorphism sites related to fertility in sheep. To increase fertility, a better understanding of the regulation of Bone Morphogenetic Protein 15 in sheep is essential. This work describes the phenotype and molecular characteristics of a mutation identified in the Bone Morphogenetic Protein 15 gene of Cele black sheep in Xinjiang, China. This mutation may affect the fertility of sheep, which is very useful for breeding purposes.The Bone Morphogenetic Protein 15 (BMP15) gene is known to have multiple single-nucleotide polymorphism sites associated with sheep fecundity. This study used gene sequence analysis and mutation detection assays for BMP15 by using 205 blood samples of ewes with known lambing records. Sequence analysis showed that mutation B1 missed the CTT base in exon 1 at positions 28–30, leading to a leucine deletion in the BMP15 protein. Litter size of ewes differed significantly between BB and B+ genotypes of B1 (p < 0.05); however, the differences between wild genotype (++) and homozygous (BB) or wild genotype (++) and heterozygous (B+) were not significant (p > 0.05). Another mutation, T755C, is a T-to-C base change at position 755 of exon 2, resulting in leucine replacement by proline at this position of the BMP15 protein (p.L252P). Two genotypes were identified in the flock: heterozygous (E+) and wild-type genotype (++). Ewes with heterozygous (E+) p.L252P had significantly larger litter sizes than those with the wild-type genotype (p < 0.05). Comprehensive analysis suggests that p.L252P is a mutation that affects fecundity in Cele black sheep.

Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

BackgroundGrowth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment.MethodsA total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.ResultsCompared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P < 0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P < 0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P < 0.05).ConclusionsFor poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant.Trial registrationChinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

Ovarian Growth Factor Protein Expression in Pediatric Patients before and after Cytotoxic Therapy

The improved survival of young cancer patients results often in follicle destruction. The infertility level varies by age, with alkylating agents considered the most gonadotoxic. One option for fertility preservation is ovarian cryostorage, often conducted post-chemotherapy. Large number of follicles remain in ovaries of pediatric patients post-chemotherapy. Anti-Mullerian hormone (AMH), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are ovarian markers. Their ovarian expression in post-chemotherapy girls might provide an indication of follicle normality, and if it is worthwhile to cryopreserve such tissue. AMH, GDF9 and BMP15 protein expression in human ovarian samples from 49 girls pre-and-post-chemotherapy was evaluated by regular immunohistochemistry (IHC) and immunofluorescence (IF) for confocal laser microscopy. Protein expression for the three growth factors in follicles of pediatric patients pre-and-post-chemotherapy (including alkylating agent exposure) was identified by both methods from primordial stages onwards in oocytes and granulosa cells, and in irregularly shaped follicles. In the pre-chemotherapy group, IHC AMH staining seemed strongest at age ≤ 8 years, and was more abundant in pubertal-than-pre-pubertal patients by IF. Post-chemotherapy patients had significantly more immunostaining for AMH and BMP15 in atretic follicles after exposure to alkylating agents. IHC immunostaining for all three proteins seemed fuller before than after chemotherapy; there were more follicles containing growth factor-expressing granulosa cells post-chemotherapy; BMP15 staining seemed stronger pre-than-post-chemotherapy. The growth factor distribution post-chemotherapy as well as the high numbers of remaining follicles, suggests that it is worthwhile to cryostore ovarian tissue from pediatric patients even after chemotherapy initiation, including alkylating agent exposure.

Use of Bone Morphogenetic Protein 15 Polymorphisms to Predict Ovarian Stimulation Outcomes in Infertile Brazilian Women.

