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Background: Naturally occurring mutations in growth and differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) genes are associated with increased ovulation rate (OR) and litter size (LS) but also sterility. Observing the Tunisian Barbarine ewes of the "W" flock selected for improved prolificacy, we found prolific and infertile ewes with streaky ovaries. Blood genomic DNA was extracted from a subset of low-ovulating, prolific and infertile ewes of the "W" flock, and the entire coding sequences of GDF9 and BMP15 were sequenced. Results: We evidenced a novel polymorphism in the exon 1 of the BMP15 gene associated with increased prolificacy and sterility. This novel mutation called FecXBar is a composite polymorphism associating a single nucleotide substitution (c.301G > T), a 3 bp deletion (c.302_304delCTA) and a C insertion (c.310insC) in the ovine BMP15 cDNA leading to a frame shift at protein position 101. Calculated in the "W" flock, the FecXBar allele increased OR by 0.7 ova and LS by 0.3 lambs (p = 0.08). As for already identified mutations, homozygous females carrying FecXBar exhibited streaky ovaries with a blockade at the primary stage of folliculogenesis as shown by histochemistry. Conclusions: Our investigation demonstrates a new mutation in the BMP15 gene providing a valuable genetic tool to control fecundity in Tunisian Barbarine, usable for diffusion program into conventional flocks looking for prolificacy improvement. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 15 (BMP15) is specifically expressed in oocytes in pigs at all stages from early stages to ovulation and has an important role in oocyte maturation. However, there are few reports on the molecular mechanisms by which BMP15 affects oocyte maturation. In this study, we identified the core promoter region of BMP15 using a dual luciferase activity assay and successfully predicted the DNA binding motif of the transcription factor RUNX1. The effect of BMP15 and RUNX1 on oocyte maturation was examined using the first polar body extrusion rate, a reactive oxygen species (ROS) assay and total glutathione (GSH) content at three time points of 12, 24 and 48 h of in vitro culture of porcine isolated oocytes. Subsequently, the effect of the transcription factor RUNX1 on the TGF-β signaling pathway (BMPR1B and ALK5) was further verified using RT–qPCR and Western blotting. We found that the overexpression of BMP15 significantly increased the first polar body extrusion rate (P < 0.01) and total glutathione content of oocytes cultured in vitro for 24 h and decreased reactive oxygen levels (P < 0.01), whereas interference with BMP15 decreased the first polar body extrusion rate (P < 0.01), increased reactive oxygen levels in oocytes cultured in vitro for 24 h (P < 0.01), and decreased glutathione content (P < 0.01). The dual luciferase activity assay and online software prediction showed that RUNX1 is a potential transcription factor binding to the core promoter region (−1203/−1423 bp) of BMP15. Overexpression of RUNX1 significantly increased the expression of BMP15 and oocyte maturation rate, while inhibition of RUNX1 decreased the expression of BMP15 and the oocyte maturation rate. Moreover, the expression of BMPR1B and ALK5 in the TGF-β signaling pathway increased significantly after overexpression of RUNX1 , whereas their expression decreased after inhibition of RUNX1. Overall, our results suggest that the transcription factor RUNX1 positively regulates the expression of BMP15 and influences oocyte maturation through the TGF-β signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-β signaling pathway to regulate mammalian oocyte maturation. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is an endocrine disorder seen in women of reproductive age and has been gradually increasing over the years. The mechanism of the syndrome has still not been clearly understood. In this study, the possible effects of exogenously administrated melatonin on melatonin (MT1) receptor, Growth Differentiation Factor-9 (GDF9), and Bone Morphogenetic Protein-15 (BMP15) in experimental PCOS were investigated. Thirty-two 6–8-week-old Sprague-Dawley rats were randomly divided into four groups (n = 8 in each) as Sham control (Group 1), Melatonin (Group 2), PCOS (Group 3), and PCOS + Melatonin (Group 4) groups. At the end of the 21st day, the experiment was terminated, the ovary tissues were taken, and Hematoxylin-Eosin staining, MT1, GDF9, BMP15 immunohistochemical labeling, western blot, and quantitative real-time polymerase chain reaction (qPCR) analyses were performed. Serum Luteinizing Hormone (LH)/Follicle Stimulating Hormone (FSH) levels and colpo-cytological examinations were also carried out. The results revealed that melatonin administration increased the expression levels of the MT1 receptor, GDF9, and BMP15 in PCOS at protein and mRNA levels. It was determined that melatonin administration reduced the microscopic symptoms of PCOS. Melatonin was found to be effective via the MT1 receptor in the pathogenesis of PCOS, and it suppressed the transport pathways of GDF9 to granulosa cells in antral follicles. • Melatonin increased MT1R, GDF9 and BMP15 levels in PCOS. • Melatonin administrations reduced the micrscopic symptoms of PCOS. • Melatonin was found to be effective in the PCOS pathogenesis via MT1R. • Melatonin suppress the transport pathways of GDF9 to granulosa cells in antral follicles. [ABSTRACT FROM AUTHOR]

