Bone Morphogenetic Protein 15 Gene Research Articles

Detection of genetic variations in the GDF9 and BMP15 genes in Kazakh meat-wool sheep.

Kazakh meat-wool sheep are of great interest because of the intrabreed multifetal type's high productivity of 140 %-160 %. Genes encoding growth differentiation factor-9 (GDF9) and bone morphogenetic protein 15 (BMP15) are promising candidates for studying sheep productivity, as they affect fertility in mammals, including sheep. Thus, the purpose of this study was to assess the fertility of the Kazakh meat-wool sheep breed based on GDF9 and BMP15 candidate genes of fecundity for the selection of animals with valuable genotypes. We selected 300 heads of the Kazakh meat-wool sheep breed from two populations for PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis, 15 of which were subsequently used for sequencing of exon regions of the GDF9 and BMP15 genes. The sheep populations were tested for G1 and G8 mutations of the GDF9 gene and B2 and B4 mutations of the BMP15 gene. The PCR-RFLP analysis revealed that 59 (19.7 %) of the 300 Kazakh meat-wool breed sheep were heterozygous carriers of the G1 mutation (genotype AG) of the GDF9 gene, and sequencing analysis supported these results. The comparative phylogenetic analysis showed a clear separation of Kazakh meat-wool sheep wild types and carriers of the G1 mutation. This mutation was reported to have a relationship with the animals' litter size in other sheep breeds. For this reason, similar relationships should be investigated in Kazakh meat-wool sheep. However, G8, B2, and B4 mutations were not detected among the studied animal populations, showing that these mutations are not characteristic of the Kazakh meat-wool sheep breed.

Implication of Novel BMP15 and GDF9 Variants in Unexpected Poor Ovarian Response.

Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.

Investigating mutations in the genes GDF9 and BMP15 in Pelibuey sheep through the amplification-refractory mutation system with tetra-primers

Single Nucleotide Polymorphisms (SNP) or mutations are variations with a broad distribution in the genome and, as part of genetic studies, SNP allow the identification of allelic variants related to characteristics of economic importance in sheep production. However, the identification of SNP and their genotypes through sequencing is expensive, as it requires specialized materials and equipment. The objective of this study was to identify polymorphisms and their genotypes in the growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in Pelibuey sheep using the tetra-primer amplification-refractory mutation system through polymerase chain reaction (T-ARMS-PCR). DNA extraction and amplification of BMP15 and GDF9 were conducted from blood samples contained in WhatmanTM FTATM cards from 60 multiparous Pelibuey ewes with reproductive records. The T-ARMS-PCR methodology allowed the identification of wild-type genotypes and mutated homozygous genotypes in polymorphisms G4 and G6 of GDF9, whereas mutations in the BMP15 gene were not found. These results were confirmed by sequencing. In conclusion, the T-ARMS-PCR methodology allowed the identification of mutated and wild-type genotypes in SNP G4 and G6 of GDF9, although no mutations were found in BMP15 in Pelibuey sheep. This technique was found to be reliable, rapid, and easily applied to identify polymorphic genotypes.

Polymorphisms Analysis of BMP15, GDF9 and BMPR1B in Tibetan Cashmere Goat (Capra hircus).

The Tibetan cashmere goat is a prolific goat breed in China. In sheep breeds, natural mutations have demonstrated that the transforming growth factor beta (TGF-β) super family ligands, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor (BMPR1B), are essential for ovulation and increasing litter size. In this study, 216 female Tibetan cashmere goats were sampled, and candidate genes with fecundity traits were detected via restriction fragment length polymorphism (RFLP) and sequenced. Four polymorphic loci were found in specific amplification fragments of BMP15 and GDF9. Two SNP sites of the BMP15 gene were discovered, namely G732A and C805G. The G732A mutation did not cause the change in amino acids, and the frequencies of each genotype were 0.695 for the GG type, 0.282 for the GA type and 0.023 for the AA type. The C805G mutation caused amino acids to change from glutamine to glutamate. The genotype frequencies were 0.620 for the CC type, 0.320 for the CG type and 0.320 for the CG type. For the GG type 0.060, the G3 and G4 mutations of the GDF9 gene were all homozygous mutations. Two known SNP sites, C719T and G1189A, were detected in the Tibetan cashmere goat GDF9 gene, of which the C719T mutation caused a change of alanine to valine, with a genotype frequency of 0.944 for the CC type and 0.056 for the CT type, whereas no TT type was found. The G1189A mutation caused valine to become isoleucine, and the frequencies of each genotype were 0.579 for the GG type, 0.305 for the GA type and 0.116 for the AA type; G1, B2, B3, B4, FecXH, FecXI, FecXL, G2, G5, G6, G7, G8, FecGE, FecTT and FecB mutations were not found in Tibetan cashmere goats. The results of this study provide a data basis for future studies of BMP15, GDF9 and BMPR1B gene mutations in goats.

