瘦素和FSH对绵羊卵泡颗粒细胞孕激素分泌的影响

试验旨在探明瘦素和卵泡刺激素(FSH)对绵羊卵泡颗粒细胞孕激素分泌的影响并建立瘦素和FSH之间的相互作用。从屠宰场收集健康母羊的卵巢,机械分离卵泡,收集卵泡颗粒细胞。在细胞培养液中加入不同浓度的FSH(0 U/mL,2.5 U/mL,5 U/mL,7.5 U/mL,10 U/mL)培养24 h和48 h,通过MTT检测细胞增殖,以确定后续试验FSH的使用浓度。在细胞培养液中加入瘦素(0 ng/mL,25 ng/mL,50 ng/mL)和FSH(5 U/mL)单独或联合作用48 h后,通过ELISA检测细胞培养液中孕酮(P_4)的浓度。收集细胞,通过qPCR和Western blotting检测CYP11A1、STAR、3β-HSD的表达。结果表明:5 U/mL的FSH浓度可显著促进细胞生长,使类固醇相关基因(CYP11A1、STAR、3β-HSD)的表达升高(P<0.05),但对其蛋白无显著影响,对P4分泌无刺激作用。单独添加瘦素时,25 ng/mL瘦素降低了CYP11A1、STAR、3β-HSD基因的表达(P<0.05),但相应蛋白无显著性差异,同时对P_4的分泌无影响;50 ng/mL瘦素降低了STAR、CYP11A1基因的表达,但相应蛋白表达和P4分泌未受影响。瘦素和FSH联合使用时,P4分泌显著降低,STAR表达降低(P<0.05),但对其他类固醇合成基因和蛋白(CYP11A1、3β-HSD)的表达无显著影响(P>0.05)。综上所述,FSH对绵羊卵泡颗粒细胞增殖和类固醇合成基因表达有促进作用;瘦素对FSH诱导下P_4的分泌有抑制作用,且具有剂量依赖性,提示瘦素参与卵泡的发育过程。

Effects of Leptin and FSH on Progesterone Secretion of Granulosa Cells in Sheep

The aim of this study was to investigate the effects of leptin and follicle stimulating hormone(FSH)on progesterone secretion of sheep ovarian follicular and to establish the interaction between leptin and FSH.The ovaries of healthy ewes were collected from slaughterhouse and follicular granulosa cells(GCs)were collected by mechanical separation.The GCs were cultured in vitro for 24 h and 48 h in the medium with different concentrations of FSH(0 U/mL,2.5 U/mL,5 U/mL,7.5 U/mL,10 U/mL).MTT was used to detect cell proliferation to determine added concentration of FSH in the culture medium in the follow-up tests.Leptin(0 ng/mL,25 ng/mL,50 ng/mL)and FSH(5 U/mL)were added to the cell culture medium for 48 h and the progesterone(P4)concentration in the cell culture medium was detected by ELISA.The expression of CYP11A1,STAR and 3β-HSD were detected by qPCR and Western blotting.The results showed addition of 5 U/mL FSH in the medium significantly promoted cell growth and increased the expression of steroid related genes(CYP11A1,STAR,3β-HSD)(P<0.05),but had no significant effect on its protein and no stimulation on P4 secretion.Leptin(25 ng/mL)alone decreased the expression of CYP11A1,STAR and 3β-HSD genes(P<0.05),but had no significant effect on its protein and no stimulation on P4 secretion.Addition of 50 ng/mL leptin in the culture medium decreased the expression of STAR and CYP11A1,but had no significant effect on the corresponding protein and secretion of P4.When leptin and FSH were used together used in the culture medium,P4 secretion and STAR expression were significantly decreased(P<0.05),but the expression of other steroidal synthesis genes and proteins(CYP11A1,3β-HSD)was not affected(P>0.05).In conclusion,FSH could promote the proliferation of GCs and promote the synthesis of steroids.Leptin has an inhibit effect on the secretion of P4 induced by FSH,suggesting that leptin is involved in the sheep ovarian follicular development.