Patterns of expression of messenger RNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development and characterization of ovarian follicular populations in ewes carrying the Woodlands FecX2W mutation

Woodlands sheep have a putative genetic mutation (FecX2(W)) that increases ovulation rate. At present, the identity of FecX2(W) is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2(W) could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2(W) may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2(W) ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2(W) ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2(W) ewes. FecX2(W) ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2(W) mutation.     

Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep

The aim of this study was to test the hypothesis that both growth differential factor 9 (GDF9) and bone morphogenetic protein (BMP15; also known as GDF9B) are essential for normal ovarian follicular development in mammals with a low ovulation rate phenotype. Sheep (9-10 per group) were immunized with keyhole limpet hemocyanin (KLH; control), a GDF9-specific peptide conjugated to KLH (GDF9 peptide), a BMP15-specific peptide conjugated to KLH (BMP15 peptide), or the mature region of oBMP15 conjugated to KLH (oBMP15 mature protein) for a period of 7 mo and the effects of these treatments on various ovarian parameters such as ovarian follicular development, ovulation rate, and plasma progesterone concentrations evaluated. Also in the present study, we examined, by immunohistochemistry, the cellular localizations of GDF9 and BMP15 proteins in the ovaries of lambs. Both GDF9 and BMP15 proteins were localized specifically within ovarian follicles to the oocyte, thereby establishing for the sheep that the oocyte is the only intraovarian source of these growth factors. Immunization with either GDF9 peptide or BMP15 peptide caused anovulation in 7 of 10 and 9 of 10 ewes, respectively, when assessed at ovarian collection. Most ewes (7 of 10) immunized with oBMP15 mature protein had a least one observable estrus during the experimental period, and ovulation rate at this estrus was higher in these ewes compared with those immunized with KLH alone. In both the KLH-GDF9 peptide- and KLH-BMP15 peptide-treated ewes, histological examination of the ovaries at recovery (i.e., approximately 7 mo after the primary immunization) showed that most animals had few, if any, normal follicles beyond the primary (i.e., type 2) stage of development. In addition, abnormalities such as enlarged oocytes surrounded by a single layer of flattened and/or cuboidal granulosa cells or oocyte-free nodules of granulosa cells were often observed, especially in the anovulatory ewes. Passive immunization of ewes, each given 100 ml of a pool of plasma from the GDF9 peptide- or BMP15 peptide-immunized ewes at 4 days before induction of luteal regression also disrupted ovarian function. The ewes given the plasma against the GDF9 peptide formed 1-2 corpora lutea but 3 of 5 animals did not display normal luteal phase patterns of progesterone concentrations. The effect of plasma against the BMP15 peptide was more dramatic, with 4 of 5 animals failing to ovulate and 3 of 5 ewes lacking surface-visible antral follicles at laparoscopy. By contrast, administration of plasma against KLH did not affect ovulation rate or luteal function in any animal. In conclusion, these findings support the hypothesis that, in mammals with a low ovulation rate phenotype, both oocyte-derived GDF9 and BMP15 proteins are essential for normal follicular development, including both the early and later stages of growth.     

Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes

Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3-4 days following mating. Subsequently, about one-half of the ewes in each group underwent an embryo transfer procedure 4-6 days following mating, with the embryos being transferred to synchronized, nonimmunized recipients. The remaining ewes were allowed to carry their pregnancies to term. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals.     

A 17 bp deletion in the Bone Morphogenetic Protein 15 (BMP15) gene is associated to increased prolificacy in the Rasa Aragonesa sheep breed

