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目的:研究培养不同时间鸡卵泡颗粒细胞孕酮和雌激素的分泌水平,促卵泡素受体(FSHR)和促黄体素受体(LHR)的基因表达水平,推断体外培养时间对颗粒细胞激素分泌及相关受体基因表达的影响。方法:通过细胞体外培养的方法,分别于0 h、24 h、48 h、72 h、96 h收集鸡卵泡颗粒细胞上清液,采用ELISA法测定细胞上清液内的孕酮及雌激素分泌水平,并采用荧光定量PCR技术检测颗粒细胞内FSHR和LHR基因表达情况。结果:在培养初期0 h~48 h孕酮和雌激素分泌量显著降低(P<0.05),随着培养时间增加到72 h两种激素的分泌量又开始增加,并达到培养初期水平,培养至96 h细胞内孕酮和雌激素分泌量再次降低;颗粒细胞FSHR和LHR m RNA的表达水平则随着培养时间的增加而降低(P<0.05)。结论:体外培养的卵泡颗粒细胞内孕酮和雌激素的分泌量随体外培养时间的延长呈先降低后升高的趋势,可能与体外培养细胞的生长状态相关,从整体上看随着培养时间的延长,细胞内孕酮和雌激素的分泌量均降低,可能与两种促性腺激素受体FSHR和LHR基因表达量下降相关。
OBJECTIVE: To study the gene expression levels of progesterone and estrogen, follicle stimulating hormone (FSHR) and luteinizing hormone receptor (LHR) in cultured chicken follicular granulosa cells at different time points, and to infer the relationship between the secretion of progesterone and estrogen, Receptor gene expression. Methods: The supernatant of granulosa cells of chicken follicles was collected at 0 h, 24 h, 48 h, 72 h, 96 h by cell culture in vitro. The contents of progesterone and estrogen in the supernatant of the cells were measured by ELISA Level, and quantitative PCR was used to detect the expression of FSHR and LHR in granulosa cells. Results: The secretion of progesterone and estrogen decreased significantly from 0 h to 48 h after culture (P <0.05), and the secretion of two hormones began to increase after 72 h of culture and reached the initial level of culture. The level of progesterone and estrogen secretion decreased again after 96 hours. The expression of FSHR and LHR m RNA in granulosa cells decreased with the increase of culture time (P <0.05). CONCLUSION: The secretion of progesterone and estrogen in follicular granulosa cells cultured in vitro firstly decreases and then increases with the prolongation of culture time, which may be related to the growth of cultured cells in vitro. As a whole, , The secretion of progesterone and estrogen decreased, which may be related to the decrease of FSHR and LHR gene expression of two gonadotropin receptors.