Growth Differentiation Factor 9 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.

DNA methylation of GDF-9 and GHR genes as epigenetic regulator of milk production in Egyptian Zaraibi goat

BackgroundDNA methylation is an epigenetic mechanism that takes place at gene promoters and a potent epigenetic marker to regulate gene expression.ObjectiveThe study aimed to improve the milk production of Zaraibi goats by addressing the methylation pattern of two milk production-related genes: the growth hormone receptor (GHR) and the growth differentiation factor-9 (GDF-9).Methods54 and 46 samples of low and high milk yield groups, respectively, were collected. Detection of methylation was assessed in two CpG islands in the GDF-9 promoter via methylation-specific primer assay (MSP) and in one CpG island across the GHR promoter using combined bisulfite restriction analysis (COBRA).ResultsA positive correlation between the methylation pattern of GDF-9 and GHR and their expression levels was reported. Breeding season was significantly effective on both peak milk yield (PMY) and total milk yield (TMY), where March reported a higher significant difference in PMY than November. Whereas single birth was highly significant on TMY than multiple births. The 3rd and 4th parities reported the highest significant difference in PMY, while the 4th parity was the most effective one on TMY.ConclusionThese results may help improve the farm animals' milk productive efficiency and develop prospective epigenetic markers to improve milk yield by epigenetic marker-assisted selection (eMAS) in goat breeding programs.

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.

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.

The relationship between egg production, reproductive hormones, and the GDF9 gene in three different chicken strains

Egg production is the most important commercial attribute of layer hens since it has a direct impact on the efficiency of the poultry industry. This research aimed to study the age at first egg (AFE), egg number (EN), egg quality, and some plasma hormones within the period from the start of laying eggs until day 90 of production in three strains of laying hens (Lohman Brown (LB), Golden Sabahia (GS)) and White Leghorn (WL) as the reference breed for egg production. In addition, a time series expression profile of growth differentiation factor 9 (GDF9) gene was investigated to get a better understanding of the genetic basis of egg production. The LB laying hens recorded the highest improved estimates of egg production and egg quality traits. Further, GDF9 was highly expressed in LB tissues (magnum and isthmus) compared to WL except in the uterus. These results suggest that there is a direct relationship between number of eggs produced, quality of eggs and the expression level of the GDF9 gene in the LB strains. Besides, the LB strain is superior with candidate productive traits, therefore they can be used as proven ancestors to improve laying hens.

Introduction of the FecGF mutation in GDF9 gene via CRISPR/Cas9 system with single-stranded oligodeoxynucleotide

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecGF mutation (g. G1111A, p. V371 M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecGF mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 μM, 1 μM, and 2 μM) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 μM Scr7 were significantly lower than that for 0 μM Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 μM Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.

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.

Candidate genes for litter size in Xinjiang sheep identified by Specific Locus Amplified Fragment (SLAF) sequencing

The aim of this study was to investigate the selection signatures at a genome-wide level in ‘Pishan’ sheep using Specific Locus Amplified Fragment (SLAF)-seq. Blood samples from 126 ewes were sequenced using SLAF tags, and the ovarian tissues from 8 ewes (Bashbay sheep, a single litter size group (SG group); ‘Pishan’ sheep, double litter size group (DG group)) were collected to detect expression levels by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Selection signature analysis was performed using global fixation index (Fst) and nucleotide diversity (π) ratio. A total of 1,192,168 high-quality SLAFs were identified. Notably, 2380 candidate regions under selection using two approaches were identified. A total of 2069 genes were identified, which were involved in dopaminergic synapses, thyroid hormone synthesis, ovarian steroidogenesis and thyroid hormone signalling pathways. Furthermore, Growth Differentiation Factor 9 (GDF9), Period Circadian Regulator 2 (PER2), Thyroid Stimulating Hormone Receptor (TSHR), and Nuclear Receptor Coactivator 1 (NCOA1) reside within these regions and pathways. The expression levels of GDF9 and PER2 genes in sheep tissue of the DG group were significantly higher than those in the SG group. These genes are interesting candidates for litter size and provide a starting point for further identification of conservation strategies for ‘Pishan’ sheep.

