Novel bone morphogenetic protein 15 (BMP15) gene variants implicated in premature ovarian insufficiency.

BACKGROUND: Bone morphogenetic protein 15 (BMP15) is expressed in oocytes and plays a crucial role in the reproduction of mono-ovulating species. In humans, BMP15 gene mutations lead to imperfect protein function and premature ovarian insufficiency. Here we investigated the BMP15 gene variants in a population of Iranian women with premature ovarian insufficiency. We conducted predictive bioinformatics analysis to further study the outcomes of BMP15 gene alterations. METHODS: Twenty-four well-diagnosed premature ovarian insufficiency cases with normal karyotype participated in this study. The entire coding sequence and exon-intron junctions of the BMP15 gene were analyzed by direct sequencing. In-silico analysis was applied using various pipelines integrated into the Ensembl Variant Effect Predictor online tool. The clinical interpretation was performed based on the approved guidelines. RESULTS: By gene screening of BMP15, we discovered p.N103K, p.A180T, and p.M184T heterozygous variants in 3 unrelated patients. The p.N103K and p.M184T were not annotated on gnomAD, 1000 Genome and/or dbSNP. These mutations were not identified in 800 Iranians whole-exome sequencing that is recorded on Iranom database. We identified the p.N103K variant in a patient with secondary amenorrhea at the age of 17, elevated FSH and atrophic ovaries. The p.M184T was detected in a sporadic case with atrophic ovaries and very high FSH who developed secondary amenorrhea at the age of 31. CONCLUSIONS: Here we newly identified p.N103K and p.M184T mutation in the BMP15 gene associated with idiopathic premature ovarian insufficiency. Both mutations have occurred in the prodomain region of protein. Despite prodomain cleavage through dimerization, it is actively involved in the mature protein function. Further studies elucidating the roles of prodomain would lead to a better understanding of the disease pathogenesis.

MicroRNA-21 plays a pivotal role in the oocyte-secreted factor-induced suppression of cumulus cell apoptosis.

It is known that oocytes and cumulus cells (CCs) are more resistant to apoptosis than other compartments of the antral follicle. However, although oocyte-secreted factors (OSFs) have been found to be involved in suppressing bovine CC apoptosis, little is known about the intracellular mechanisms by which OSFs render CCs resistant to apoptosis. Here, we show that coculture with mouse or pig cumulus-denuded oocytes, culture with recombinant mouse growth differentiation factor-9 (GDF-9), or culture in pig oocyte-conditioned medium (POCM) significantly inhibited CC apoptosis of mouse oocytectomized cumulus oophorus complexes (OOXs). The POCM contained both GDF-9 and bone morphogenetic protein-15, and their levels remained constant during culture of OOXs. The level of microRNA-21 (miR-21) was significantly lower in OOXs than in COCs after culture in a simplified α-MEM medium, but increased significantly when OOXs were cultured with GDF-9 or in POCM. The level of miR-21 in OSF-treated CCs was correlated with that of Dicer1 but not that of Drosha mRNA. Inhibiting activin receptor-like kinase 5 or SMAD3 completely abolished the beneficial effects of GDF-9 or POCM on CC apoptosis and miR-21 levels. Up- and downregulating miR-21 expression significantly reduced and increased CC apoptosis, respectively. The OSF-upregulated miR-21 expression suppressed CC apoptosis with activation of the PI3K/Akt signaling. In conclusion, miR-21 plays a pivotal role in the OSF suppression of CC apoptosis. OSFs upregulated miR-21 expression through the TGF-ß superfamily signaling, which worked through DICER. MicroRNA-21 prevented apoptosis via the PI3K/Akt signaling.

Molecular forms of ruminant BMP15 and GDF9 and putative interactions with receptors.

Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted factors with demonstrable effects on ovarian follicular development and ovulation rate. However, the molecular forms of BMP15 and GDF9 produced by oocytes remain unclear. The aims herein, using Western blotting (WB) procedures with specific monoclonal antibodies (mabs), were to identify the molecular forms of BMP15 and GDF9 synthesised and secreted by isolated ovine (o) and bovine (b) oocytes in vitro The mabs were known to recognise the biological forms of BMP15 or GDF9 since they had previously been shown to inhibit their bioactivities in vitro and in vivo Using recombinant variants of oBMP15 and oGDF9, including a cysteine mutant form of oBMP15 (S356C) and a human (h) BMP15:GDF9 heterodimer (cumulin), it was established that the mabs were able to identify monomeric, dimeric, promature and higher-molecular-weight forms of BMP15 and GDF9 and cumulin (GDF9 mab only). After using non-reducing, reducing and reducing + cross-linking conditions, the major oocyte-secreted forms of o and b BMP15 and GDF9 were the cleaved and uncleaved monomeric forms of the promature proteins. There was no evidence for dimeric or heterodimeric forms of either mature BMP15 or GDF9. From in silico modelling studies using transforming growth factor beta (TGFB), activin or BMP crystal templates, and both present and previously published data, a model is proposed to illustrate how the monomeric forms of BMP15 and GDF9 may interact with their type II and type I cell-surface receptors to initiate the synergistic actions of these growth factors.

Repertoire of bone morphogenetic proteins and growth/differentiation factors in ovary of the Indian wall lizard (Hemidactylus flaviviridis) with emphasis on differential expression and gonadotropic regulation of bmp15 and gdf9.

Analysis of ovarian transcriptome of Indian wall lizard demonstrates the existence of several bone morphogenetic proteins (bmp1, 2, 3, 3b, 7, 8, 15) and growth/differentiation factors (gdf5, 9) for the first time in reptilian ovary. The characterization of putative full-length/partial protein sequences of BMPs (BMP2, 3, 3b, 7, 15) and GDF9 showed high homology of their TGF-ß domain with that of other vertebrates while BMP1 bore homology to zinc-dependent metalloprotease. Phylogenetic analyses showed clustering of BMPs and GDF9 from wall lizards with that of squamates lying in close proximity to chelonia, crocodilia and aves. This study also correlates the expression of ovarian bmp15 and gdf9 with folliculogenesis. Level of bmp15 dramatically increased with the onset of follicular growth in early recrudescence and attained peak during late recrudescence whereas gdf9 sharply decreased during recrudescence as compared to regression. Nonetheless, expression of these growth factors decreased appreciably with the formation of vitellogenic follicle during breeding phase. Ovarian expression of bmp15 and gdf9 appeared to be regulated by gonadotropin as bmp15 considerably increased while gdf9 decreased in parallel to follicular development after administration of 3 injections of FSH. Expression of both the growth factors declined with the prolongation of treatment that led to formation of early/late vitellogenic follicle. Our in vitro study revealed stimulatory effect of FSH on expression of bmp15 and gdf9 in early growing, previtellogenic and early vitellogenic follicles. In light of in vitro results, FSH-induced in vivo decline in gene expression seems to be due to some other FSH-induced factor.

Anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor 9 (GDF9), and bone morphogenic protein-15 (BMP15) mRNA and protein are influenced by photoperiod-induced ovarian regression and recrudescence in Siberian hamster ovaries.

Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with their transfer to long photoperiods (LD). To investigate the mechanism of photo-stimulated recrudescence, we assessed key folliculogenic factors-anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor-9 (GDF9), and bone morphogenic protein-15 (BMP15)-across the estrus cycle and in photo-regressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks, which respectively represent functional and regressed ovaries. Select regressed hamsters were transferred back to LD for 2 (post-transfer week 2; PTw2) or 8 weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-α, GDF9, and BMP15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (P < 0.05) ovarian AMH, GDF9, and BMP15, but not inhibin-α mRNA levels as compared to LD. Transfer of regressed hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF9 mRNA levels to LD-treated levels, and further increased mRNA levels for inhibin-α and BMP15. Immunostaining for AMH, inhibin-α, GDF9, and BMP15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-α generally mirrored the mRNA data, though no changes were observed for GDF9 or BMP15 immunostaining. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photo-stimulated recrudescence via potential regulation of follicle recruitment, preservation, and development.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice.