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) can result in inhibited secretion or lowered bioactivity of the BMP15 protein. BMP15 levels are associated with follicle-stimulating hormone receptor (FSHR) action on granulosa cells, wherein FSHR increases the sensitivity of ovarian follicles to follicle-stimulating hormone (FSH). In this study we evaluated the BMP15 polymorphisms A905 > G/rs3897937, C901 > T/rs17003221, and C-9 > G/rs3810682 in infertile Brazilian women in terms of anti-Mullerian hormone (AMH), FSH, and estradiol serum levels, as well as controlled ovarian hyperstimulation response and assisted reproduction outcomes. A cross-sectional study comprising 186 infertile women who underwent the first cycle of high complexity assisted reproduction treatment was conducted using the TaqMan assay for quantitative polymerase chain reaction genotyping. Serum AMH, FSH, and estradiol levels were measured by enzyme-linked immunosorbent assay. For C901 > T (rs17003221) carriers, there was a statistically significant difference among carriers of a polymorphic BMP15 genotype (TT) and the estradiol concentration. These women had higher estradiol levels than women who had homozygous wild type or heterozygous genotypes. There was also a positive correlation between serum AMH and the C-9 > G (rs3810682) polymorphism, wherein women carrying both polymorphic alleles (homozygous, GG) had higher average AMH levels than heterozygous women. However, none of the three polymorphisms studied showed a statistically significant correlation with assisted reproduction outcome. Oocytes are known to secrete factors that regulate follicular development and oocyte maturation. Abnormal expression of these factors may thus be involved in follicular development disorders. A recent study highlighted the importance of BMP15 in regulating ovulation rates in sheep and that heterozygous deletions in the -9C > G polymorphism reduced BMP15 concentrations, increased granulosa cell FHSR mRNA levels, elevated estrogen secretion, and activated production of stem cell factors. In this study we found that BMP15 polymorphisms affected estrogen and AMH levels. BMP15 polymorphisms are not correlated with ovarian stimulation and assisted reproduction outcomes in infertile Brazilian women.

Oocyte expression, secretion and somatic cell interaction of mouse bone morphogenetic protein 15 during the peri-ovulatory period

Bone morphogenetic protein 15 (BMP15) is a key intraovarian growth factor regulating mammalian fertility, yet expression and localisation of different BMP15 protein forms within ovarian follicles around the time of the preovulatory LH surge remains unclear. Using immunoblotting and immunocytochemistry, the present study identified that post-translationally processed BMP15 proregion and mature proteins are increasingly expressed and localised with cumulus and granulosa cells from mice treated with pregnant mare's serum gonadotropin (PMSG) + human chorionic gonadotrophin (hCG). However, this increased expression was absent in cumulus-oocyte complexes matured in vitro. Pull-down assays further revealed that the recombinant BMP15 proregion is capable of specific interaction with isolated granulosa cells. To verify an oocyte, and not somatic cell, origin of Bmp15 mRNA and coregulated growth differentiation factor 9 (Gdf9), in situ hybridisation and quantitative polymerase chain reaction results confirmed the exclusive oocyte localisation of Bmp15 and Gdf9, regardless of treatment or assay method. Relative oocyte expression levels of Bmp15 and Gdf9 decreased significantly after PMSG + hCG treatment; nevertheless, throughout all treatments, the Bmp15:Gdf9 mRNA expression ratio remained unchanged. Together, these data provide evidence that the preovulatory LH surge leads to upregulation of several forms of BMP15 protein secreted by the oocyte for putative sequestration and/or interaction with ovarian follicular somatic cells.

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

Effects of species differences on oocyte regulation of granulosa cell function

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate (3)H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R (2)=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

Impaired production of BMP-15 and GDF-9 mature proteins derived from proproteins WITH mutations in the proregion

Mutations in the bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) genes have been identified in women with primary ovarian insufficiency (POI) and mothers of dizygotic twins. Here, we show that biological activities of the conditioned media from human embryonic kidney 293F cells transfected with two representative BMP-15 and GDF-9 mutants identified in the affected women have significantly reduced biological activities compared with the corresponding wild-type. Moreover, this difference is due to decreased production of the mature proteins, attributed most likely to impaired posttranslational processing of the proprotein. As genetic studies of the BMP-15 and/or GDF-9 genes in ewes established that a reduction of these proteins is associated with an increased ovulation rate, it is conceivable that women affected with these mutations may have an increased probability of bearing dizygotic twins during active reproductive ages before diagnosis with POI at later ages due to an earlier exhaustion of ovarian reserve.

DEHP impairs zebrafish reproduction by affecting critical factors in oogenesis.

