孕酮受体膜组分1基因在性成熟雌性半滑舌鳎(Cynoglossus semilaevis)的组织学定位定量分析

摘要

运用Western blotting、免疫组化和原位杂交方法检测性成熟半滑舌鳎(Cynoglossus semilaevis)孕酮受体膜组分1(Progesterone receptor membrane component 1,PGRMC1)蛋白和mRNA在不同组织的分布和表达特征.原位杂交结果发现,PGRMC1 mRNA主要分布在成熟的卵母细胞膜上,在脑组织神经元和分散的垂体细胞中也有表达.利用制备的半滑舌鳎PGRMC1多克隆抗体,对不同组织中的PGRMC1蛋白表达量进行Western blotting检测,发现在半滑舌鳎卵巢、脑、肝脏中PGRMC1蛋白表达量相对较高,在垂体、头肾、肾也有表达,但表达量相对较少.免疫组化结果表明,半滑舌鳎PGRMC1蛋白在成熟的卵母细胞膜上显著表达,进一步证明PGRMC1为卵膜上的受体基因,推测其主要在卵膜上行使相关的生理功能.研究结果为探究PGRMC1在半滑舌鳎卵母细胞成熟过程中的生理功能提供了重要参考.%Progesterone receptor membrane component 1 (PGRMC1) may play an important role in oocyte maturation of the female half-smooth tongue sole (Cynoglossus semilaevis).To better understand the underlying mechanisms,here we measured the mRNA expression of PGRMC1 in different tissues with in situ RNA hybridization and immunohistochemistry,and analyzed the protein expression with western blotting.According to the results of in situ RNA hybridization,the mRNA of PGRMC 1 was detected in the membrane of oocytes,in scattered cells of the entire pituitary,and in the neurons of the brain.Immunohistochemistry showed strong signals of PGRMC1 in the oocyte membrane of the mature ovary,and signals were also observed in the liver,kidney,and head kidney of C.semilaevis.In the liver,PGRMC 1 was mainly expressed in the bile duct and hepatic veins that were connected to the outer tissue.In the kidney,the PGRMC1 was expressed in the vicinity of the renal tubules.The results above demonstrated that PGRMC1 was a membrane receptor that functioned on the cell membrane.We also analyzed the protein sequence of PGRMC1,selected epitope of the sequence,and synthesized the corresponding immune polypeptide.We obtained the polyclonal antibody of PGRMC1 by immunizing New Zealand rabbit with the recombinant protein.The antibody was applied to measure the expression level of PGRMC 1.Western blotting results showed that the expression of PGRMC 1 protein was higher in the ovary,liver,and brain,and was relatively low in the kidney,head kidney,and pituitary.Our results may provide valuable information on the physiological function of PGRMC 1 in the oocyte maturation and reproductive endocrinal regulation of C.semilaevis