Expression of caspase-3, caspase-9, GDF-9 and IGF-1 genes in ovine oocytes cultured with optimum and elevated doses of Amphiregulin, Neuregulin-1 and Tumour necrosis factor-α in-vitro

The present study was conducted to evaluate the expression of caspase-3, caspase-9, growth differentiation factor-9 (GDF-9) and insulin growth factor-1 (IGF-1) genes in oocytes cultured in vitro with optimum and elevated doses of amphiregulin (50 ng and 150 ng), neuregulin-1 (25 ng and 150 ng) and tumor necrosis factor-α (25 ng and 150 ng) during in vitro maturation of oocytes based on the results of effects of AREG or NRG-1 or TNF-α concentration on maturation of oocytes which caused significant effect, were selected for gene expression studies along with the basal/control level. Total RNA was extracted from in vitro matured oocytes using Trizol method and Real-time reverse transcription polymerase chain reaction was used to evaluate the expression of genes. There was an upregulation of caspase-3 at 150 ng of TNF-α, caspase-9 at 50 ng of AREG, GDF-9 at 150 ng of AREG and IGF-1 at 150 ng of AREG and 25 ng of TNF-α. These results suggested that AREG at elevated dose and TNF-α at optimum dose enhanced the expression of GDF-9 and IGF-1, while the presence of elevated dose of TNF-α and optimum dose of AREG activated caspase-3 and caspase-9, respectively in oocytes cultured in vitro.

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.

Akkaraman Koyun Irkında Kalpastatin (CAST) ve Büyüme Farklılaşma Faktörü 9 (GDF9) Genlerin Polimorfizmi

Objective: In this study, the gene polymorphisms of Calpastatin (CAST) and Growth Differentiation factor 9 (GDF9) were determined in Akkaraman sheep breed.Material and Methods: Genomic DNA was obtained from blood samples of 50 Akkaraman sheep. All samples for CAST and GDF9 were genotyped by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method using the enzymes MspI and Hhal.Results: Three (MM, MN, and NN) and two (GG and GA) genotypes, respectively, were detected at the CAST and GDF9 genes. The frequencies of the genotypes MM, MN and NN were determined to be 44 %, 54 % and 2 %, respectively. The frequencies of the GG and GA genotypes were found to be 84 % and 16 %, respectively. The AA genotype was not found.Conclusion: The study showed for the first time that the Akkaraman breed carries the GDF9 G1 mutation and has sufficient variation in the CAST. Previous studies provided that both genes can be used as biomarkers for increasing lambing yield and growth rates. Therefore, in order for the studied genes to be used as biomarkers in the Akkaraman breed, association studies should first be performed. 

Effect of vitrification on the expression of genes in porcine blastocysts derived from in vitro matured oocytes

This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6–7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.

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.

Regulation of GDF9 and CDKN1B expression in Tibetan sheep testes during different stages of maturity

Normal spermatogenesis is heavily dependent on the balance of germ cell proliferation, differentiation and apoptosis. Growth differentiation factor 9 (GDF9) and cyclin-dependent kinase inhibitor 1 B (CDKN1B) are strongly associated with cell cycle transition from G0/G1 to S and G2/M phase and hence regulating the growth and development of testicular germ cells and somatic cells. The current study was aimed at seeking out scientific evidence to determine if GDF9 and CDKN1B gene expression functions in the development of Tibetan sheep testes. To this end, developmental testes were derived from three-month-old (pre-puberty), one-year-old (sexual maturity), and three-year-old (adult) Tibetan sheep and then the expression and localization patterns of GDF9 and CDKN1B in these testes were evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunofluorescence. qRT-PCR and Western blot results showed that GDF9 and CDKN1B were detected in the testes throughout the different developmental stages. The abundance of GDF9 mRNA and protein in the testes of one- and three-year-old Tibetan sheep were higher than that in the testes of three-month-old Tibetan sheep; the mRNA and protein abundance of the CDKN1B gene in three-month-old Tibetan sheep testes were higher than that in the testes of the one-and three-year-old sheep. Moreover, immunofluorescence results suggested that the GDF9 protein was expressed in spermatogonia and Leydig cells, and that the CDKN1B protein was localized mainly in Leydig cells with some in the seminiferous epithelium throughout developmental stages. This indicated a novel role of the GDF9 and CDKN1B genes in Leydig cell development over and above their known roles in germ cell development. These findings have significant implications for our understanding of the molecular mechanisms of GDF9 and CDKN1B genes in Tibetan sheep spermatogenesis.