Utilization of fecundity genes such as GDF9 and BMP15 can help improve reproductive traits in sheep breeding programme. To evaluate effects of missense mutations on protein function, the polymorphisms of GDF9 and BMP15 genes were screened in twelve mehraban sheep using DNA sequencing, followed by protein structure modeling. Six single nucleotide polymorphism (SNPs) known as FecG mutations (G1-G6), were detected in exons 1 and 2 of GDF9 gene. Mutations of G1 (GDF9 exon 1 g.2118 G>A), G4 (GDF9 exon 2 g.3451 T>C) and G6 (GDF9 exon 2 g.3974 G>A) have shown amino acid substitution. None polymorphism was detected in exon 1 and exon 2 of BMP15 gene. Based on identified polymorphisms, individuals were classified into three haplotypes of wild haplotype (without mutation), haplotype A (simultaneous mutations of G1, G2, G3 and G4) and haplotype B (simultaneous mutations of G5 and G6). The 3D-structure of GDF9 protein in A and B haplotypes was rotated 90° and 45° than wild haplotype, respectively. The missenses G1/p.Arg87His, G4/p.Glu241Lys and G6/p.Val332Ile variants were benign. However both the missenses of G7/p.Val371Met and G8/p.Ser315Phe were probably damaging. Phylogenetic tree of GDF9 gene revealed that individuals with A and B haplotypes were distinct from wild haplotypes with bootstrapping values of 63 and 76, respectively. In conclusion, GDF9 protein in A and B haplotypes showed a higher performance than wild haplotype due to synergism effects of simultaneous mutations. These types of mutations with effect on turn and helix of GDF9 conservative regions showed physical and functional interaction with TGFβ proteins. [ABSTRACT FROM AUTHOR]

Background: Bone morphogenetic protein-15 (BMP15) is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM) was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1)/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi) method was used to inhibit the expression of (MCP-1)/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR) and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1)/CCL2 and fibrillin1 (FBN1) were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.

Ovarian dysfunction is one of the most common features of women with Turner syndrome. In these women, oocyte apoptosis is markedly accelerated from the early stage of fetal life. Reduction in the number of germ cells disturbs primordial follicle development and thereby leads to the formation of streak gonads. There are three possible causes of accelerated germ cell loss in 45,X ovaries. First, chromosomal pairing failure due to X chromosomal aneuploidy is believed to induce meiotic arrest. Indeed, it has been suggested that the dosage of the X chromosome is more critical for the survival of the oocytes than for other cells in the ovary. Second, impaired coupling between oocytes and granulosa cells may also contribute to germ cell apoptosis. Previous studies have shown that 45,X ovaries may tend to lose tight junctions which are essential for intercellular interactions. Lastly, ovarian dysfunction in women with Turner syndrome is partly attributable to the reduced dosage of several genes on the X chromosome. Specifically, BMP15 , PGRMC1 , and some other genes on the X chromosome have been implicated in ovarian function. Further studies on the mechanisms of ovarian dysfunction are necessary to improve the reproductive outcomes of women with Turner syndrome., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fukami.)

TheBMP15gene is a growth factor and a member of the transforming growth factor β (TGFβ) superfamily, specifically expressed in oocytes. In the present study, polymorphism ofBMP15gene exon 1 was studied using single strand conformational polymorphism (SSCP) and direct DNA sequencing methods in 170 Mehraban and Lori sheep ewes. A 231-bp fragment inBMP15exon 1 was amplified by PCR reactions. Two genotypes (GG and AG) with a new point mutation at position 121 bp of the studied fragment (c.379G>A in reference GenBank number AF236078.1 sequence), deducing an amino acid exchange in the codified amino acid sequence (p.Glu41Lys) were identified in the studied populations. The AG and GG frequencies were 74.4% and 25.6% in Mehraban and 44.7% and 55.3% in Lori sheep, respectively. Frequencies of the A and G alleles were 37.2% and 62.8% in Mehraban and 22.4% and 77.6% in Lori sheep, respectively. Two different secondary structures of protein were predicted for encoded precursor protein. The genotypes GG and AG did not have any significant association with the studied reproductive traits, but the AA genotype is likely to have a lethal or sterility effect. [ABSTRACT FROM AUTHOR]

In the present work we present the main productive studies carried out to characterize ROA sheep (R+, Rasa Oviaragón®), ewes of the Rasa Aragonesa breed carrying in heterozygosity a natural genetic prolific variant found in 2007 (FecXR allele, BMP15 gene). Its effect on prolificacy leads to an increase of 0.35 lambs/lambing ewe when compared with non-carrier ewes, with an additive effect over the standard hormonal treatments used in farms. In artificially inseminated ewes, an increased fertility was also found in a set of farms (71.1 vs. 59.5 %; p<0.01) as well as an advanced puberty (around 30 days). On the contrary, no differences between genotypes were found in male’s fertility. The studies on the preovulatory LH peak did not show differences between genotypes, which allows for the application of the same protocols used in non-carrier ewes concerning Artificial Insemination (AI) at fixed time. The increased ovulation rate of R+ ewes could be related with the smaller size of their ovarian follicles, with no further influence on fertility, as sown in farms as well as on in vitro embryo production studies. Regarding lamb marketing, the presence of the FecXR allele does not affect morphology, lamb growth or the characteristics of the carcass or meat quality of their offspring. Finally, it has been observed that the productive advantages of the FecXR (ROA) variant result in an increase in profitability of farms that use this variant, which confirms its interest for most farmers. [ABSTRACT FROM AUTHOR]

