Expression Of The Bcl2-Associated Of Cell Death Genes In The Sheep Reproductive Organs And The Functional Analysis Of Bad Gene During The Reproductive Cycle

Most ewes in China are seasonally polyestrous with normal ovulatory cycles,which is controlled by photoperiod(length of the daily light phase).These ewes are estrous in the short-day season and anestrus in the long-day season and cannot mate during anestrus.Thus seasonal breeding limits both diversification and intensification of production.If sheep can estrus all round year,it can be mated twice per year,which can greatly improve the economic benefits.To change seasonal estrus at the genetic level and cultivating new sheep breeds,it is important to understand the molecular mechanisms of seasonal breeding trait in sheep.Year-round estrus is an excellent reproductive characteristic in sheep(Ovis aries),which is controlled by cell apoptosis.Apoptosis is mainly regulated by the apoptosis-related genes(Bad,Bax,Bcl-2 and Bcl-xL)of the Bcl-2 family.In order to identify the differential expression levels of Bad,Bax,Bcl-2 and Bcl-xL in hypothalamus-pituitary-gonadal axis tissues of Small Tail Han sheep,and to study the association of these genes with year-round estrus in sheep,year-round estrous Small Tail Han ewes were selected as tested subjects,and real-time PCR method was used to detect Bad,Bax,Bcl-2 and Bcl-xL mRNA expression in hypothalamus-pituitary-gonadal axis tissues during luteal and follicular periods,to analyze the relationship between the cell apoptosis of the reproductive organs and the reproductive state of Small Tail Han sheep,which lays a foundation for revealing the molecular mechanism of the perennial estrus in Small Tail Han sheep.In order to investigate the specific mechanism of Bad gene regulates oestrus in sheep,combined with the results of RNA-Seq in our previous research,we characterized the ovine Bad gene,including its complete cDNA sequence,predicted protein sequence and expression profiling in Small-tailed Han(STH)sheep.Furthermore,functional analysis of the Bad gene was performed in primary sheep GCs using RNA interference(RNAi)and lentivirus-mediated overexpression techniques.The expression of Bad,Bax and Bcl-2 genes in cells was detected,as well as the secretion of hormones by granulosa cells.Apoptotic characteristics of granulosa cells after gene overexpression were detected.The results of this study are as follow:The expression levels of Bad and Bcl-2 genes in pituitary and ovary tissues in the luteal phase were higher than those in the follicular phase(P<0.05).The expression levels of Bad and Bcl-2 genes in hypothalamus had no significant differences between the luteal phase and the follicular phase(P>0.05).The expression levels of Bax and Bcl-xL genes in hypothalamus,pituitary and ovary tissues had no significant differences between the luteal phase and the follicular phase(P>0.05).The concentration of progesterone in the luteal phase was significantly higher than that in the follicular phase(P<0.05),while the estradiol concentration had no significant difference between the two stages(P>0.05).The results indicated that the expression levels of Bad and Bcl-2 genes had great differences during the luteal and follicular periods of Small Tail Han sheep,which may be regulated the stage transition from luteal to follicular periods,and the two genes were associated with estrus onset in Small Tail Han sheep.Bad and Bcl-2 genes showed great expression differences during the luteal and follicular periods in ovary of Small Tail Han sheep(Ovis aries),and may be the main regulatory genes of follicular development and atresia during estrous cycle in Small Tail Han sheep(Ovis aries);Bcl-xL gene plays an important regulatory role in cell apoptosis in uterus.The full-length cDNA sequence,predicted protein sequence and expression of Bad gene in Small-tailed Han sheep were identified.It was found that the expression level of Bad gene was highest in hypothalamus,lung and pituitary of sheep,while lowest in liver.In functional validation of Bad in sheep ovarian granulosa cells,it was found that the concentration of progesterone increased significantly after RNAi interference of Bad,while the change of progesterone concentration after overexpression of Bad was contrary.It has been found that Bad can reduce progesterone levels by promoting ovarian GC apoptosis,which might be involved in regulating the estrus cycle in sheep.