Polymorphism Of BMP15 In Sheep And Effects Of Short-Term Feed Restriction During The Luteal Phase On Expression Of BMP15 Pathway Genes

Bone morphogenetic protein 15(BMP15), which maps to the X chromosome, is a member of the TGFP superfamily. As an oocyte-derived growth factor, BMP15 regulates the proliferation and differentiation of granulosa cells via paracrine signaling pathways so as to carefully control its own microenvironment, which plays a central role in the regulation of early folliculogenesis. This research detected the genetic polymorphism of the exonl and exon2 of BMP15 gene in Hu sheep and Chinese Merino up to 348 individuals by PCR-SSCP. Moreover, the relationship between polymorphic sites and sheep litter size was also analyzed in order to study the gene sites controlling sheep reproduction, which would provide theoretical basis for selection and preservation of Hu sheep. Furthermore, the amino acid composition and the secondary structure of BMP 15 protein were predicted by bioinformatics softwares assay. In addition, short-term feed withdrawal suppressed ovarian follicle development. This study tested whether the effects of a short-term feed restriction given to ewes during the luteal phase on the relative amounts of BMP15/Smad pathway genes. Results are as follows:1. Detection of single nucleotide polymorphism of BMP15 gene in Hu SheepTwo fragments of Exonl and Exon2 of BMP15 gene were amplified from the genomes of three high prolificacy Hu Sheep and three low prolificacy Hu Sheep by PCR. The 36 positive clones were selected and sequenced. Seven mutations were found in two fragments of Exonl and Exon2 by Sequence alignment. Four pairs of primers were designed in mutation districts to identify the authenticity of mutation by PCR-SSCP. The result showed that the amplified fragment of primer 3 existed Exonl 31∧CTT, which had been reported abroad, and the primer 5 existed Exon2 T430C that were first discovered in the sheep.2. Bioinformatics analysis of the sheep BMP15 geneThe amino acid composition, analysis of signal peptides, subcellular location and the secondary structure of BMP 15 protein were also predicted by bioinformatics softwares assay. And the difference of protein secondary structure of the wild type and mutant were compared. The deletion of leucine in Exonl and the substitution of leucine to proline in Exon2 slightly changed the proportion of the secondary structure elements.3. Genetic Polymorphism of BMP15 Gene and Its Relationship with Litter Size Traits in Hu SheepSingle nucleotide polymorphism of BMP 15 gene was detected in Hu sheep and Chinese Merino by PCR-SSCP based on the two mutations. Relationship between the polymorphism of BMP 15 gene and Hu sheep's prolificacy was analyzed. The results indicated that there were two genotypes (AA and AB) detected by primer 3(Exonl 31∧CTT). Allele A was preponderant allele. The ewes with genotype AA had 0.12 lambs more than those with genotype AB in Hu sheep (P>0.05); There were three genotypes (CC, CD and DD) detected by primer 5 (Exon2 T430C) in two sheep breeds. Allele C was preponderant allele, and the difference of gene frequency between Hu sheep and Chinese Merino was very significant (P<0.01). The ewes with three genotypes had no difference in litter size (P>0.05). Haplotype analysis showed that the mean litter size of AACC haplotype combination was 2.607, but was not significantly high (P>0.05). These results showed that the two mutations of BMP 15 gene did not affect the prolificacy of Hu sheep, but T430C mutation may related to fecundity of different sheep breeds.4. Effects of short-term feed restriction during the luteal phase on mRNA levels of BMP15 pathway genes in Hu SheepThis study tested whether the effects of a short-term feed restriction given to ewes during the luteal phase on the relative amounts of BMP15/Smad pathway genes. Ewes were randomly assigned to 2 groups:1.5M group(n=5) and 0.5M group(n=5). Nutritional treatments were started over days 6 to 12 of the estrous cycle after ewes were synchronized. Expression of BMP15/Smad pathway genes in ovaries was analyzed by fluorescence quantitative PCR. The expression of Smad1, Smad4, BMPRIB and BMPRII mRNA was up-regulated in ewes fed 0.5M compared with those fed 1.5M. No differences between 1.5M and 0.5M groups were observed for five genes (P>0.05), but the P value of BMPRII gene was 0.082. The results indicated that the mechanism by which short-term feed withdrawal suppressed follicle development may involve BMP15/Smad pathway.