In the hen, optimal ovulation rate depends on selection of a single follicle into the pre-ovulatory hierarchy. Follicle selection is associated with increased oocyte growth and changes in gene expression in granulosa cells surrounding the oocyte, in preparation for ovulation. This study investigated the expression, function and regulation of bone morphogenetic protein-15 (BMP15) during follicle development in the hen. BMP15 mRNA expression was analyzed in the ooplasm and granulosa cells of 3 mm follicles and was confirmed to be primarily in the ooplasm. BMP15 was detected by immunoblotting in 6 and 8 mm follicles near the time of follicle selection. Expression of mRNA for BMP15 receptors ( BMPR1B and BMPR2 ) in granulosa cells increased with follicle size, indicating that BMP15 may play an important role around follicle selection. The function of BMP15 was examined by culturing granulosa cells from 3–5 mm and 6–8 mm follicles with recombinant human BMP15 (rhBMP15). BMP15 increased expression of follicle stimulating hormone receptor ( FSHR ) mRNA and decreased anti-Müllerian hormone ( AMH ) mRNA and occludin ( OCLN) , factors associated with follicle maturation and growth in the hen. Hormonal regulation of BMP15 was assessed by whole follicle culture with estradiol (E2) which increased BMP15 mRNA expression . The distinct expression pattern of BMP15 and its receptors, coupled with the effects of BMP15 to increase FSHR mRNA and decrease AMH mRNA and OCLN mRNA and protein expression suggest that the oocyte may have a role in follicle selection in the chicken. [ABSTRACT FROM AUTHOR]

Goats form the backbone of rural livelihood and financial security systems in India but their population is showing decreasing trend. Improvement of reproductive traits such as prolificacy offers a solution to stabilize the decreasing goat population and to meet the nutritional needs of growing human population. In the present study, six novel SNPs in three candidate genes for prolificacy (BMPR1B, BMP15, and GDF9) were genotyped in seven breeds of Indian goats to evaluate their association with litter size. Tetra primer ARMS-PCR and PCR-RFLP based protocols were developed for genotyping six novel SNPs, namely, T(-242)C in BMPR1B; G735A and C808G in BMP15; and C818T, A959C, and G1189A in GDF9 gene. The effect of breed was highly significant (p ≤ 0.01) on litter size but the effect of genotype was nonsignificant. The effect of parity on litter size was also significant in the prolific Black Bengal breed. The litter size differences observed between breeds are attributed to breed differences. Novel mutations observed at different loci in GDF9, BMP15, and BMPR1B genes do not contribute to the reproductive capability of the investigated breeds. Further studies with more number of breeds and animals exploring association of these novel SNPs with reproductive traits may be fruitful. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 15 (BMP15) is crucial factor for ovulation as well as for increasing litter size. In the present investigation efforts had been carried out to assess the genetic variations in Exon 2 region of BMP15 in goat, using polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) sequencing methods and cooperated frequency distribution to discuss its possibility of related fecundity. Across the 144samples from six breeds were identified in the A963G location of BMP15 using PCR-SSCP and sequences technology. A963A genotype was the most frequent (85.4%) and G963G was the least frequent with a frequency of 4.2% and A963G is 10.4%.. It revealed non significant different between high and low fecundity breed. Therefore, this single nucleotide variant is not common Bio-Marker for fecundity in Goat. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are the most abundant form of DNA polymorphism which can be used as simple genetic markers for many breeding applications as well as for population studies. SNPs in fecundity (Fec) genes viz. BMPR1B, GDF9 and BMP15 were genotyped in Indian goat breeds to assess their basic population parameters and genetic structuring, using simple and economical methods of PCR-RFLP and Tetra-primer ARMS PCR. Three hundred and ninety one animals of seven different goat breeds differing in prolificacy, utility and geographic distribution were employed for diversity assessment at six loci in these Fec genes. Four loci were polymorphic in all the breeds with varied allelic frequencies. Statistical analysis revealed that breeds differed with respect to genetic variability as observed heterozygosity varied from 0.163 (Ganjam) to 0.359 (Jakhrana). Sangamneri and Osmanabadi were least differentiated (0.003) whereas Jakhrana and Black Bengal were most differentiated (0.048) on the basis of genetic distance. The genetic distance as well as PCA analysis indicated that breeds are grouped according to their geographical distribution. The genetic diversity based on fecundity genes presented here is an essential step towards the future exploitation of the available goat genetic resources in research and breeding programs. [ABSTRACT FROM AUTHOR]

Premature ovarian insufficiency ( POI) affects approximately 1% of women before the age of 40. Genetic contribution is a significant component of POI. In this context, heterozygous mutations in NOBOX, BMP15 and GDF9 have been reported. The objective of our study was to evaluate the prevalence of these genes mutations in 125 unrelated Tunisian patients diagnosed with POI. The screening of NOBOX gene revealed three missense mutations (p. Arg117Trp; p. Gly91Trp and p. Pro619Leu) in eight patients. These mutations were not found in a 200 ethnically matched women without fertility problem. The sequencing of BMP15 and GDF9 gene revealed only previously reported variants. In contrast to previous studies, the prevalence of BMP15 variations is not higher than in the control population. Conversely, 6.4% of the cases present a NOBOX mutations; this high prevalence strengthens the consideration of NOBOX gene as strong autosomal candidate for POI. [ABSTRACT FROM AUTHOR]

