BMP15对藏鸡等级卵泡颗粒细胞孕酮分泌的影响

Effects of Bone Morphogenic Protein(BMP15)on Progesterone Secretion in Primary Granulosa Cells of Tibetan Chicken

为探究骨形态发生蛋白15(Bone Morphogenic Protein 15,BMP15)对体外培养的藏鸡等级卵泡颗粒细胞孕酮分泌的影响,研究体外分离、培养并鉴定藏鸡等级卵泡F1颗粒细胞后,用0、25、50、100 ng/mL四种不同浓度的BMP15蛋白单独处理或者联合25 ng/mL促卵泡素(FSH)处理细胞,72 h后采用ELISA法检测不同处理组细胞培养液中的孕酮含量。同时,采用实时荧光定量PCR(RT-qPCR)法检测不同处理组细胞中孕酮合成关键基因StAR、CYP11A1和HSD3B1 mRNA相对表达量。结果显示:免疫组化法鉴定体外分离培养的藏鸡F1卵泡颗粒细胞卵泡刺激素受体(FSHR)抗体阳性率95%以上;BMP15单独处理细胞后,随着BMP15浓度的升高,孕酮分泌量呈下降的趋势,且100 ng/mL处理组的孕酮分泌量极显著低于其他组(P<0.01);BMP15联合FSH处理细胞后,随着BMP15浓度的升高,孕酮分泌量呈现上升的趋势,其中100 ng/mL处理组的孕酮分泌量极显著高于其他组(P<0.01);不同处理组细胞中孕酮合成关键基因StAR、CYP11A1和HSD3B1 mRNA相对表达量与孕酮分泌量趋势基本一致。结果表明,体外BMP15蛋白单独处理抑制藏鸡等级卵泡颗粒细胞孕酮的分泌,但是联合FSH处理促进孕酮的分泌。

To investigate the effects of Bone Morphogenic Protein 15(BMP15)on the progesterone secretion of hierarchical follicles Granulosa Cells(GCs)in vitro,the F1hierarchical follicles GCs of Tibetan chickens were isolated and cultured in vitro and were treated with different concentrations of BMP15 protein(0,25,50 and 100 ng/mL)or four different concentrations of BMP15 combined with 25 ng/mL FSH.After 72 hours,the content of progesterone in cell culture medium of different treatment groups was detected by ELISA.Meanwhile,the relative mRNA expression of the key genes of progesterone synthesis like StAR,CYP11A1 and HSD3B1 in different groups of cells were detected by real-time fluorescence quantitative PCR(RT-qPCR).The results showed that more than 95%of the cultured F1follicles GCs were positive to FSHR antibody;After treated by BMP15,the secretion of progesterone decreased with the increase of concentration,and the secretion of progesterone in 100 ng/mL treatment group was significantly lower than that in other groups(P<0.01);After treated by BMP15 combined with FSH,the secretion of progesterone increased with the increase of BMP15 concentration.The secretion of progesterone in 100 ng/mL treatment group was significantly higher than that in other groups(P<0.01);The relative mRNA expression of StAR,CYP11A1 and HSD3B1 of progesterone synthesis key genes in different treatment groups were basically consistent with the trend of progesterone secretion.The results indicated that BMP15 inhibited progesterone secretion of Tibetan chicken F1follicles GCs in vitro,but promoted the progesterone secretion when combined with FSH.