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.

Effects of novel variants in BMP15 gene on litter size in Mongolia and Ujimqin sheep breeds

Reproductive traits, such as ovulation rate and litter size, are important factors influencing the sheep industry. The bone morphogenetic protein 15 (BMP15) is a major gene affecting the reproductive traits in sheep, and multiple mutations in BMP15 gene could affect the ovulation rate and litter size in many sheep breeds, showing high breed specificity. However, identification of novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are still important. In this study, we sequenced the BMP15 gene of Mongolia sheep, and 12 novel variants were detected by direct sequencing and whole-genome resequencing. Among them, the g.50985975 G > A polymorphism in intron and synonymous c.755 T > C (Leu252Pro) in exon 2 of BMP15 were significantly associated with the litter sizes of Mongolia ewes (P < 0.01 and P < 0.01, respectively), as well as the g.50988478C > A and g.50987863G > A in the promoter region of BMP15 were significantly associated with the litter sizes of Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The c.755 T > C mutation is predicted to change the tertiary structure of BMP15. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

Identification of polymorphisms in GDF9 and BMP15 genes in Jamunapari and crossbred goats in Bangladesh.

Polymorphisms in growth differentiation factor 9 (GDF9)andbone morphogenetic protein 15 (BMP15)genes have been found to beassociated with litter size in goats across the globe. Our previous study detected single-nucleotide polymorphisms (SNPs) inGDF9andBMP15genes associated with litter size in Black Bengal, Bangladesh's primary native goat breed. However, Jamunapari and crossbred goats in Bangladesh are yet to be investigated for litter size-associated polymorphisms. In this study, we screened Jamunapari and crossbred (50% Black Bengal × 50% Jamunapari) goats to identify polymorphisms inthe GDF9and BMP15genes and to assess the association between identified SNPs and litter size. The genomic DNA from 100 female goats (50 Jamunapari and 50 crossbred) was used in polymerase chain reactions (PCRs) to amplify exon 2 ofthe GDF9and exon 2 ofthe BMP15genes. PCR products were sequenced employing the BigDye Terminator cycle sequencing protocol to identify SNPs. We used a generalized linear model to perform the association analysis for identified SNPs and litter size. Seven SNPs were identified, of which four, C818CT, G1073A, G1189A, and G1330T, were inthe GDF9gene and three, G616T, G735A, and G811A, were inthe BMP15gene. G735A was a synonymous SNP, whereas the remaining were non-synonymous SNPs. Identified SNP loci in GDF9 were low polymorphic (PIC < 0.25), while loci in BMP15 were moderately polymorphic (PIC ≥ 0.25). The genotypes at the G1330T locus had a significant (p < 0.05) difference in litter size in Jamunapari goats, but no significant difference was observed for all genotypes at other loci. Therefore, the G1330T loci could be useful as a marker in marker-assisted selection for litter size traits in goats of Bangladesh.

Altered Expression of GDF9 and BMP15 Genes in Granulosa Cells of Diminished Ovarian Reserve Patients: A Case-Control Study.