Quantitative patterns of expression of the growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) genes in different development stages of in vivo and in vitro grown ovarian follicles in sheep were studied for the first time. Both GDF9 and BMP15 were expressed in the cumulus cells and oocytes at all the development stages of in vivo and in vitro grown ovarian follicles. Growth differentiation factor 9 and bone morphogenic protein 15 exhibited stage-specific undulations in the expression in the cumulus cells and oocytes isolated from in vivo grown ovarian follicles. These undulations could be related to discrete development events during the ovarian follicle development. The expression of GDF9 and BMP15 was highest (3.38 ± 0.02 and 2.69 ± 0.06, respectively; P ≤ 0.05) in the primordial follicles compared with preantral, early antral, antral, and large antral stages. Similarly, GDF9 and BMP15 expression in the cumulus cells (0 ± 0.16 and 0 ± 0.07) and oocytes (1.47 ± 0.07 and 1.32 ± 0.03) was lowest (P ≤ 0.05) in the in vivo grown antral follicles. In the cultured follicles, the stage-specific undulations observed in the expression of GDF9 and BMP15 in the in vivo grown follicles were either different or abolished. For example, in the oocytes from in vitro grown follicles, the expression of BMP15 did not change as the development progressed all the way from preantral to large antral follicle stage although in the oocytes from in vivo grown follicles BMP15 expression exhibited stage-specific variations. It is concluded that GDF9 and BMP15 follow a stage-specific pattern of expression during the in vivo development of ovarian follicles in sheep, and in vitro culture altered the stage-specific changes in the expression of these two genes. [ABSTRACT FROM AUTHOR]

Aim of this work was to analyze the fecundity genes BMPRIB ( FecB allele), BMP15 (exon 2) and GDF9 (both exons) in two autochthonous sheep breeds, Laticauda and Bagnolese, reared in Campania region and characterized by a high prolificacy rate. Both breeds have an economic relevance due to the production of meat and milk. The analyses of fecundity genes ruled out the presence of the main alleles ( FecB , FecX G , FecX H , FecX I ), reported earlier to be associated with a high prolificacy rate in other breeds. Eleven SNPs were found in GDF9 exon 2, five of these for the first time, in particular at nt 531, 953 and 1203 in Bagnolese sheep and at nt 617 and 1358 in Laticauda sheep. In Bagnolese breed the association analysis of these SNPs with prolificacy rate showed a significantly higher value of litter size of GG individuals at nt 750 when compared with GA ewes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Black Bengal goats are an interesting genetic material to underpin the genetic mechanism of reproduction due to high prolificacy and sexual precocity. In the present study, novel SNPs in BMPR1B, BMP15 and GDF9 genes were genotyped to evaluate their association with the reproductive traits. PCR-RFLP and Tetra primer ARMS-PCR based protocols were developed for genotyping the six novel SNPs viz. T(-242)C in BMPR1B , G735A and C808G in BMP15 and C818T, A959C and G1189A in GDF9 . Linear mixed model for association of these SNPs with litter size and linear fix model for other traits were employed. The effect of season and parity was highly significant ( p ≤ 0.01) on litter size which varied with change in locus combination. However, there was no significant effect of genotype and year of birth on the litter size. Similarly, age at first heat, age at first service and age at first kidding were not affected by the year of birth as well as the genotype. Season of kidding did not affect the age at first heat and age at first service. However, significant ( p ≤ 0.05) association was observed between the season of kidding and the age at first kidding. The regression of age at sexual maturity on the age at first service and regression of age at first service on the age at first kidding was highly significant ( P ≤ 0.01). Further studies involving more number of breeds as well as animals may be fruitful for exploring the association of novel SNPs with caprine reproductive traits. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study was to determine the effect of a high-fat diet (HFD) on oocyte maturation and quality in a mouse model.Methods: Female BALB/c mice were allocated to one of the following groups: (a) control group (n = 40), which received a controlled diet; or (b) HFD group (n = 40), which received an HFD for 12 weeks. Sections of the ovary were examined histologically. The number of follicles and corpora lutea were counted. In vitro maturation and in vitro fertilisation (IVF) were assessed in germinal vesicle (GV) and metaphase II (MII) oocytes, respectively. The expression of bone morphogenetic protein 15 (BMP15) and leptin receptor genes in GV and MII oocytes was evaluated using reverse transcription real-time polymerase chain reactions.Results: In the HFD group, there was a decreased number of primordial and Graafian follicles, as well as corpora lutea (p < 0.05). The rate of oocyte development to the MII stage was also reduced (p < 0.001). Cumulus expansion was observed more frequently in the control group than the HFD group (p < 0.05). The IVF rate in the HFD group was lower than that in the control group (p < 0.05). In the HFD group, BMP15 and leptin receptor genes were upregulated in the GV stage (p > 0.05) and MII stage (p < 0.05), compared to the control group.Conclusion: An HFD reduces folliculogenesis in the primordial and Graafian stages, in vitro maturation and in vitro fertilisation rates, as well as oocyte quality in mice. [ABSTRACT FROM AUTHOR]