Different mutations in the Bone Morphogenetic Protein 15 (BMP15) and the Growth Differentiation Factor 9 (GDF9) genes cause increased ovulation rate and infertility in a dosage-sensitive manner in sheep. They cause increased ovulation rate and twin and triplet births in heterozygotes, and complete primary ovarian failure in homozygotes resulting in total infertility. We are here presenting a novel mutation in the second exon of the ovine BMP15 gene, found in the Spanish breed Rasa Aragonesa. It consists of a 17 bp deletion resulting in displacement of the open reading frame and premature stop codons. As a consequence, nearly 85% of the sequence of the wild type aminoacidic chain in the second exon of the BMP15 pro-protein is modified or suppressed as only the first 45 amino acids are conserved of the 245 original. The mature peptide is lost. The ewes heterozygous for this deletion present very high prolificacy (2.66 lambs/birth) when compared to a mean flock prolificacy of 1.36 lambs. The deletion causes a complete lack of functionality of the second exon of BMP15, comparable to the effect of premature stop codons in other mutations. Therefore, homozygous females for the deletion are expected to present primary ovarian failure. DNA sequence analysis of the GDF9 coding regions detected only a synonymous Single Nucleotide Polymorphism (SNP), apparently not linked to changes in prolificacy.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown 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 crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

Prolificacy genotypes at BMPR 1B,BMP15 and GDF9 genes in North African sheep breeds

The aim of this research was to investigate the genetic structure at BMPR 1B, BMP15 and GDF9 prolificacy genes in five sheep breeds reared in Tunisia: Barbarine, Queue Fine de L’Ouest, Noire de Thibar, Sicilo-Sarde and D’man. Genomic DNA of 204 sheep was investigated for the FecB^B (BMPR 1B), FecX^R, FecX^H, FecX^I, FecX^L, FecX^G, FecX^B (BMP15) and FecG^H (GDF9) mutations. The sequence variability of the different DNA fragments utilised for genotyping was further investigated by Single Stranded Conformation Polymorphism (SSCP) and sequencing. All the above-mentioned mutations were absent in the five sheep breeds examined. SSCP analysis and sequencing allowed the detection of two nucleotide variations. A non-functional mutation (T/C transition at nt 747 of BMP15 cDNA known as B3) was found at the BMP15 gene, in the Noire de Thibar breed; this mutation was first detected in the Belclare sheep. A new nucleotide change G/A at nt 1159 of BMP15 cDNA, causing the amino acid change A119T in the mature peptide, was detected in the Barbarine breed for the first time. The highly prolific D’man ewes were monomorphic for the absence of all the known prolificacy alleles.     

贵州白山羊Bmp15和Gdf9基因多态性及其编码蛋白的进化分析(英文)

BMP15和GDF9是转化生长因子β（TGFβ）超家族的成员,对绵羊的繁殖性状有直接的调节作用,从中发现的多个高产突变位点直接提高了排卵数和产羔数。在之前的研究中,作者从贵州白山羊中找到了一个高产突变位点。为了进一步揭示Bmp15和Gdf9基因突变与繁殖性状之间的关系,对贵州白山羊Bmp15和Gdf9基因编码区进行了克隆,以人BMP7的晶体结构为模板构建了贵州白山羊BMP15和GDF9成熟肽的三维模型。贵州白山羊Bmp15和Gdf9基因分别编码394和453个氨基酸的蛋白前体。对BMP15和GDF9成熟肽序列进行分析发现,除了之前确认的BMP15中的FecX^B 突变（S99I）和GDF9中的V79I突变之外,还从贵州白山羊的BMP15和GDF9成熟肽分别发现7个和3个位点突变。其中,BMP15成熟肽的S32G、N66H、S99I/P99I和G107R突变可能影响二聚体与受体的结合;GDF9成熟肽的P78Q和V79I影响二聚体与I型受体的亲和力,将值得进一步深入研究。对Bmp15和Gdf9基因编码的蛋白前体序列进行聚类分析,结果显示在鱼类到哺乳类的进化过程中,BMP15出现长度逐渐增加的现象,以BMP15成熟肽N端长度增加为主。这种演变可能使BMP15对低排卵哺乳动物繁殖力的控制更为灵敏。该文的研究结果为贵州白山羊Bmp15和Gdf9基因变异与繁殖力的关系提出了合理的解释,并支持这两个因子是贵州白山羊高产性状重要调节因子的观点。     

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep

Brazilian Santa Inês (SI) sheep are very well‐adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG^E (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG^E allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.     

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.     

Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells

The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23-31, which is syntenic to human chromosome 4q21-25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-beta (TGF-beta) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-beta pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.     

The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate

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

绵羊GDF9和BMP15基因多态性检测

以绵羊GDF9基因FecG^H突变和BMP15基因FecX^B和FecX^G突变为候选基因,采用PCR—RFLP方法研究其在湖羊、夏洛来、陶赛特、萨福克、中国美利奴肉用多胎品系、中国美利奴羊和罗米丽羊7个品种中的多态性．结果发现,湖羊群体中存在GDF和BMP15基因的FecG^H和FecX^B突变,但发生率极低,分别为0．645％(2／310)和0．968％(3／310)：而其它品种中则没有发现GDF9和BMP15基因的相应突变．这一发现对于建立绵羊基因标记辅助选择方法具有重要意义．     

A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep

Bone morphogenetic protein 15 (BMP15) is a member of the transforming growth factor beta superfamily, is specifically expressed in oocytes and is essential for sheep prolificacy. Reported mutations in this gene cause increased ovulation rate and infertility in a dosage-sensitive manner. In this work, a new naturally occurring mutation in the BMP15 gene from the ovine Rasa Aragonesa breed is described. This mutation is a deletion of 17 bp that leads to an altered amino acid sequence and introduces a premature stop codon in the protein. Highly significant associations (P < 0.0001) were found between the estimated breeding value for prolificacy and the genotype of BMP15 in Rasa Aragonesa animals with high and low breeding values for this trait. As for other mutations in BMP15, this new mutation is associated with increased prolificacy and sterility in heterozygous and homozygous ewes respectively.     

GDF9 as a candidate gene for prolificacy of Small Tail Han sheep

Growth differentiation factor 9 (GDF9) which controls the fecundity of Belclare, Cambridge, Santa Ines, Moghani, Ghezel and Thoka ewes was studied as a candidate gene for the prolificacy of Small Tail Han sheep. According to the sequence of ovine GDF9 gene, six pairs of primers were designed to detect single nucleotide polymorphisms of two exons of GDF9 gene in both high fecundity breed (Small Tail Han sheep) and low fecundity breed (Dorset sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Only the products amplified by primers 2-1 and 2-2 displayed polymorphisms. For primer 2-1, three genotypes (AA, AB and BB) were detected in both sheep breeds. Sequencing revealed one silent mutation (G477A) in exon 2 of GDF9 gene in the BB genotype in comparison with the AA, which was known as G3 mutation of GDF9 gene in Belclare and Cambridge ewes. The relationship of least squares means for litter size was AA?>?AB?>?BB in Small Tail Han sheep (P?>?0.05). For primer 2-2, two genotypes (CC and CD) were detected in both sheep breeds. Sequencing revealed one novel single nucleotide mutation (G729T) in exon 2 of GDF9 gene in the CD genotype in comparison with the CC, which resulted in an amino acid change (Gln243His). The ewes with mutation heterozygous genotype CD had 0.77 (P?相似文献   

Nucleotide sequencing and DNA polymorphism studies of BMP 15 gene in Corriedale and local Kashmir valley sheep (Ovis aries)