Objective Diminished ovarian reserve (DOR) is a challenging issue encountered during assisted reproductive technology. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) belong to the transforming growth factor-beta (TGF-β) superfamily which are essential for folliculogenesis. We aimed to the evaluation of the GDF9 and BMP15 expression in the granulosa cells (GCs) of DOR patients. Materials and Methods This case-control study included 14 women with DOR and 12 controls, who were between 28- 40 years of age undergoing controlled ovarian stimulation with a gonadotropin releasing hormone (GnRH) antagonist protocol. DOR patients were selected by the Bologna criteria. The GCs were extracted from the aspirated follicular fluids and RNA isolated from this. The fold change of gene expressions was assessed by real-time polymerase chain reaction (PCR). Results GDF9 expression in patients was 0.23 times lower than the control group, which was significant (P<0.0001). BMP15 expression in patients was 0.32 times lower than the control group, which was significant (P<0.0001). The number of archived oocytes, MII, and two pronuclei (PN) embryos was higher in the control group and these differences were statistically significant (P<0.05). ConclusionGiven that GDF9 and BMP15 are specifically involved during follicular recruitmen., we expect expression of these two genes in DOR patients which is greatly reduced by reducing follicular reserve.

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

BackgroundBone 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.MethodsTwenty-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.ResultsBy 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.ConclusionsHere 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.

The effect of gold nanoparticles synthesized with Achillea biebersteinii on gene expression in Cultured preantral Follicles derived from NMRI mice ovary

Infertility is one of the global public health problems facing society today. Studies on the causes of infertility have greatly expanded with the advancement of new technologies. The increasing use of nanomaterials has raised concerns about nanoparticles' potential dangers. The use of plant extracts is an easy and cost-effective approach to synthesize metal nanoparticles. In this comparative study, the effects of green and chemically synthesized Gold nanoparticles (GNPs), as well as Achillea biebersteinii (AM) (medicinal plant) extract, were investigated on the maturity of preantral Follicles (PF) of mice in vitro. The expression of GDF-9, BMP-15, GPX, and SOD-1 genes was analyzed by real-time polymerase chain reaction (Real Time-PCR). Twenty PF in control and experimental groups were treated with 50 μg/ml of AM extract, green GNPs, and GNPs, and were then used to determine intracellular ROS (Reactive oxygen species) in the first and fifth days after culture. AM extract also increased the expression of genes that stimulate preantral follicle growth (PF), including Growth differentiation factor-9 (GDF-9)and Bone morphogenetic protein 15 (BMP-15). Expression of the glutathione peroxidase (GPX) gene was observed in the group of NPs synthesized by a green method. Due to its antioxidant properties, this gene expression was also higher in the plant extract group than commercially synthesized GNPs. Overall, this study's findings showed that AM extract, due to its antioxidant effects, improves PF development, and AM-GNPs did not significantly impact PF growth, but GNPs inhibited PF growth. Findings suggest that the AM extract that covered the surface of AM-GNPs may be responsible for reducing the toxic effects of GNPs.

Screening for Inverdale (FecXI) Mutation in BMP15 Gene in Prolific Turkish Awassi Sheep

In the early 1980s, by the determination of the Boorola gene (FecB) in Booroola sheep with high ovulation rate, there has been a great interest for identification of genes that are responsible for prolificacy and their use in breeding programs. The mutation occurred in Bone morphogenetic protein 15 (BMP15), Bone morphogenetic protein-1B (BMPR-1B) and Growth differentiation factor 9 (GDF9) genes have been found to increase the ovulation rate. Additionally some others gene are known to exist based on expressed inheritance patterns although the location of mutations has yet to be found. In the BMP15 gene, with almost the same phenotypic expression eight distinct mutations have been recognized, among them Hanna (FecXH) and Inverdale (FecXI) were identified first in Romney sheep in New Zealand. X-linked dominant genes with sterility in homozygous females are the modes of inheritance of the BMP15 gene. A total of 88 prolific Awassi sheep were screened for the presence of the FecXI mutation in the BMP15 gene and FecXI mutation was not found in any of the sheep tested.

An investigation of the effects of BMPR1B, BMP15, and GDF9 genes on litter size in Ramlıç and Dağlıç sheep.