We hypothesised that advanced mare age is associated with follicle and oocyte gene alterations. The aims of the study were to examine quantitative and temporal differences in mRNA for LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells, phosphodiesterase (PDE) 4D in cumulus cells and PDE3A, G-protein-coupled receptor 3 (GPR3), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and mitochondrial (mt) DNA in oocytes. Samples were collected from dominant follicles of Young (3-12 years) and Old (≥20 years) mares at 0, 6, 9 and 12 h after administration of equine recombinant LH. LHR mRNA declined after 0 h in Young mares, with no time effect in Old mares. For both ages, gene expression of AREG was elevated at 6 and 9 h and EREG was expression was elevated at 9 h, with higher expression in Old than Young mares. Cumulus cell PDE4D expression increased by 6 h (Old) and 12 h (Young). Oocyte GPR3 expression peaked at 9 and 12 h in Young and Old mares, respectively. Expression of PDE3A increased at 6 h, with the increase greater in oocytes from Old than Young mares at 6 and 9 h. Mean GDF9 and BMP15 transcripts were higher in Young than Old, with a peak at 6 h. Copy numbers of mtDNA did not vary over time in oocytes from Young mares, but a temporal decrease was observed in oocytes from Old mares. The results support an age-associated asynchrony in the expression of genes that are essential for follicular and oocyte maturation before ovulation. Maternal aging is related to reduced fertility in various species, including the mare. We compared gene expression in follicle cells and oocytes from Young and Old mares after an ovulation-induction signal. Differences were observed in the timing and amount of expression for key regulatory genes between Young and Old mares, suggesting age-associated changes in follicles and oocytes that could impact fertility. [ABSTRACT FROM AUTHOR]

Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2ʹ-O-dibutyryladenosine 3′:5′ cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2 , TNFIPA6 and PTGS2 . Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence. [ABSTRACT FROM AUTHOR]

Fertility traits, such as the ovulation rate and the number of offspring at birth, are genetically regulated by fecundity genes. This study was performed to identify DNA polymorphisms in Booroola (FecB), Galway (FecXG), Inverdale (FecXl) and Calpastatin (CAST) genes in Sakiz sheep. A total of 71 ewes were genotyped for gene or allelic polymorphisms in the genes listed above using the PCR-RFLP method. The results obtained from this study indicated that all of the Sakiz ewes sampled were non-carriers for FecB, FecXG or FecXl mutations. However, genotypic frequencies in the CAST gene were 0.59,0.36 and 0.05 for AA, AB and BB, respectively. A significant deviation from Hardy-Weinberg equilibrium for the CAST gene was not observed in the investigated breed (P>0.05). As a result, more extensive screening is required as tests for newly discovered mutations are developed. Additionally, this study is the first to report a genetic polymorphism in the CAST gene in Sakiz sheep. [ABSTRACT FROM AUTHOR]

Some mutations in bone morphogenetic protein 15 ( BMP15) gene have been known to be associated with prolificacy in various breeds of sheep. Polymorphism of BMP15 gene exon 2 was studied in 100 Mehraban and 100 Lori ewes, using PCR-SSCP and DNA sequencing methods. A new point mutation (G → A) was found at position 57 of the amplified 312-b fragment of BMP15 gene exon 2. The frequencies of the AG and GG genotypes were 69.4 and 30.6 % in Mehraban and 44.7 and 55.3 % in Lori ewes, respectively. No individual carrying the AA genotype was found in the studied population. The allelic frequencies for A and G alleles were 34.7 and 65.3 % in Mehraban and 22.4 and 77.6 % in Lori ewes, respectively. Average litter size in the AG genotype (1.56) was significantly ( P < 0.01) higher than the GG ewes (1.08). The results of the present study indicated the potential of the observed SNP in exon 2 of BMP15 for further exploitation in marker-assisted selection to improve reproduction efficiency of Mehraban and Lori sheep. [ABSTRACT FROM AUTHOR]

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding Site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF. [ABSTRACT FROM AUTHOR]

Objective Premature ovarian failure (POF) is a complex, heterogeneous disorder that is influenced by multiple genetic components. This meta-analysis aimed to investigate the association between gene variants and susceptibility to POF. Study design MEDLINE and CNKI were searched for studies published from inception (1950) to June 2014. Meta-analysis was performed when three or more studies reported genetic data on the same polymorphism or mutation. Additive and dominant models were analyzed using RevMan Version 5.1. Results The literature search yielded 575 articles, of which 59 studies on the association between POF and gene variants were identified for meta-analysis. Five genes were selected for analysis, including 10 common gene polymorphisms [BMP15 (−9C>G, 788insTCT and 852C>T), ESR1 (−351A>G and −397C>T), FMR1 CGG repeat, FSHR (919A>G and 2039A>G), INHA (−16C>T and −124A>G)] and two mutations (BMP15 538G>A and INHA 769G>A). BMP15 538G>A was found to be significantly more common in patients with POF compared with controls. No significant associations were found between the other variants of BMP15 and POF. With respect to ESR1, the accumulative results were not significant, although the findings of the individual studies were controversial. The incidence of FMR1 premutation was significantly higher in patients with POF compared with controls [odds ratio (OR) 9.2, 95% confidence interval (CI) 5.42–15.61; p < 0.001] in the overall population, as well as in both Caucasian and Asian subgroups. Stratified analysis was applied for INHA 769G>A by ethnicity; a significant association with POF was only found in the Asian subgroup (allelic frequency: OR 8.89, 95% CI 2.1–5.52; p = 0.004). No significant associations were found between the other variants of INHA and POF. Conclusions BMP15 538A, FMR1 premutation and INHA 769A (in Asians alone) may indicate susceptibility to POF. Further well-designed studies and larger samples are required to confirm the association between gene variants and POF. [ABSTRACT FROM AUTHOR]

