The Temporal-spatial Expression Of ADRA1B And PPARGC1B Genes In The Ovarian Follicles And Effect Of Their Novel SNPs On The Egg Production Traits In Hens

Gene ADRA1 B encodes alpha-1B adrenergic receptor that is a member of the G-protein-coupled family of transmembrane receptors. Not only has the ADRA1 B been shown to activate mitogenic response and regulate growth, but also it can regulate growth and proliferation of many cells. PPRAGC1 B is a regulating transcription coactivation, the protein encoded by this gene stimulates the activity of several transcription factors and nuclear receptors. It is also involved in fat oxidation,non-oxidative glucose metabolism, and the regulation of energy expenditure. Previous studies have demonstrated adrenergic, alpha-1B-, receptor(ADRA1B) and Peroxisome proliferator-activated receptor gamma, coactivator 1 beta(PPARGC1B) genes are involved in the regulation of ovarian follicular development, selection and maturation in mammal. However, little is known regarding regulation of hen ovarian development in chicken ADRA1 B and PPARGC1 B genes. The temporal-spatial expression of ADRA1 B and PPARGC1 B genes in the ovarian follicles were investigated using the methods of semi-quantitative RT-PCR and immunohistochemistry. To screen potential molecular makers associated with chicken egg production trait, ADRA1 B and PPARGC1 B fragments were examined for novel sequence polymorphism using a PCR-SSCP approach and screening SNPs, with the association with egg production traits in chicken. It provides the basis for breeder in the breeding objective of molecular maker assisted selection.The first part, the expression of ADRA1 B and PPARGC1 B were examined in undifferentiated prehierarchichal follicles(1-8 mm in diameter) and preovulatory(post-selection) follicles(F6-F1) in 150-day-old Hy-Line Brown hens by RT-PCR.The results showed that ADRA1 B gene is expressed in all of the examined follicles at the various development stages, and with a relatively low level expression. The expression level in F2 and compared with 1-4mm、6-7mm、7-8mm、F4 and F5 was difference significantly(P ﹤ 0.05), the expression in stroma was difference significantly in 6-7mm and F4, other follicles were not significant( P > 0.05).PPARGC1 B gene is expressed in all of the examined follicles at the various development stages. While, the expression of PPARGC1 B mRNA were significant differences(P﹤0.05), and with a progressive increasing and then decreasing both atprehierarchichal follicles(1-8 mm in diameter) and preovulatory follicles(F6-F1).The expression level in stroma and compared with F4 and F3 was difference significantly(P﹤0.05), other follicles were not significant(P>0.05).The second part, this study localized ADRA1 B and PPARGC1 B proteins in prehierarchichal follicles of the adult hen ovary by immunohistochemistry in150-day-old Hy-Line Brown hens. It was shown that ADRA1 B proteins were predominantly expressed in oocytes, stroma and granulosa cells in the prehierarchichal follicles at different developing stages, and it predispose to expression in granulosa cells with strong immunostaining in the medium and large follicles than small in the prehierarchichal follicles. The PPARGC1 B protein was found to be predominantly expressed in oocytes and stroma cells from the primary follicles and prehierarchichal follicles examined.The third part,ADRA1 B and PPARGC1 B fragments were examined for novel sequence polymorphism using a PCR-SSCP approach and sequencing analysis, with the association with egg production traits in the Dagu chicken. Among them, an A/G transition at base position 1915 in exon 2 of ADRA1 B gene was found to be polymorphic. The SNP A1915G(ADRA1B) leads to a nonsynonymous substitution(aspartic acid 489-to-glycine) and then the 360 birds from the Dagu population were divided into genotypes AA and AG, genotypes AA was found to be present at a higher frequency. Furthermore, the AG genotype correlated with significantly higher hen-housed egg production(HHEP) at 30, 43, 57 and 66 weeks(wks) of age and with a higher egg weight(EW) at 30 and 43 wks(P﹤0.05). T/C mutation at base position6146 in the 3’- untranslated region(UTR) of PPARGC1 B gene were found to be polymorphic. For the SNP T6146C(PPARGC1B), the hens were typed into TT and TC genotypes, with the T allele shown to be dominant. The TC genotype was also markedly correlated with higher HHEP at 57 and 66 wks of age and EW at 30 and 43wks(P ﹤0.05). Collectively, the results of the present study strongly suggest that the two novel ADRA1 B and PPARGC1 B polymorphisms can be associated with egg production and egg weight, thus acting as potential molecular markers for egg productivity in Chinese Dagu chicken breeding.