The transforming growth factor ß (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Signalling pathways involved in the cooperative effects of ovine and murine GDF9+BMP15-stimulated thymidine uptake by rat granulosa cells.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors known to be involved in regulating the proliferation and differentiation of granulosa cells during follicular growth. The aims of this study were to determine the signalling pathways used by recombinant forms of murine and ovine GDF9 and BMP15 in combination (GDF9+BMP15) and the molecular complexes formed by combinations of these factors. Differences in the molecular forms of combinations of murine and ovine GDF9+BMP15 were observed by western blot analysis. Ovine GDF9+BMP15-stimulated (3)H-thymidine uptake was completely blocked by SMAD2/3 and nuclear factor-κB pathway inhibitors and partially blocked by a p38-mitogen-activated protein kinase (MAPK) inhibitor. Thymidine uptake by murine GDF9+BMP15 was reduced by the SMAD2/3 and extracellular signal-regulated kinase-MAPK pathway inhibitors and increased after addition of a c-Jun N-terminal kinase inhibitor. Stimulation of (3)H-thymidine uptake by GDF9+BMP15 from either species was not affected by the SMAD1/5/8 pathway inhibitor. In conclusion, both murine and ovine GDF9+BMP15-stimulated thymidine incorporation in rat granulosa cells was dependent on the SMAD2/3 signalling pathway but not the SMAD1/5/8 pathway. Divergence in the non-SMAD signalling pathways used by murine and ovine GDF9+BMP15 was also evident and may be due to the differences observed in the molecular complexes formed by these factors. These results are consistent with the hypothesis that the disparate cooperative functions of GDF9 and BMP15 in different species are mediated by divergent non-SMAD signalling pathways.

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

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

Serum Concentrations of Oocyte-Secreted Factors BMP15 and GDF9 During IVF and in Women With Reproductive Pathologies.

Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.

Variation in bone morphogenetic protein 15 is not associated with spontaneous human dizygotic twinning.

BACKGROUND: Spontaneous dizygotic (DZ) twinning in humans is under genetic control. In sheep, heterozygous loss of function mutations in bone morphogenetic protein 15 (BMP15) increase ovulation and hence twinning rates. METHODS: To investigate the role of BMP15 in human twinning, we typed 14 common variants, 4 rare novel variants initially detected by sequencing 279 mothers of DZ twins (MODZT) and 17 variants previously associated with premature ovarian failure (POF) in 933 DZ twinning families. We also typed five additional POF associated GDF9 variants. RESULTS: There was some evidence for association between DZ twinning and a common intronic BMP15 variant (rs3897937), but this was not significant after correction for multiple testing. Three of the four novel variants (p.Pro174Ser, p.Ala311Thr and p.Arg392Thr) occurred in 1-5 MODZT but were not detected in 1512 controls. We also detected three POF associated mutations in both BMP15 and GDF9 at low frequencies in MODZT and controls. CONCLUSIONS: We conclude that neither rare nor common BMP15 variants play a significant role in the variation in human DZ twinning.

Molecular Aspects and Clinical Relevance of GDF9 and BMP15 in Ovarian Function.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors with a leading role in the control of ovarian function in female reproduction, modulating both the cell fate of the somatic granulosa cells and the quality and developmental competence of the egg. This short review aims to consolidate the molecular aspects of GDF9 and BMP15 and their integral actions in female fertility to understand particularly their effects on oocyte quality and fetal growth. The significant consequences of mutations in the GDF9 and BMP15 genes in women with dizygotic twins as well as the clinical relevance of these oocyte factors in the pathogenesis of primary ovarian insufficiency and polycystic ovary syndrome are also addressed.

