Zhang Baole, Wei Quanwei, Shi Shuai, Dong Fulu, Shi Fangxiong, Xu Yinxue
College of Animal Science & Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
J Reprod Dev. 2012;58(4):410-9. doi: 10.1262/jrd.2012-050. Epub 2012 Apr 13.
Gpr3, a member of the G protein-coupled receptor superfamily, was known as a critical factor for the maintenance of meiotic prophase arrest in oocytes via a Gs protein-mediated pathway. The present studies were conducted to examine the ovarian immunolocalization of Gpr3, its expression pattern in different stages of fetal, postnatal and developmental pigs and its effect on proliferation of ovarian granulosa cells in pigs. Immunohistochemical analysis indicated that Gpr3 was localized in egg nests, oocytes and granulosa cells (GCs) of the follicle ranging from the primordial to Graafian stages and the corpora lutea. Staining was faintly present in the corpora lutea and weak in GCs but was strong in oocytes. Real-time PCR and Western blotting indicated that Gpr3 mRNA and protein were both present in the different ages of ovaries, and there were wavy changes in the expression levels from postpartum 1 to 180 days. Moreover, both the mRNA and protein levels of Gpr3 were upregulated significantly during follicle growth, suggesting that Gpr3 might play potential roles in regulating ovarian follicle development in the pig. MTT and flow cytometry analyses indicated that Gpr3 knockdown significantly promoted proliferation of porcine GCs while increasing the proportion of cells in the S phase and the expression of Cyclin B1 and Cyclin D2, providing new insights into how Gpr3 signaling regulates the proliferation of porcine GCs. In conclusion, the stage- and cell-specific expression pattern of Gpr3 in the porcine ovary suggested that Gpr3 might play an important role during the entire process of follicular development and luteinization.
Gpr3是G蛋白偶联受体超家族的成员，通过Gs蛋白介导的途径，它被认为是维持卵母细胞减数分裂前期阻滞的关键因素。本研究旨在检测Gpr3在卵巢中的免疫定位、其在胎儿期、出生后及发育中的猪不同阶段的表达模式及其对猪卵巢颗粒细胞增殖的影响。免疫组织化学分析表明，Gpr3定位于原始卵泡至格拉夫卵泡阶段的卵泡的卵母细胞巢、卵母细胞和颗粒细胞以及黄体。黄体中的染色较淡，颗粒细胞中的染色较弱，但卵母细胞中的染色较强。实时PCR和蛋白质免疫印迹表明，Gpr3的mRNA和蛋白在不同年龄的卵巢中均有表达，产后1至180天表达水平呈波浪式变化。此外，在卵泡生长过程中，Gpr3的mRNA和蛋白水平均显著上调，这表明Gpr3可能在调节猪卵巢卵泡发育中发挥潜在作用。MTT和流式细胞术分析表明，敲低Gpr3可显著促进猪颗粒细胞的增殖，同时增加S期细胞比例以及细胞周期蛋白B1和细胞周期蛋白D2的表达，这为Gpr3信号如何调节猪颗粒细胞的增殖提供了新的见解。总之，Gpr3在猪卵巢中的阶段和细胞特异性表达模式表明，Gpr3可能在卵泡发育和黄体化的整个过程中发挥重要作用。
