Expression Of GnRH And E2 And Receptors At The Onset Of Puberty In Duolang Sheep

In mammals,the puberty is the critical period for first estrus,ovulation and the acquisition of fertility.Accelerating the time of female to puberty can improve the utilization rate of female and accelerate the breeding process.Prepuberty estrogen can inhibit puberty initiation through negative feedback.Estrogen can play a positive feedback role to promote the synthesis and release of gonadotropin to complete the initiation of puberty.In this study,the CDS sequences of GnRH,GnRHR,ERα and ERβ genes in Duolang sheep were obtained by clone sequencing.Real-time quantitative PCR(q PCR)and granulosa cells culture were performed.The expression of each gene in the hypothalamic-pituitary-gonadal axis related tissues at different pubertal stages were detected,and the role of exogenous GnRH and estrogen in sheep ovarian granulosa cells was analyzed,providing theoretical supplement for studying the role of GnRH and estrogen in sheep puberty initiation process.In this study,CDS of GnRH,GnRHR,ERα and ERβ genes were obtained by clone sequencing.Sequence alignment and homology analysis showed that GnRH,GnRHR,ERα and ERβ genes CDS of Duolang sheep had the highest homology with Goat.The homology of ERα and ERβ protein was 43.29%,and the homology of C region and E region was 68.37% and 56.35%,respectively.Bioinformatics analysis showed that GnRHR protein was a typical G protein-coupled receptor with seven transmembrane structures and was hydrophobic.ERα and ERβ proteins were hydrophilic proteins,which mainly exist in cells and had no transmembrane structure.The qPCR showed that ERα m RNA of hypothalamus and pituitary decreased significantly at the beginning of estrus,and increased significantly after estrus(P < 0.05).The m RNA of ERβ in ovary was significantly increased in puberty and still significantly increased in post-puberty(P < 0.05).Western blot showed that the expression trend of ERα protein in hypothalamus and pituitary was consistent with its m RNA.The expression of GnRH m RNA was the highest in the hypothalamus of Duolang sheep in puberty,which was significantly higher than that in prepuberty and post-puberty(P < 0.05).GnRHR m RNA widely existed in HPG axis related tissues.GnRHR m RNA in pituitary was significantly increased in puberty,which was significantly higher than the two periods in prepuberty and post-puberty(P < 0.05),and had the same expression trend with GnRH m RNA in hypothalamus.GnRH and GnRHR m RNA in ovary were significantly increased in estrus,but significantly decreased after estrus(P < 0.05).The expressions of LH,LHR and FSHR in ovine ovarian granulosa cells were significantly inhibited at500 ng/m L by exogenous GnRH for 24 h(P < 0.05).GnRH could up-regulate FSH,FSHR and GnRHR at100 ng/m L,E2 and ERβ m RNA expression at 250 ng/m L.ERβ and GnRH m RNA in ovarian granulosa cells increased with the increase of exogenous estrogen concentration.In conclusion,the differential expressions of GnRH,GnRHR,ERα and ERβ genes in various tissues and the expression of related hormones and receptors can be regulated by exogenous GnRH and estrogen in ovarian granulosa cells during the initiation of puberty of Duolang sheep.These results suggested that GnRH and estrogen and their receptors played a key role in the initiation of puberty in sheep.