Highly efficient enrichment of porcine cells with deletions induced by CRISPR/Cas9 using dual fluorescence selection.

Production of nuclear donor cells with a high percentage of desired modifications is a critical step in the successful generation of genetically modified pigs through somatic cell nuclear transfer (SCNT). The CRISPR/Cas9 system has been used for efficient modification of the nuclear DNA in eukaryotic cells, including porcine cells. However, in vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their enrichment. Here we investigate a dual fluorescence selection system for the efficient enrichment of porcine embryonic fibroblasts (PEFs) with CRISPR/Cas9-induced chromosomal deletions. Enrichment of cells with 170 bp deletions reached a frequency of 74%, whilst enrichment of cells with a larger 5 kb deletions achieved a frequency of 46%. This demonstrates the utility of a dual fluorescence reporter as an attractive tool for improving the efficiency of generating genome edited pigs.

Characterization of bmp15 and its regulation by human chorionic gonadotropin in the follicle of gibel carp (Carassius auratus gibelio).

Bone morphogenetic protein (BMP15) is a member of the transforming growth factor ß (TGF-ß) superfamily with a key role in regulating follicle development in mammals and birds. However, potential ovarian roles of BMPs remain unexplored in teleosts. In this study, the full-length sequences of bmp15 were obtained using rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of bmp15 is 2217 bp which contained 214 bp 5'-UTR and 845 bp 3'-UTR. The open reading frame (ORF) sequence of bmp15 is 1158 bp, encoding a predicted protein of 385 amino acid residues. BMP15 has a specific RXXR protease cleavage site of TGF-ß superfamily (is RIRR) and six conserved cysteine residues. Using real-time quantitative PCR revealed that bmp15 mRNA was largely expressed in the ovary and testis and mostly in oocytes within the follicle, slightly expressed in muscle, liver and pituitary. BMP15 is mainly present at stage I follicles by real-time quantitative PCR and immunohistochemistry. Phylogenetic analysis showed that gibel carp bmp15 was similar to bmp15 of zebrafish and other fish species. Treatment with human chorionic gonadotropin (hCG) in isolated follicles of gibel carp in vitro showed altered bmp15 mRNA expression: when treated with 10 ng/mL hCG for 10h, the expression level of bmp15 was significantly increased. However, with proceeding cultivation, the expression level of BMP15 mRNA decreased. The results of this study indicate that bmp15 may play a key role during development of follicles in gibel carp, especially in early stage follicles.

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region.

Oocyte-derived bone morphogenetic protein-15 (BMP15) is critical for the regulation of mammalian fertility. Previously we have found that a C-terminal His(6)-tag destroys the bioactivity of growth differentiation-9 (GDF9, a homolog of BMP15). In this study we found that recombinant human BMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by C-terminal modification, specifically, fusion to a Flag tag. After purification the mature BMP15 wt is active in transcriptional reporter assays specific for Smad1/5/8 in human granulosa-luteal (hGL) and COV434 granulosa tumor cells, whereas BMP15 with a carboxy-terminal Flag tag remains inactive. Using these same cell models we found that treatment with purified mature BMP15 wt causes a rapid phosphorylation of Smad1. The purified BMP15 wt is a potent stimulator of rat granulosa cell DNA synthesis, which could be antagonized by the BMPRII ectodomain-Fc fusion molecule, whereas the BMP15C-Flag was completely inactive. Further, the BMP15 wt form is a potent stimulator of inhibin B production in hGL cells. We found that the purified BMP15 wt consists of P16 and -17, both of which are post-translationally modified forms. This is the first characterization of a purified untagged human BMP15 mature region, which is stable and highly bioactive in human and rodent granulosa cells and as such is of importance for studies on human fertility.