| Granulosa cells(GCs)are one of the important follicular cells,and their proliferation and differentiation are closely related to follicular development.The proliferation and metabolism of granulosa cells are mainly regulated by follicle-stimulating hormone(FSH).But whether FSH regulates the glycolysis of granulosa cells and its mechanism,is unclear.In this study,the correlation between glycolysis and FSH in granulosa cells from different size of buffalo follicle was investigated,and the effect of FSH on glycolysis metabolism of in vitro cultured granulosa cells and its mechanism were also explored.This study will lay a foundation to understand the mechanism of FSH controlling follicle development in buffalo.1.To investigate the correlation between glycolysis of granulosa cells and FSH in buffalo follicle of different sizes.ELISA and q RT-PCR were used to detect the concentrations of FSH and lactic acid in follicles with different diameters(small follicles: diameter of 2-5mm,medium follicles: diameter of 5-8mm,large follicles: diameter of follicle>8mm),and the expression level of FSHR and glycolytic related genes in granulosa cells.The results showed that concentrations of FSH and lactate in follicular fluid from medium follicles were significantly higher than that of small and big follicles;the expression level of FSHR,and glycolytic related genes HK,PFK,MCT1,GLUT1,and HIF-1? in granulosa cells from medium follicles were also significantly higher than that of small follicle and large follicles(P<0.01).Western blot analysis showed the protein expression level of the glycolytic related genes PKM2 and LDHA in the middle follicle group was the highest,which was consistent with the concentration trend of FSH and lactic acid in different follicles with different diameters.2.The effects of FSH on glycolysis of follicular granulosa cells cultured in vitro was investigated.When follicular granulosa cells were cultured in medium with different concentrations of FSH(0 IU/m L,1 IU/m L,2 IU/m L,4 IU/m L,8 IU/m L)for 12 h,the m RNA expression levels of the glycolytic related genes GLUT1,HK,PFK,PKM2,LDHA,MCT1 showed a gradually increasing trend with the increase of concentration,and reached the highest when FSH was 4 IU/m L(P<0.05).Western blot results showed that the expression levels of HIF-1?,GLUT1,PKM2 and LDHA proteins also increased with the concentration of FSH,and reached the highest level when FSH was 4 IU/m L.When granulosa cells were cultured in medium containing 4 IU/m L FSH for 0h,12 h,24h and 48 h respectively,the m RNA expression levels of glycolytic related genes were the highest under FSH culture treatment for 12h(P<0.05),and then decreased with the extension of treatment time.With the FSH concentration was 4 IU/m L,the lactic acid production and glucose uptake of granulosa cells were detected after 12 h of culture.The results showed that the lactic acid production level and glucose uptake the in the treatment group increased significantly(P<0.05).3.The mechanism of FSH regulating glycolysis of granulosa cells was preliminarily studied.The expression of AMPK/SIRT1 signaling pathway related genes in granulosa cells from different diameters of follicles were detected.The results showed m RNA and protein expression level of AMPK and the protein expression level of p-AMPK were the highest in the medium follicles,which was consistent with the change trend of FSH and lactate in follicles with different diameters.The expression of SIRT1 protein and glycolytic related protein(HIF-1?,GLUT1)in granulosa cells from different size of follicles showed the same trend.Granulosa cells treated with different concentrations of FSH for 12 h.The result showed the expression of AMPK and SIRT1 in m RNA and protein increased with the increase of FSH concentration.When SRT1720(SIRT1 activator),FSH + sirtinol(SIRT1 inhibitor)and AICAR(AMPK activator)were used to culture granulosa cells,the expression level of glycolytic key proteins and the production of lactate in SIRT1 activated group were increased,while that was opposite to the SIRT1 inhibited group.However,the change of lactate level in AMPK activated group was not significant(P > 0.05).These results showed that the glycolysis of buffalo granulosa cells in follicles and in vitro cultured,were positively correlated with the concentration of follicle-stimulating hormone.Activation of AMPK/SIRT1 pathway,especially SIRT1,plays an important role in the regulation of follicle-stimulating hormone on glycolysis of granulosa cells. |
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