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Purpose: We hypothesised that varying native oocyte-secreted factor (OSF) exposure or using different recombinant OSF peptides would have differential effects on post-in vitro maturation (IVM) embryo and fetal development. Methods: Mouse cumulus oocyte complexes (COCs) were treated with the purified mature domain of GDF9 and/or BMP15 or were co-cultured with denuded oocytes (DOs) from 0 h or 3 h of IVM. DOs were matured for 3 h as either intact COCs+/-FSH before denuding, or as DOs + FSH. COCs were fertilised and blastocyst development was assessed on days 5 and 6, and either differentially stained for ICM numbers or vitrified/warmed embryos were transferred to recipients to assess implantation and fetal rates. Results: No improvement in embryo development was observed with the addition of GDF9 and/or BMP15 to IVM. In contrast, embryos derived from COCs co-cultured with DOs had significantly improved blastocyst rates and ICM numbers compared to controls ( P < 0.05). The highest response was obtained when DOs were first added to COCs at 3 h of IVM, after being pre-treated (0-3 h) as COCs + FSH. Compared to control, co-culture with DOs from 3 h did not affect implantation rates but more than doubled fetal yield (21 % vs 48 %; P < 0.05). GDF9 Western blot analysis was unable to detect any differences in quantity or form of GDF9 (17 and 65 kDa) in extracts of DO at 0 h or 3 h. Conclusions: This study provides new knowledge on means to improve oocyte quality in vitro which has the potential to significantly aid human infertility treatment and animal embryo production technologies. [ABSTRACT FROM AUTHOR]

STUDY QUESTION What is the burden of X chromosome mosaicism in the occurrence of spontaneous menarche (SM) in Turner syndrome (TS)? SUMMARY ANSWER SM was significantly associated with X chromosome mosaicism in the TS patients; a mosaicism with around 10% euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques on uncultivated tissues. WHAT IS KNOWN ALREADY Spontaneous puberty can be observed in a minority of patients with TS, more frequently, but not exclusively, in those with a high level of 46,XX/45,X mosaicism at standard karyotype. The genetic mechanisms contributing to ovarian function in TS patients are still not determined. However, submicroscopic X-linked and autosomal copy number variations (CNVs) have recently emerged as an important genetic risk category for premature ovarian insufficiency and may be involved in modulating the TS ovarian phenotype. STUDY DESIGN, SIZE, DURATION A group of 40 patients with a diagnosis of TS at conventional karyotyping participated in the study; 6 patients had SM and 34 patients had primary amenorrhoea (PA). All clinical data and the patients’ DNA samples were collected over the years at a single paediatric clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS The patients' samples were used to perform both genetic (Copy Number Assay) and molecular-cytogenetic (array-CGH and iFISH, interphase-FISH) analyses in order to evaluate the X chromosome mosaicism rate and to detect possible rare CNVs of genes with a known or predicted role in female fertility. MAIN RESULTS AND THE ROLE OF CHANCE All TS patients showed variable percentages of the 46,XX lineage, but these percentages were higher in the SM group (P < 0.01). A mosaicism around 10% for the euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques performed in uncultivated tissues. A few CNVs involving autosomal and X-linked ovary-related loci were identified by array-CGH analysis and confirmed by real-time quantitative PCR, including a BMP15 gene duplication at Xp11.22, a deletion interrupting the PAPPA gene at 9q33.1, and an intragenic duplication involving the PDE8A gene at 15q25.3. LIMITATIONS, REASONS FOR CAUTION This is a pilot study on a relatively small sample size and confirmation in larger TS cohorts may be required. The ovarian tissue could not be studied in any patients and in a subgroup of patients, the mosaicism was estimated in tissues of different embryonic origin. WIDER IMPLICATIONS OF THE FINDINGS The combined determination of X chromosome mosaicism by molecular and molecular-cytogenetic techniques may become useful for the prediction of SM in TS. The detection of CNVs in both X-linked and autosomal ovary-related genes further suggests gene dosage as a relevant mechanism contributing to the ovarian phenotype of TS patients. These CNVs may pinpoint novel candidates relevant to female fertility and generate further insights into the mechanisms contributing to ovarian function. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Telethon Foundation (grant no: GGP09126 to L.P.), the Italian Ministry of the University and Research (grant number: 2006065999 to P.F.) and a Ministry of Health grant ‘Ricerca Corrente’ to IRCCS Istituto Auxologico Italiano (grant number: 08C704-2006). The authors have no conflict of interest to declare. [ABSTRACT FROM AUTHOR]

Abstract: Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P <0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P <0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles. [Copyright &y& Elsevier]

