Cloning Expression Profile Of Yak IFN-τ Gene And IFN-τ Effect On The Process Of Granulosa Cell Luteinization

Reproductive disorder is one of the important factors that hinder the reproductive development of yaks.Corpus luteum is an important temporary endocrine tissue in female animals.Abnormal function and structure of corpus luteum is one of the important causes for female infertility.IFN-τ is a pregnancy-related type I interferon found in ruminants.It is secreted by monocytes in embryonic trophoblast and can prolong the time of corpus luteum.Luteinized granulosa cells are similar to ovarian luteal cells in morphology and function.They can be used as a cell model of follicle to luteal development in the estrous cycle,and play a positive role in the study of luteinization of granulosa cells.In this experiment,the IFN-τ sequence of Maiwa Yak was cloned by reverse transcription PCR(RT-PCR),and the m RNA expression levels of IFN-τ in heart,liver,spleen,lung,kidney,stomach,small intestine,muscle tissue and ovary tissue in different estrous cycles(follicular phase,erythroid phase and luteal phase)of Maiwa Yak were detected by quantitative real-time PCR(q PCR),the cloned sequence was analyzed by different bioinformatics software.Yak luteinized granulosa cells were isolated and cultured in this study.On this basis,the effects of IFN-τ on the luteinization process and function of yak granulosa cells were studied by CCK-8,ELISA and q PCR.The results are as follows:(1)In this experiment,the IFN-τ of Maiwa Yak was successfully cloned.The IFN-τ sequence is 598 bp,the open reading frame is 564 bp,and encodes 187 amino acids.It has high homology with the IFN-τ sequence of large forehead cattle and yellow cattle.The largest proportion of IFN-τ protein secondary structure is α-Helix(50.8%).It contained 15 phosphorylation sites and was a negatively charged and unstable hydrophilic protein.The results of tissue expression profile showed that the m RNA expression levels of IFN-τ in small intestine and liver of Maiwa Yak was significantly higher than that in other tissues that were detected.The mRNA expression levels of IFN-τ in ovarian tissue in follicular phase was significantly higher than that in erythroid phase and luteal phase.(2)IFN-τ Promote the proliferation of luteinized granulosa cells.At 48 h,72h and 96 h,the cell proliferation ability of 0.1 ng/ml IFN-τ group was significantly higher than that of control group without IFN-τ.The cell proliferation ability of 1 ng/ml IFN-τ group was significantly up-regulated compared with that of control group at 48 h,72h and 96 h.The cell proliferation ability of 10 ng/ml IFN-τ group was significantly higher than that of control group at 12 h,24h,48 h,72h and 96 h.In the treatment group supplemented with 10ng/ml IFN-τ,the m RNA expression levels of PCNA related to the proliferation in luteinized granulosa cells was significantly up-regulated at 12 h,24h and 48 h,and the m RNA expression levels of BAX related to apoptosis was significantly down-regulated at96 h.(3)IFN-τ promotes progesterone secretion in luteinized granulosa cells.The progesterone secretion level of the treatment group added with 10 ng/ml IFN-τ was significantly higher than that of the control group at 24 h and 96 h,and the progesterone level was significantly higher than that of the control group at 12 h,48h and 72 h.The m RNA expression levels of progesterone synthesis related HSD3 B was significantly up-regulated at 12 h,48h,72 h and 96 h,the m RNA expression levels of CYP11A1 was significantly up-regulated at 24 h,48h,72 h and 96 h,while the m RNA expression levels of STAR was significantly up-regulated at 48 h and 72 h and 96 h.This study explored the effect of IFN-τ on the fate and function of yak granulosa cells during the process of luteinization.It was found that IFN-τ might promote cell proliferation by up regulating the m RNA expression levels of PCNA and down regulating the m RNA expression levels of BAX.It could also promote the secretion of progesterone by promoting the m RNA expression levels of progesterone synthesis related genes HSD3 B,STAR and CYP11A1.This study provides a theoretical basis for the treatment of reproductive diseases caused by corpus luteum abnormalities,and provides some research data for using IFN-τ to improve the reproductive performance of yaks at the cellular level.