| Studies have shown that Wnt/β-Catenin signaling pathway has been implicated in the regulation of the synthesis of estradiol and progesterone in granulosa cells.To this end,this research was intended to explore the effects and mechanisms of Wnt/β-Catenin signaling affecting estradiol and progesterone synthesis in granulosa cells to understand the regulatory role of Wnt/β-Catenin signaling pathway in buffalo follicle development.1.The expression of FSHR and cell growth in granulosa cells cultured with or without serum,and the protein expression of Wnt3 a in granulosa cells from different sized follicles were compared.The results showed that serum-free culture did not affect the expression of FSHR in granulosa cells except for a lower cell attachment rate than culture with serum;The change trend of growth curve under serum-free culture was basically consistent with serum culture,and both growth curves were basically “S”-shaped;Wnt3a expression was detected in granulosa cells from different sized follicles and was significantly higher in large than in medium and small follicles(P < 0.05).2.To explore the effects of Wnt3 a,an activator of the Wnt/β-Catenin signaling pathway,on estradiol and progesterone synthesis in granulosa cells and its mechanism.First,granulosa cells were treated with various concentrations(0,50,100,and 150 ng/ml)of Wnt3 a serum-free medium for 48 h,and granulosa cell viability,estradiol and progesterone secretion,and estradiol and progesterone synthesis key enzymes gene m RNA and protein expression were examined by MTT,ELISA,RT q PCR,and Western blot,respectively.The results showed that the viability of granulosa cells was not significantly altered at Wnt3 a concentrations of 50 and 100 ng/ml,while significantly reduced when the concentration was 150 ng/ml(P < 0.05).Compared with the control(0 ng/ml Wnt3a),50 ng/ml Wnt3 a group did not show a significant decrease in estradiol(P > 0.05),and the 100 and 150 ng/ml Wnt3 a groups significantly decreased estradiol secretion in granulosa cells(P < 0.05);Progesterone concentrations in50,100,and 150 ng/ml Wnt3 a groups were all significantly up-regulated(P <0.05);100 and 150 ng/ml Wnt3 a significantly downregulated the m RNA expression of estradiol synthesis key enzymes CYP19A1 and HSD17B1(P <0.05),while significantly upregulated the m RNA expression levels of progesterone synthesis key enzymes St AR and HSD3B1(P < 0.05);50,100 and150 ng/ml Wnt3 a significantly down regulated the protein expression of estradiol synthesis key enzymes CYP19A1 and HSD17B1(P < 0.05),on the contrary,significantly upregulated the protein expression of progesterone synthesis key enzymes St AR and HSD3B1(P < 0.05).Next,the apoptosis of granulosa cells and the expression of proteins related to apoptosis as well as Wnt/β-Catenin signaling pathway were compared between control(0 ng/ml Wnt3a)and Wnt3 a group(100 ng/ml Wnt3a)by flow cytometry and Western blot.The results showed that Wnt3 a promoted granulosa cell apoptosis(P < 0.05),upregulated the expression of Bax but downregulated the expression of Bcl2(P < 0.05);Wnt3a potently activated Wnt/β-Catenin signaling pathway,and promoted the protein expression of endogenous Wnt3 a and Foxo3a(P < 0.05).3.To study the effects of Wnt/β-catenin signaling pathway inhibitor XAV939 on estradiol and progesterone synthesis in granulosa cells and its mechanism.First,granulosa cells were treated with different concentrations(0,2.5,5,10 μM)XAV939 serum-free medium for 48 h,and cell proliferation,secretion of estradiol and progesterone,and the expression of genes and proteins of estradiol and progesterone synthesis key enzymes were examined by MTT,ELISA,RT q PCR,and Western blot.We found that different concentrations of XAV939 did not significantly affect granulosa cell proliferation(P > 0.05).Compared with the control group(0μM XAV939),the estradiol concentration in 2.5μM XAV939 group was not significantly different(P > 0.05).The estradiol concentrations in the 5μM and 10μM XAV939 groups were significantly increased(P < 0.05),and the estradiol concentration in the 10μM XAV939 group was significantly higher than that in the 5μM group(P < 0.05).The progesterone concentrations in the5μM and 10μM XAV939 groups were significantly decreased(P < 0.05),and the differences between the 2.5μM group and the control group as well as the 5μM group were not significant(P > 0.05).The progesterone concentration in the10μM group was the lowest and significantly lower than that in the control group,the 2.5μM group and the 5μM group(P < 0.05).The 5μM and 10μM XAV939 could significantly up-regulate the m RNA expression levels of estradiol synthesis key enzymes CYP19A1 and HSD17B1(P < 0.05),but significantly downregulated the m RNA expression levels of progesterone synthesis key enzymes St AR and HSD3B1(P < 0.05).5 μM and 10 μM XAV939 could significantly upregulate the protein expressions of estradiol synthesis key enzymes CYP19A1 and HSD17B1(P < 0.05),while 10 μM XAV939 could significantly down-regulate the protein expressions of progesterone synthesis key enzymes St AR and HSD3B1(P< 0.05).And then,the apoptosis of granulosa cells and the expression of proteins related to apoptosis as well as Wnt/β-Catenin signaling pathway were compared between control(0 μM XAV939)group and the XAV939(10 μM XAV939)group were detected by flow cytometry and Western Blot.The results showed that XAV939 could inhibit apoptosis of granulosa cells(P < 0.05),down-regulate the expression of Bax and up-regulate the expression of Bcl2(P < 0.05).XAV939 inhibited the Wnt/β-Catenin signaling pathway and weakened the expression of Wnt3 a and Foxo3a(P < 0.05).In summary,Wnt/β-Catenin signaling pathway is related to the synthesis of estradiol and progesterone in buffalo follicular granulosa cells.The activation and inhibition of Wnt/β-Catenin signaling pathway inhibits and promotes the synthesis of estradiol,and promotes and inhibits the synthesis of progesterone,respectively,thus participating in the regulation of buffalo follicular development. |
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