Bone Morphogenetic Protein 15 (BMP15), also known as Growth and Differentiation Factor 9B (GDF9B), is a member of the superfamily of Transforming Growth Factor β (TGFβ) and it expression in the oocyte is essential for follicular development and growth. Polymorphisms in the BMP15 gene are associated to the increase in ovulation rate or prolificacy in some sheep breeds. The objectives of this study were to determine the FecXG, FecXL and FecXH polymorphisms with a mutation of a single base (SNP– single nucleotide polymorphisms) on the BMP15 gene, and to estimate the association of the three polymorphisms with the lambing type in Pelibuey sheep. Blood samples were taken from the jugular vein of 253 adult Pelibuey ewes in reproductive age to determine the SNPs, according to the most probable producing ability (MPPA) in prolificacy. Through the tetraprimer ARMS-PCR technique (Amplification Refractory Mutation System-Polymerase Chain Reaction), the polymorphisms were determined for the first time in Pelibuey sheep. Of the 253 ewes, 1050 births were used, where the wild (CC) and mutated (TT) homozygous genotypes of polymorphism FecXG with 45 % and 43 %, respectively, and the wild (GG) and mutated (AA) homozygous genotype of polymorphism FecXL with 66 % and 29 %, respectively, were associated to a higher number of double lambing (p≤0.01). In polymorphism FecXH only CC genotype was found, with 52 % double lambing (p≤0.01). In conclusion, polymorphisms FecXG and FecXL of the BMP15 gene were determined for the first time in Pelibuey ewes, and they are associated to a higher number of double lambing. [ABSTRACT FROM AUTHOR]

The use of exogenous gonadotropins to increase oocyte yields for better pregnancy outcomes remains unpredictable and inefficient. The objectives of this review are to address two questions concerning this issue: (1) are there any alternatives for improving consistency in oocyte yields? (2) is it possible to develop a molecular diagnostic test for oocytes with blastocyst potential? Studies in sheep with a heterozygous inactivating mutation in the oocyte-derived growth factor, bone morphogenetic protein 15 (BMP15), report increased oocyte yields and significantly more offspring. Moreover, partial immuno-neutralization of BMP15 bioactivity increases oocyte yield without modifying gonadotropin or ovarian steroid secretion. Therefore, it is hypothesized that the development of BMP15 antagonists may prove them to be suitable alternatives to exogenous gonadotropins. Recent evidence from analyses of candidate gene expression in human cumulus cells separated from individual oocytes before IVF indicates a potential non-invasive molecular screen for oocytes with improved blastocyst and live-birth outcomes. [ABSTRACT FROM AUTHOR]

Aims: To investigate the regulatory mechanism of SCF expression in human GCs of PCOS related follicles., Materials and Methods: SCF, BMP15 and HIF-1α were evaluated in human serums, follicular fluids (FFs) and GCs, which were collected from 69 PCOS patients and 74 normal ovulatory patients. KGN cell line was used in this study., Results: Our results showed that the rate of MII oocyte and 2PN fertilization was lower in PCOS group, though PCOS patients retrieved much more oocytes. The level of BMP15 in FF and the level of SCF in serum and FF were also lower in PCOS patients. We found a weakened expression of HIF-1α and SCF in GCs from PCOS patients when compared with the non-PCOS patients. The expression of HIF-1α and SCF was significantly increased in KGN cells after treating cells with rhBMP15, however, this promotion effects of BMP15 on HIF-1α and SCF expression were obviously abolished by co-treatment with BMP-I receptor inhibitor (DM). Moreover, knock down of HIF-1α expression in KGN cells significantly reduced the expression of SCF in human GCs, in spite of activating BMP15 signaling pathway., Conclusions: The present study suggest that BMP15 could induce SCF expression by up-regulating HIF-1α expression in human GCs, the aberrance of this signaling pathway might be involved in the PCOS related abnormal follicular development.

Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

The aim of this study was to investigate the effects of sitagliptin/metformin (sitaformin), metformin and sitagliptin in PCOS patients. PCOS is a hormonal disorder and therefore the use of treatments that modulate hormone levels Like AMH, testosterone, insulin, leptin and especially FAI and HOMA-IR can reclaim the symptoms of PCOS. PCOS also increases oxidative stress and lipid peroxidation. Therefore, in clinical and research trials, the level of these factors is usually examined to reduce patients' symptoms. Participants were randomly assigned to receive metformin, sitagliptin, sitaformin or placebo Treatment was carried out 2 months before the start of the ovulation cycle and continued until the day of ovum pick up. The serum levels of HOMA-IR and FAI decreased significantly in the treated groups compared to the placebo. The serum and the FF levels of leptin also decreased significantly in the sitaformin group when compared to the metformin and sitagliptin groups. Moreover, the serum and FF levels of AMH and MDA had a significant decrease in the sitaformin and sitagliptin group compared to the placebo. The mRNA expression and protein levels of GDF9 and BMP15 in the cumulus cells increased significantly in the sitaformin compared to metformin and sitagliptin groups. The expression level of GDF9 and BMP15 mRNA were positively correlated with the fertilization rate and grade I embryos. Sitaformin improves levels of GDF9 and BMP15 in PCOS compared to metformin and sitagliptin, which can increase the rate of fertilization and grade I embryos. • Treatment with sitagleptin/metformin can be better in PCOS than metformin monotherapy. • Sitagliptin/meforminb, as a combination of metformin and sitagliptin, improves the rate of fertilization and grade I embryos in PCOS patients. • The expression level of GDF9 and BMP15 mRNA were positively correlated with the fertilization rate and grade I embryos. [ABSTRACT FROM AUTHOR]

