膜孕激素受体(mPRα)在半滑舌鳎(Cynoglossus semilaevis)卵母细胞成熟过程中的表达特征

The Expression Patterns of Membrane Progestin Receptor α (mPRα) During Oocytes Maturation in Cynoglossus semilaevis

中文关键词: 膜孕激素受体  半滑舌鳎  原位杂交  免疫组化  Western blotting  实时荧光定量PCR

英文关键词: Membrane progesterone receptor  Cynoglossus semilaevis  in situ hybridization  Immunohistochemistry  Western blotting  qRT-PCR

中文摘要:

      通过实时荧光定量PCR方法(qRT-PCR)检测半滑舌鳎(Cynoglossus semilaevis)雌性性成熟不同时期、不同时相卵母细胞中膜孕激素受体(mPRα)的表达，通过qRT-PCR和Western blotting方法检测促性腺激素(HCG)对成熟期半滑舌鳎卵母细胞mPRα表达的影响，同时，采用原位杂交、免疫组化和Western blotting方法研究雌性性成熟半滑舌鳎mPRα mRNA和蛋白在各组织中的表达。对半滑舌鳎性成熟不同时期、不同时相卵母细胞中mPRα mRNA的定量研究结果显示，在成熟时期半滑舌鳎Ⅴ时相卵母细胞的mPRα mRNA表达量最高。HCG对成熟期半滑舌鳎卵母细胞mPRα表达的影响研究结果显示，20 IU/ml HCG比10 IU/ml HCG对成熟时期半滑舌鳎卵母细胞mPRα mRNA和蛋白表达的促进作用更明显，进一步证明mPRα介导孕激素参与了卵母细胞成熟的调控。对mPRα组织表达的定量和定位研究中，Western blotting结果显示，在半滑舌鳎的卵巢、垂体、脑、头肾、肾、肝脏组织中均有mPRα蛋白表达，且在脑、垂体和卵巢中表达量较高，表明mPRα在不同组织中都发挥着一定的作用，且在内分泌相关组织如脑、垂体和卵巢中的作用更明显。原位杂交和免疫组化结果显示，mPRα mRNA和蛋白表达明显定位在卵母细胞膜上，且在其他组织的外周和管腔结构表达。本研究为进一步研究mPRα在膜上的信号转导机制提供了理论基础和形态学依据。

英文摘要:

      In this study we investigated the expression patterns of the mRNA and proteins of membrane progestin receptor alpha (mPRα) in different tissues and oocyte phases using quantitative real-time PCR (qRT-PCR), in situ hybridization, immunohistochemistry, and western blotting analysis. Mature tissue samples were collected from female half smooth tongue sole Cynoglossus semilaevis. After in vitro incubation with or without human chorionic gonadotropin (HCG), the levels of mPRα mRNA and proteins in the oocytes at different developmental stages were measured with qRT-PCR and western blotting analysis. It was shown that the highest level of mPRα mRNA appeared in Phase V oocyte of mature C. semilaevis. 20 IU/ml HCG promoted the mRNA and protein expression more effectively than 10 IU/ml HCG. This further implied that mPRα was involved in the regulation of oocyte maturation through guiding progesterone. The western blotting analysis confirmed that the expression of mPRα proteins was higher in the brain, pituitary, and ovary than in other tissues. These results indicated that mPRα could play certain roles in different tissues, especially the endocrine-related ones. Results of in situ hybridization and immunohistochemistry clearly showed that the mPRα mRNA and proteins were expressed on the oocyte membrane and the peripheral or bureaucratic structures of other organs. Our study enriched the knowledge about mechanisms of membrane receptor signal transduction.