This study was carried out to determine the presence of polymorphisms in genes affecting litter size. The SNPs in bone morphogenetic protein receptor type 1B (BMPR1B), bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) genes were detected in 60 uniparous and 60 multiparous ewes from Ramlıç and Dağlıç breeds. The ewes are maintained in nine public herds at the breeding station of the Afyonkarahisar Sheep and Goats Breeders' Association and lambed in two consecutive breeding seasons. PCR and DNA sequencing analyses were conducted, and 36, 4, and 11 SNPs in Ramlıç and 40, 3, and 11 SNPs in Dağlıç were detected in BMPR1B, BMP15, and GDF9 genes, respectively. A total of 16 SNPs in Ramlıç and 10 SNPs in Dağlıç breeds for three genes were found to be significant (). The resulting analyses showed that four SNPs (g.49496G>A, c.1658A>C, c.2037C>T, c.2053C>T) of the BMPR1B gene and one deletion mutation (c.28_30delCTT) in the BMP15 gene of the Ramlıç breed as well as five SNPs (c.1487C>A, c.2492C>T, c.2523G>A, c.2880A>G, and c.2763G>A) of the BMPR1B gene of the Dağlıç breed have significant positive regression coefficients in the desired direction of the rare allele. The observed mutations have potential to be used as genetic markers in the selection of prolific animals for both breeds.

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro.

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4 ) and estradiol (E2 )) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.

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.

Expression Profiles of the Progesterone Receptor, Cyclooxygenase-2, Growth Differentiation Factor 9, and Bone Morphogenetic Protein 15 Transcripts in the Canine Oviducts during the Oestrous Cycle.

Simple SummaryThe oestrous cycle in canines is specifically more extended than that in other mammals. This implies that the oocytes do not reach maturity within the ovarian follicle but undergo final maturation in the oviducts. Besides oocyte maturation, the oviduct provides the necessary milieu for fertilization and preimplantation embryonic development. Consequently, the oviductal environment presumably changes in the postovulatory period and throughout the entire reproductive cycle to provide a suitable condition for supporting different functions. In this study, we evaluated the gene expression of different genes associated with oocyte-embryo development, such as progesterone receptor, cyclooxygenase-2, growth differentiation factor 9, and bone morphogenetic protein 15 in the canine oviductal cells at different phases of the oestrous cycle. Using quantitative PCR (qPCR) analysis in bitch oviductal cells, this study revealed the ovarian cycle’s influence on the oviductal essential transcripts in the bitch. It also assessed the influence of the ovulated cumulus-oocytes complexes on the expression of GDF-9 and BMP-15 genes. Thus, the oestrous-cycle-dependent gene expression pattern of PR, COX-2, GDF-9, BMP-15 in the canine oviduct was found to execute the oviductal cell interactions necessary for the development and function of the canine reproductive tract.The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs.

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.

Allelic polymorphism in exon 1 of GDF9 and exon 2 of BMP15 genes and its impact on litter size at lambing in Iran-Black sheep

The main purpose of this study was to assess the presence of the previously reported single nucleotide polymorphisms (SNPs) in the sheep growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes and their association with litter size at lambing in Iran-Black sheep breed. Blood samples were taken from 120 Iran-Black ewes. DNA extraction was conducted using a modified salting-out method. DNA fragments with sizes of 462bp and 141bp for the GDF9 and BMP15 genes were amplified using PCR with specific primers, respectively. The PCR-RFLP approach was adopted for detecting the genotypes. The results indicated that the SNP in the exon 2 of BMP15 is a monomorphic locus in Iran-Black sheep.  However, the substitution of G to A nucleotide was determined in the GDF9 locus. Digestion of the 462bp PCR product from exon 1 of theGDF9 using the HhaI restriction enzyme produced fragments of 52, 156, and 254bp. However, DNA fragments containing the A nucleotide yielded only two fragments (52 and 410bp). The heterozygous animals for this mutation in GDF9 locus had fragments of all four sizes (52, 156, 254, and 410bp). The frequency (0.75) of the wild type allele (+) in GDF9 locus was higher than the frequency (0.25) of mutant allele (G). The observed frequencies for the GG, G+ and ++ genotypes were 0.05, 0.40 and 0.55, respectively.  The association results indicated that the mutation of GDF9 gene has a substantial impact on lambing rate and the Iran-Black ewes with the GG and G+ genotypes had higher lambing rate than those with the ++ genotype. Thus, a gene assisted selection program to improve lambing rate in this breed can be designed based on the GDF9 gene mutation. Key words: BMP15, GDF9, Litter Size, Iran-Black Sheep