Background: Bone morphogenetic protein-15 (BMP15) is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM) was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1)/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi) method was used to inhibit the expression of (MCP-1)/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR) and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1)/CCL2 and fibrillin1 (FBN1) were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The present study was undertaken to explore the genetic basis of caprine prolificacy and to screen indigenous goats for prolificacy associated markers of sheep in BMPR1B, GDF9 and BMP15 genes. To detect the associated mutations and identify novel allelic variants in the candidate genes, representative samples were collected from the breeding tract of indigenous goat breeds varying in prolificacy and geographic distribution. DNA was extracted and PCR amplification was done using primers designed or available in literature for the coding DNA sequence of candidate genes. Direct sequencing was done to identify the genetic variations. Mutations in the candidate genes associated with fecundity in sheep were not detected in Indian goats. Three non-synonymous SNPs (C818T, A959C and G1189A) were identified in exon 2 of GDF9 gene out of which mutation A959C has been associated with prolificacy in exotic goats. Two novel SNPs (G735A and C808G) were observed in exon 2 of BMP15 gene. [Copyright &y& Elsevier]

Collichthys lucidus ( C. lucidus ) is an economically important fish species, exhibiting sexual dimorphism in its growth rate. However, there is a lack of research on its underlying sex-related mechanisms. Therefore, small RNA sequencing was performed to better comprehend these sex-related molecular mechanisms. In total, 171 differentially expressed miRNAs (DE-miRNAs) were identified between the ovaries and testes. Functional enrichment analysis revealed that the target genes of DE-miRNAs were considerably enriched in the p53 signaling, PI3K-Akt signaling, and TGF-beta signaling pathways. In addition, sex-related miRNAs were identified, and the expression of miR-430c-3p and miR-430f-3p was specifically observed in the gonads compared with other organs and their expression was markedly upregulated in the testes relative to the ovaries. Bmp15 was a target of miR-430c-3p and was greatly expressed in the ovaries compared with the testes. Importantly, miR-430c-3p and bmp15 co-expressed in the ovaries and testes. This research provides the first detailed miRNA profiles for C. lucidus concerning sex, likely laying the basis for further studies on sex differentiation in C. lucidus ., Competing Interests: Author JC was employed by Fuding Seagull Fishing Food Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Song, Gan, Xie, Chen and Wang.)

Context: The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome., Objective: Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)?, Design and Setting: Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation., Patients: Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size., Main Outcome Measures: Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays., Results: The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ± 189 pg/mL [mean ± SEM], n = 188; non-PCO: 3498 ± 199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ± 40 pg/mL, n = 125; non-PCO: 573 ± 55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO., Conclusions: Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Assisted reproductive technology has important clinical applications and commercial values in the horse industry. However, this approach is limited largely by the low efficiency of oocyte in vitro maturation (IVM), especially cytoplasmic maturation. To improve the efficiency of mare oocyte IVM, we evaluated the effects of co-culture with cumulus-oocyte complexes (COCs) and granulosa cells (GCs) from follicles with small (<15 mm) and large diameters (>35 mm). Our results showed that oocyte nucleus maturation was not significantly improved by co-culturing with GCs. Interestingly, the cytoplasmic maturation of oocytes, defined by the distribution of cortical granules and mitochondria, as well as reactive oxygen species (ROS) levels, improved dramatically by co-culture with GCs, especially those derived from small follicles. Moreover, GCs promoted cumulus cell expansion by upregulating the expression of BMP15 in oocytes. To determine the mechanism underlying the effects of GCs, the transcriptomes of GCs from large and small follicles were compared. Expression levels of COL1A2 , COL6A1 , and COL6A2 were significantly higher in GCs from small follicles than in those from large follicles. These three genes were enriched in the extracellular matrix proteins-receptor interaction pathway and were involved in the regulation of collagens. Taken together, our results suggest that co-culture with GCs is beneficial to oocyte cytoplasmic maturation, and the increased expression of COL1A2 , COL6A1 , and COL6A2 improve the mare oocyte IVM system via the regulation of collagen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Zhao, Xu, Zhang, Zhu, Pan and Huang.)

Abstract Genetic mutations with major effects on ovulation rate in sheep were recently identified in two genes of the transforming growth factor (TGFβ) superfamily and a TGFβ receptor, namely bone morphogenetic protein 15 (BMP15), otherwise known as the growth differentiation factor 9b (GDF9b), GDF9 and activin-like kinase 6 (ALK6) otherwise known as the BMP receptor type IB (BMPRIB). Animals homozygous for the BMP15 or GDF9 mutations are anovulatory whereas animals heterozygous for BMP15 or GDF9 or heterozygous or homozygous for ALK6 have higher than normal ovulation rates. Immunisation of ewes against BMP15 or GDF9 shows that both are essential for normal follicular development and control of ovulation rate. Common features of fertile animals with the BMP15, ALK6 (and possibly GDF9) mutations are changes in oocyte development during early preantral follicular growth, earlier maturation of granulosa cells and ovulation of mature follicles at smaller diameters. In summary, these findings have led to a new paradigm in reproductive biology, namely that the oocyte plays a key role in regulating the ovulation rate.