The Localized Expression Of HAS2 And PTGS2 In Yak Ovary And The Effect Of FGF10 On Their Expression

The yak(Bos grunniens)originated in China and is a primitive species in China.It is mainly distributed in the Qinghai-Tibet Plateau and surrounding plateau areas above 3000 m.The yak has a strong ability to adapt to high cold and low oxygen,but its fertility is relatively low and the abortion rate is high.It seriously affected the reproduction of yak and the local economic development.In this study,yak ovary was selected as the research object to explore the localization and expression of hyaluronic acid synthase 2(HAS2)and prostaglandin endoperoxide synthase 2(PTGS2)in yak ovary at different stages,and to explore the effect of FGF10 treatment of yak granulosa cells on the expression of HAS2 and PTGS2.Collecting and using clinically healthy yak ovarian tissues in different periods(follicular phase,luteal phase,pregnancy period)and using FGF10 at concentrations of 0,0.5,1,5,10,20 ng/m L to act on granulosa cells as the research object,extract total RNA and protein from ovarian tissue and granular cells,using Quantitative real time PCR(q RT-PCR)and Western blotting(WB)method was used to detect the expression of HAS2 and PTGS2 genes in the ovary of yak at different stages and the effect of adding exogenous FGF10 on the expression of HAS2 and PTGS2 during the process of culturing granulosa cells in vitro.Immunohistochemistry(IHC)method was used to detect the distribution of HAS2 and PTGS2 in the ovaries at different stages,and immunofluorescence method was used to detect the localized expression of HAS2 and PTGS2 in granulosa cells.The results are as follows:(1)The expression of HAS2 gene in the ovaries during the luteal phase was significantly higher than that during the follicular phase and pregnancy(P<0.05),and the expression of the ovaries during the follicular phase was significantly higher than that during pregnancy(P<0.05).The expression of PTGS2 gene in the ovarian phase in the luteal phase was significantly higher than that in the other two periods(P<0.05),and the expression in the ovarian phase in the follicular phase was significantly higher than that in the pregnancy period(P<0.05).The HAS2 protein is expressed in the ovaries of the three stages.The ovarian expression in the luteal phase is significantly higher than that in the other two stages(P<0.05),and the gestation period is significantly higher than the follicular phase(P<0.05);The expression of PTGS2 in the luteal phase was significantly higher than the other two periods(P<0.05),and the follicular phase was significantly higher than that in the pregnancy period(P<0.05).IHC experiments showed that HAS2 and PTGS2 proteins are expressed in corpus luteum,granular layer and cumulus cells.(2)After using FGF10 at different concentrations of 0,0.5,1,5,10 and 20 ng/m L on granulosa cells,the expression of cumulus expansion-related genes HAS2 and PTGS2 was positively correlated with the concentration of FGF10.The HAS2 gene expression level was the highest when the FGF10 concentration was 1ng/m L and was significantly higher than other treatment groups(P<0.05).The difference in expression level was not significant compared with 0.5ng/m L,5ng/m L and 10ng/m L(P>0.05),but compared with the control group(0ng/m L),the difference was significant(P<0.05).The expression of PTGS2 gene was the highest in the 10ng/m L group(P<0.05).Compared with the control group(0ng/m L),the expression difference between 0.5ng/m L and 1ng/m L treatment groups was not significant(P>0.05).The expression level of HAS2 protein at 1ng/m L was significantly higher than that of other treatment groups,and there was no significant difference in expression between0.5ng/m L,5 ng/m L and 10 ng/m L treatment groups(P>0.05)The expression level of PTGS2 protein in the 1ng/m L treatment group was significantly higher than that in the other groups(P<0.05).Immunofluorescence results showed that HAS2 and PTGS2 were both expressed in the cytoplasm and nucleus.(3)The yak granulosa cells were cultured in vitro.After identification,both FSHR and LHR were positively expressed in the yak granulosa cells.In summary,the results show that HAS2 and PTGS2 are expressed in yak ovary at different stages and have significant differences(P<0.05),and the expression in luteal phase is the highest compared with other groups and has significant difference(P<0.05)It is speculated that HAS2 and PTGS2 may be involved in the development and maturation of yak oocytes,luteinization and degeneration,and early embryonic development.The expression of HAS2 and PTGS2 in granular cells with different concentrations of FGF10 is different(P<0.05),and they are expressed in a concentration-dependent manner.The effect is related to the concentration of FGF10.The HAS2 and PTGS2 genes have the best effect when FGF10 is 1ng/m L,which proves that FGF10 participates in the regulation of the expression of HAS2 and PTGS2 in yak,and participates in the physiological activities of cumulus expansion and reproduction.The above research results provide a basis for further research on the mechanism of growth factors related to the expansion and reproduction of yak cumulus.