Expression of PTX3, GHR and GDF9 genes in cumulus cells and oocytes in relation to developmental competence of Bos indicus

The present study was conducted to evaluate the level of expression (mRNA) of specific developmental marker genes, viz. Growth Differentiation Factor 9 (GDF9), Growth Hormone Receptor (GHR) and Pentraxin 3 (PTX3) in bovine cumulus cells and oocytes in relation to in vitro developmental competence. Good quality Cumulus- Oocytes- Complexes (COCs) were selected for in vitro maturation and fertilization. The mean percentage of in vitro fertilization performance was 59.84 ± 4.28. The relative quantification values of GDF9 were 2.38, 98.13, 1 and 132.58; GHR were 4.31, 1.47, 1 and 43.58; PTX3 were 0.28, 0, 1 and 0.5 in immature cumulus cells, immature oocyte, mature cumulus cells and mature oocyte respectively when compared to the reference control. The expression of GDF9 gene was found to be up-regulated in oocytes compared to cumulus cells. The expression pattern of GDF9 was relatively more in in vitro matured oocytes compared to cumulus groups. The expression of GHR gene was up-regulated in oocytes. The relative abundance of PTX3 was slightly up-regulated in mature cumulus cells as compared to immature counterparts. Thus, GDF9 and GHR genes could be considered as embryonic developmental markers for bovine oocytes, while PTX3 gene for bovine cumulus cells.

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.

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.

G1 point mutation in growth differentiation factor 9 gene affects litter size in Sudanese desert sheep.

Background and Aim:Sudanese desert sheep encompass different sheep breeds named according to the different Sudanese tribes that rear them such as the Dubasi, Shugor, and Watish sheep. The objectives of this study were to screen for G1 point mutation in the polymorphic growth differentiation factor 9 (GDF9) gene, investigate its association with litter size, and construct the phylogeny of the different tribal breeds that belong to the Sudanese Desert sheep tribal types.Materials and Methods:Genomic DNA was extracted from whole blood of three tribal Desert sheep breeds (Dubasi, Watish, and Shugor) using the guanidine chloride method. Polymerase chain reaction-restriction fragment length polymorphism with HhaI restriction enzyme and sequencing techniques was used for genotyping the GDF9 locus for possible mutations associated with litter size in the three desert sheep tribal types.Results:G1 mutation in GDF9 caused the replacement of Arginine by Histidine at residue 87. The wild type allele (A) had the highest frequency, whereas the mutant type allele (a) had the lowest in all the sequenced subtypes. The genotype frequencies of the wild type ewes (AA) were higher than the heterozygous (Aa) and the mutant type (aa) frequencies in the three studied desert sheep types. No significant differences were found in the allele frequency between the three tribal types. Litter size was significantly influenced by the genotypes of GDF9 gene, parities, and subtypes (p≤0.01, 0.01, and 0.05, respectively). In the Watish sheep type, heterozygous sheep in their second parity recorded the highest litter size. Sequence alignment of GDF9 gene samples with the database entry indicated that all three tribal types were similar and identical to the reference sequence. The phylogenetic tree revealed that Shugor is the common ancestor of the studied types and Watish is more closely related to Shugor than Dubasi. This result mi ght partly explain the lower reproductive performance of Dubasi compared to Watish and Shugor.Conclusion:The presence of one copy of GDF9 gene increased litter size in the studied Sudanese Desert sheep. This locus may be used as a biomarker for litter size improvement through genotypic selection and allele or gene introgression.

Genetic variants of the BMP2 and GDF9 genes and their associations with reproductive performance traits in Barki ewes

• Two BMP2 variants and three GDF9 variants were identified in Barki ewes. • BMP2 variants were associated with TNLW and TWLW; however GDF9 variants were associated with twining rate, TNLB and TWLB. • Variation in ovine BMP2 and GDF9 affects reproductive performance traits of Barki ewes. The aim of this study was to investigate variation in the bone morphogenetic protein 2 ( BMP2 ) and growth differentiation factor 9 ( GDF9 ) genes, and ascertain whether it was associated with reproductive traits in Barki ewes. Records for conception rate of ewe, lambing number of ewe, lambing rate of ewe, twining rate of ewe, total number of lambs born per ewe (TNLB), total weight of lambs born per ewe (TWLB), total number of lambs weaned per ewe (TNLW), total weight of lambs weaned per ewe (TWLW) and ewe rearing ability were obtained for 296 Barki ewes. Polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) analyses were used to detect variation in exon 2 of BMP2 and exon 1 of GDF9 . The SSCP banding patterns for BMP2 revealed two variants ( A1 and A2 ), which contained one nucleotide sequence difference (c.962A > T). Three SSCP banding patterns ( C1 , C2 and C3 ) were observed for GDF9 and these contained two nucleotide-sequence differences (c.25C > T and c.260 G > A). Association analysis between the variation in BMP2 and GDF9 and reproductive performance traits revealed that BMP2 genotype was associated ( P < 0.05) with TNLW and TWLW, while GDF9 genotype was associated with twining rate of ewe ( P < 0.001), TNLB ( P < 0.001) and TWLB ( P < 0.05). This suggests that selection for the BMP2 and GDF9 genotypes might increase reproductive performance traits in Barki ewes.

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