Cyclooxygenase 2 messenger RNA levels in canine follicular cells: interrelationship with GDF-9, BMP-15, and progesterone

Cyclooxygenase 2 (COX-2) encoded by the Cox-2 gene within the periovulatory follicles is a critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been characterized in canines. This study aimed to examine the possible relationships among BMP-15, GDF-9, progesterone, and Cox-2 gene expression in granulosa-cumulus cells in dogs. Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult bitches at estrus (n = 15) and proestrus (n = 13) after ovariohysterectomy. Total RNA extraction was performed in follicular cells, and Cox-2 gene expression was assessed by quantitative PCR analysis. Progesterone, BMP-15, and GDF-9 were determined in the FF samples using ELISA assays. Cumulus-oocyte complexes were subjected to in vitro maturation (IVM) with or without (control) recombinant GDF-9 and BMP-15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via quantitative PCR. Data were evaluated by ANOVA. An increase (P < 0.05) in Cox-2 messenger RNA levels was observed in follicular cells from follicles at estrus with respect to those at proestrus. However, the levels of BMP-15 and GDF-9 in FF decreased (P < 0.05), whereas progesterone increased (P < 0.05) from the proestrus phase to the estrus phase. The expression of Cox-2 gene in cumulus cells was 4-fold greater (P < 0.01) than that in the control when both growth factors were added to the IVM culture. In conclusion, although BMP-15 together with GDF-9 appears to upregulate the levels of Cox-2 transcripts during IVM, the inverse relationship of these paracrine factors with Cox-2 gene expression and the positive correlation of progesterone with Cox-2 transcripts suggest that the high progesterone levels could be more relevant in the local mechanisms regulating the Cox-2 gene expression.

Association between polymorphism in BMP15 and GDF9 genes and impairing female fecundity in diabetes type 2

BackgroundA shortened reproductive period and earlier menopause have been associated with type 2 diabetes. Growth differentiation factor 9(GDF9) and bone morphogenetic protein 15 (BMP15) gene mutations have been associated with earlier menopause. Therefore, this study aimed to evaluate the association between BMP15 and GDF9 mutations with impairing female fecundity in diabetic patients. The study subjects comprised 90 female diabetic patients and 60 female healthy controls. The physio-biochemical analysis was measured using enzymatic determination. A single-strand conformation polymorphism (SSCP) protocol was utilized to assess the pattern of genetic variations.ResultsGenotyping analysis of the BMP15 gene showed a heterogeneous pattern with the presence of two genotypes: AA and AC genotypes. Five novel missense single nucleotide polymorphisms (SNPs) were identified in the BMP15 gene: four SNPs detected in both genotypes, and Met4Leu, a specific SNP, was detected only in the AC genotype. Cumulative in silico tools indicated a highly deleterious effect for the Met4Leu on the mutant protein structure, function, and stability. Diabetes patients showed a significantly higher frequency of genotype AC. The physio-biochemical analysis of fasting plasma glucose (FBG), glycosylated hemoglobin (HbA1c), and luteinizing hormone (LH) were significantly higher (P < 0.05) in AC genotype than AA genotype.ConclusionsThe current research provides the first indication regarding the tight association of BMP15 polymorphism with the impairing female fecundity in the diabetic. A pivotal role is played by the novel (Met4Leu) SNP that can be used as a predictor for the impairing female fecundity of diabetes, while no polymorphism was found in exon 4 of the GDF9 gene.

GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process.

Simple SummaryThe knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte–cumulus cells’ regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation.The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.