Effects Of VEGF On Proliferation And Function Of Yak Granulosa Cells

Yak(Bos grunniens)is an endemic livestock species living in an environment with many unfavorable factors such as high cold,low oxygen and high ultraviolet radiation,which can provide local herders with milk,meat and other daily production resources and is an indispensable and important domestic animal on the Tibetan plateau.However,the reproductive efficiency of yaks is low,and research on improving the reproductive efficiency of yaks has received much attention.Ovarian vascular endothelial growth factor(VEGF),which is mainly secreted by granulosa cells(GC),can promote follicle development and maintain luteal function,and plays an important role in regulating reproductive activity in female animals.Therefore,the aim of this experiment was to investigate the tissue expression characteristics of VEGF in yak and the effects of adding VEGF to in vitro culture medium on yak GC viability,hormone secretion,reactive oxygen species(ROS),cell cycle and apoptosis.(1)Expression and localization of VEGF and its receptor in the reproductive system of female yaksThe relative expression of VEGF and its receptor(VEGFR2)in different tissues of yaks was detected by q PCR,and the distribution of VEGF and VEGER2 protein in reproductive organs of female yaks was investigated by immunohistochemical technique(IHC).q PCR results showed that both VEGF and VEGFR2 were expressed in the heart,liver,spleen,lung,kidney,oviduct,ovary and uterus of yaks,and the expression in lung and ovary was significantly higher than in other tissues(P<0.01).The VEGF was highly expessed in uterus during pregnancy than in follicular and luteal phases(P<0.05),while its expression in oviduct was significantly higher during follicular phase than that in luteal phase(P<0.05).The expression of VEGFR2 in ovary during pregnancy was significantly higher than that in the follicular phase(P<0.05),and its expressionin in the uterus during pregnancy was higher than that in the follicular and luteal phases(P<0.01).In the oviduct,the expression of VEGFR2 was higher during the follicular phase than in the luteal and gestation period(P<0.01).IHC results showed that VEGF and VEGFR2 proteins were highly expressed in yak ovarian GC,endometrial cells and cells of the mucosal epithelium of the oviduct.(2)Regulation of VEGF on the function of yak granulosa cellsThe effects of VEGF addition to culture medium on the viability of yak GC and the secretion of estrogen(E2)and progesterone(P4)were examined by Elisa technique.q PCR was used to detect the changes in the expression of hormone-related genes(St AR,P450 ssc,3β-HSD,FSHR and ERβ).The results showed that VEGF(20 ng/m L)in the medium significantly increased yak GC viability(P<0.05),P4 secretion(P<0.05)and the expression of 3β-HSD,St AR and P450 scc.VEGF addition group did not affect E2 secretion at 24 h of incubation,but E2 secretion was significantly lower at 48 h(P<0.05),and ERβ relative expression was lower than that of the control group(P<0.05).(3)Effect of VEGF on apoptosis and cell cycle of yak granulosa cellsThe effect of VEGF addition in the culture medium on the cell cycle and apoptosis of yak GC was examined by flow cytometry.The expression levels of cell cycle-related genes(CCND1,CCNE1,CDK2,CDK4,PCNA and P53)and proliferation-and apoptosis-related genes(Bcl-2,GDF9,Bax and Caspase-3)were detected by q PCR.The results showed that VEGF in culture medium significantly reduced the ROS level of yak GC(P<0.05),increased the percentage of yak S-phase GC(P<0.05),increased the expression of PCNA,CCND1,CCNE1,CDK2 and CDK4(P<0.05)and decreased the expression level of P53(P<0.05).The treatment group significantly reduced the rate of yak GC apoptosis and relative expression of Bax in the treated group(P<0.05).In conclusion,VEGF was widely expressed in various tissues of yak and highly expressed in yak GC.In GC in vitro culture,a certain concentration of exogenous VEGF could improve the viability of yak GC,reduce the apoptosis rate,promote P4 secretion,reduce ROS level,increase the percentage of S-phase cells,and regulate the expression level of related genes.These findings are beneficial to reveal the regulatory mechanism of VEGF on yak reproduction and provide some theoretical basis for further improvement of yak GC in vitro culture and oocyte in vitro maturation techniques.