The families of TGF-β proteins are the most important growth factors in the ovary for growth and differentiation of early ovarian follicles. Three related oocyte-derived members of the transforming growth factor-β superfamily, namely GDF9, BMP15 and BMPR-IB have been shown to be essential for follicular growth and ovulation. The objective of the present study was to detect the incidence of mutation in intronic portion of BMP 15 gene in Corriedale and local Kashmir valley sheep breeds. Blood samples were collected from 85 ewes and genomic DNA was extracted using the modified phenol chloroform method. The quantity and quality of extracted DNA was examined using spectrophotometry and gel electrophoresis, respectively. A fragment with the size of 356 bp was amplified using polymerase chain reaction (PCR) with a pair of specific primers. The amplified PCR products were digested with Mph11031 restriction enzyme. In the presence of mutation at this locus, the Mph11031 enzyme cannot recognize the restriction site. However, here in the absence of mutations, the enzyme recognizes one restriction site and divides the amplified fragment into two fragments of 152 and 204 bp. The 356 bp fragment was also analyzed for polymorphism by SSCP technique. The results indicated two different banding patterns AA and AB for this fragment. Later on two different allelic forms A and B were confirmed by nucleotide sequencing. The 356 bp nucleotide sequence was subjected to alignment analysis and it was observed that sequence similarity of this fragment with that of other sheep and Jining grey goat was more than 97.8%. Phylogenetic analysis revealed that both designated A and B alleles as well as published sequence of sheep form a common cluster indicating their evolutionary closeness. The origin of Jining grey goat was located some distance away from the sheep. The overall frequencies of AA and AB genotypes were 0.79 and 0.21. The breed wise frequencies were 0.78 and 0.22 in Corriedale sheep and the frequencies in Kashmir valley sheep were 0.80 and 0.20 for AA and AB genotypes, respectively. The overall allelic frequencies of A and B alleles were 0.89 and 0.11 whereas allelic frequencies Corriedale sheep was 0.89 and 0.11 and that of Kashmir valley sheep were 0.90 and 0.10.     

Screening of indigenous goats for prolificacy associated DNA markers of sheep

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.     

Genotyping of Novel SNPs in BMPR1B,BMP15, and GDF9 Genes for Association with Prolificacy in Seven Indian Goat Breeds

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.     

Maximum‐likelihood approaches reveal signatures of positive selection in BMP15 and GDF9 genes modulating ovarian function in mammalian female fertility

Bone morphogenetic proteins (BMPs) and the growth factors (GDFs) play an important role in ovarian folliculogenesis and essential regulator of processes of numerous granulosa cells. BMP15 gene variations linked to various ovarian phenotypic consequences subject to the species, from infertility to improved prolificacy in sheep, primary ovarian insufficiency in women or associated with minor subfertility in mouse. To study the evolving role of BMP15 and GDF9, a phylogenetic analysis was performed. To find out the candidate gene associated with prolificacy in mammals, the nucleotide sequence of BMP15 and GDF9 genes was recognized under positive selection in various mammalian species. Maximum‐likelihood approaches used on BMP15 and GDF9 genes exhibited a robust divergence and a prompted evolution as compared to other TGFβ family members. Furthermore, among 32 mammalian species, we identified positive selection signals in the hominidae clade resulting to 132D, 147E, 163Y, 191W, and 236P codon sites of BMP15 and 162F, 188K, 206R, 240A, 244L, 246H, 248S, 251D, 253L, 254F and other codon sites of GDF9. The positively selected amino acid sites such as Alanine, Lucien, Arginine, and lysine are important for signaling. In conclusion, this study evidences that GDF9 and BMP15 genes have rapid evolution than other TGFß family members and was subjected to positive selection in the mammalian clade. Selected sites under the positive selection are of remarkable significance for the particular functioning of the protein and consequently for female fertility.     

Genome‐wide association study to identify genomic regions affecting prolificacy in Lori‐Bakhtiari sheep

Several causative mutations in candidate genes affecting prolificacy have been detected in various sheep breeds. A genome‐wide association study was performed on estimated breeding values for litter size in Lori‐Bakhtiari sheep. Prolific ewes with twinning records and others with only singleton records were genotyped using the medium‐density Illumina Ovine SNP50 array. Four single nucleotide polymorphisms (SNPs) associated with litter size were identified on chromosomes 3, 6 and 22. The region on sheep chromosome 3 between 75 739 167 and 75 745 152 bp included two significant SNPs (s52383.1 and OAR3_80038014_X.1) in high linkage disequilibrium with each other. The region that surrounds these SNPs contains a novel putative candidate gene: luteinizing hormone/choriogonadotropin receptor (LHCGR), known to be involved in ovarian steroidogenesis and organism‐specific biosystem pathways in sheep. Known prolificacy genes BMPR1B, BMP15 and GDF9 were not associated with litter size in Lori‐Bakhtiari sheep, suggesting that other biological mechanisms could be responsible for the trait's variation in this breed.