Public concerns on phthalates distributions in the environment have been increasing since they can cause liver cancer, structural abnormalities and reduce sperm counts in male reproductive system. However, few data are actually available on the effects of Di-(2-ethylhexyl)-phthalate (DEHP) in female reproductive system. The aim of this study was to assess the impacts of DEHP on zebrafish oogenesis and embryo production. Female Danio rerio were exposed to environmentally relevant doses of DEHP and a significant decrease in ovulation and embryo production was observed. The effects of DEHP on several key regulators of oocyte maturation and ovulation including bone morphogenetic protein-15 (BMP15), luteinizing hormone receptor (LHR), membrane progesterone receptors (mPRs) and cyclooxygenase (COX)-2 (ptgs2) were determined by real time PCR. The expressions of BMP15 and mPR proteins were further determined by Western analyses to strengthen molecular findings. Moreover, plasma vitellogenin (vtg) titers were assayed by an ELISA procedure to determine the estrogenic effects of DEHP and its effects on oocyte growth. A significant reduction of fecundity in fish exposed to DEHP was observed. The reduced reproductive capacity was associated with an increase in ovarian BMP15 levels. This rise, in turn, was concomitant with a significant reduction in LHR and mPRβ levels. Finally, ptgs2 expression, the final trigger of ovulation, was also decreased by DEHP. By an in vitro maturation assay, the inhibitory effect of DEHP on germinal vesicle breakdown was further confirmed. In conclusion, DEHP affecting signals involved in oocyte growth (vtg), maturation (BMP15, LHR, mPRs,) and ovulation (ptgs2), deeply impairs ovarian functions with serious consequences on embryo production. Since there is a significant genetic similarity between D.rerio and humans, the harmful effects observed at oocyte level may be relevant for further molecular studies on humans.

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor beta (TGFbeta) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of beta-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure.

Expression of bone morphogenetic protein-15 in human oocyte and cumulus granulosa cells primed with recombinant follicle-stimulating hormone followed by human chorionic gonadotropin

Bone morphogenetic protein-15 is expressed in oocytes and cumulus granulosa cells. It may play an important role in the hCG-induced oocyte final maturation and corpus luteum (CL) formation.

121. EXTRACELLULAR ROLES FOR PROREGIONS OF MOUSE BMP15 AND GDF9 IN VIVO

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are essential for normal follicular development and ovulation in mammalian species. As TGF-beta family proteins, BMP15 and GDF9 are expressed as pre-pro-mature proteins, with a signal region prompting extracellular secretion, a proregion that is post-translationally cleaved, and a mature protein that is known to be biologically active as a non-covalently interacting dimer. Until recently, the proregion was thought primarily to facilitate correct folding and dimerization of the mature proteins within the oocyte prior to processing and cleavage. However, our in vitro work with recombinant mouse BMP15 and GDF9 has shown that each of the proregion, mature protein and the non-processed promature protein are secreted from transfected 293H cells. We showed non-covalent interactions existing between the proregion and mature protein of each of BMP15 and GDF9, and between the BMP15 proregion and GDF9 mature protein. Importantly, a mouse BMP15 proregion antibody was able to abolish cooperative BMP15 and GDF9 bioactivity measured using a granulosa cell thymidine incorporation bioassay, providing strong evidence for an extracellular role for the mouse BMP15 proregion. Currently, to find out whether BMP15 and GDF9 proregions have extracellular roles in vivo, our investigation has utilised knock-down of BMP15 and GDF9 proregion proteins by mouse immunisation. Ovaries of mice immunised with the GDF9 proregion had significantly increased numbers of corpora lutea (p&lt;0.005), while ovaries from mice immunised with BMP15 proregion peptides had significantly fewer corpora lutea (p&lt;0.005). These findings provide the first evidence that the proregions of mouse GDF9 and BMP15 have different physiological roles outside the oocyte. Our future aim is to elucidate the function of these proregions, how these may differ between BMP15 and GDF9, and whether these aspects differ between species with different ovulation quota.

The Proregion of Mouse BMP15 Regulates the Cooperative Interactions of BMP15 and GDF91

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.

The Effects of Immunizing Sheep with Different BMP15 or GDF9 Peptide Sequences on Ovarian Follicular Activity and Ovulation Rate1

The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.