促卵泡素对体外培养牛卵泡类固醇合成相关基因表达的影响

Effects of FSH on Genes Expression Related to Steroidogenesis in Bovine Follicles Cultured in vitro

本研究旨在探究促卵泡素(follicle stimulating hormone,FSH)处理对体外培养的牛有腔卵泡颗粒细胞和膜细胞类固醇激素合成相关基因表达的影响。采集牛卵巢表面直径9~11 mm的有腔卵泡,用含不同浓度FSH的DMEM/F12体外培养牛有腔卵泡24 h。提取卵泡颗粒细胞、膜细胞RNA并反转录成cDNA,利用实时荧光定量PCR检测卵泡颗粒细胞、膜细胞中类固醇激素合成酶基因(CYP11A1、3β-HSD、CYP17A1、CYP19A1、17β-HSD)和促性腺激素受体基因(FSHR、LHR)的表达水平。结果显示,FSH处理上调了颗粒细胞中CYP11A 1、3β-HSD和CYP19A 1基因表达,其中,25 ng/mL FSH处理极显著上调了CYP11A 1基因表达(P<0.01),10 ng/mL FSH处理显著上调了3β-HSD基因表达(P<0.05),50 ng/mL FSH处理显著上调了CYP19A 1基因表达(P<0.05);50 ng/mL FSH处理显著或极显著上调了膜细胞中CYP11A 1、3β-HSD和CYP17A 1基因表达(P<0.05;P<0.01),但在10和25 ng/mL FSH处理组中CYP11A 1、3β-HSD和CYP17A 1基因表达显著或极显著下调(P<0.05;P<0.01)。对FSHR、LHR基因研究结果显示,不同浓度FSH处理对颗粒细胞中FSHR、LHR基因的表达均无显著影响(P>0.05),只有25和50 ng/mL FSH处理显著或极显著上调了膜细胞中LHR基因表达水平(P<0.05;P<0.01),且不同处理组之间膜细胞中CYP11A 1、3β-HSD和CYP17A 1基因的表达变化与LHR基因表达变化趋势较一致。结果表明,FSH处理可提高牛有腔卵泡颗粒细胞中CYP11A 1、3β-HSD和CYP17A 1基因的表达,膜细胞中CYP11A 1、3β-HSD和CYP17A 1基因对LH的刺激更敏感,FSH可能通过影响LHR基因的表达来调节膜细胞中类固醇合成酶基因的表达。

The purpose of this study was to investigate the effects of FSH treatment on steroidogenesis genes expression in granulosa cells and theca cells of cultured bovine antral follicles in vitro.Cavernous follicles 9-11 mm in diameter on the surface of bovine ovaries were collected and cultured in vitro with DMEM/F12 containing different concentrations of FSH for 24 hours.The total RNA of granulosa cells and theca cells were extracted,and reverse transcribed into cDNA,the expression of steroidogenesis genes(CYP11A1,3β-HSD,CYP17A1,CYP19A1 and 17β-HSD)and gonadotropin receptor genes(FSHR and LHR)in follicular granulosa cells and theca cells were detected by Real-time quantitative PCR.The results in granulosa cells showed that FSH up-regulated the expression of CYP11A 1,3β-HSD and CYP19A 1 genes,25 ng/mL FSH treatment extremely significantly increased the expression of CYP11A 1 gene(P<0.01),10 ng/mL FSH treatment significantly increased the expression of 3β-HSD gene(P<0.05),50 ng/mL FSH treatment significantly increased the expression of CYP19A 1 gene(P<0.05).The results in theca cells showed that 50 ng/mL FSH treatment significantly or extremely significantly increased the expression of CYP11A 1,3β-HSD and CYP17A 1 genes(P<0.05;P<0.01),but 10 and 25 ng/mL FSH treatment significantly or extremely significantly decreased the expression of CYP11A1,3β-HSD and CYP17A 1 genes(P<0.05;P<0.01).The results showed that FSH treatment had no significant effect on the expression of FSHR and LHR genes in granulosa cells(P>0.05),only 25 and 50 ng/mL FSH treatments significantly or extremely significantly increased the expression of LHR gene in theca cells(P<0.05;P<0.01).The expression of CYP11A 1,3β-HSD and CYP17A 1 genes in theca cells among the different treatment groups showed the same trend as that of LHR gene.The results indicated that FSH treatment could increase the expression of CYP11A 1,3β-HSD and CYP17A 1 genes in granulosa cells of bovine antral follicular,the expression of CYP11A 1,3β-HSD and CYP17A 1 genes in theca cells were more sensitive to the stimulation of LH.FSH might regulate the expression of steroidogenesis genes in theca cells by affecting the expression of LHR gene.