G蛋白偶联受体50在牦牛卵母细胞体外成熟过程中的表达与亚细胞定位研究

试验旨在研究G蛋白偶联受体50（G protein-coupled receptor 50，GPR50）在牦牛卵母细胞体外成熟过程中的表达与定位规律，为进一步解析卵母细胞成熟的分子机制及理解牦牛繁殖的特异性提供依据。通过牦牛卵母细胞体外成熟培养，利用免疫荧光染色监测不同时间点（0~24 h）纺锤丝形态和核相的变化，确定牦牛卵母细胞减数分裂4个时期，包括生发泡期（germinal vesicle，GV）、生发泡破裂期（germinal vesicle break down，GVBD）、第一次减数分裂中期（metaphase Ⅰ，MⅠ）与第二次减数分裂中期（metaphase Ⅱ，MⅡ）的时间点。在此基础上，通过实时荧光定量PCR检测GPR50基因在牦牛卵母细胞成熟过程中的动态表达量，免疫荧光染色检测GPR50蛋白在卵母细胞成熟过程中的的亚细胞动态定位情况。结果表明，牦牛卵母细胞体外成熟0 h时90%处于GV期，6 h时94%处于GVBD期，16 h时92%细胞处于MⅠ期，24 h时94%处于MⅡ期。实时荧光定量PCR结果表明，GPR50基因在牦牛卵母细胞GV期即有表达，并在GVBD、MⅠ、MⅡ期成熟过程中逐渐升高，在MⅡ期达到顶峰，且极显著高于GV与GVBD期（P<0.01）。GPR50蛋白在牦牛卵母细胞GV期时集中在膜上表达，并随着成熟进程的发展在细胞质和细胞膜均大量表达，在MⅡ期高亮度弥散表达。以上结果表明，GPR50基因参与牦牛卵母细胞减数分裂过程并发挥重要作用，为研究GPR50在牦牛卵母细胞成熟过程中的作用及机制提供了依据。

Study on the Expression and Subcellular Localization of G Protein-coupled Receptor 50 During in vitro Maturation Process of Yak Oocytes

This study was aimed to explore the site of G protein-coupled receptor 50 (GPR50) expression and localization in yak oocytes during in vitro maturation (IVM), and to provide the basis for better understanding the molecular mechanism of oocyte maturation and the specificity of yak reproduction. After yak oocytes were matured in vitro, fluorescence staining was used to monitor the changes of spindle morphology and nuclear phase at different time points (0-24 h). The time points of the four critical periods of maturity for yak oocytes, including germinal vesicle(GV), germinal vesicle break down(GVBD), metaphase Ⅰ(M Ⅰ) and metaphase Ⅱ(M Ⅱ) were determined accurately. On this basis, the dynamic expression of GPR50 gene during the maturation of yak oocytes was detected by Real-time quantitative PCR, and the subcellular dynamic site of GPR50 protein during the maturation of oocytes was detected by immunofluorescence staining. The results showed that 90% of the in vitro matured yaks oocytes were at GV stage at 0 h, 94% at GVBD stage at 6 h, 92% at MⅠ stage at 16 h, 94% at MⅡ stage at 24 h. The results of Real-time quantitative PCR showed that GPR50 gene was expressed in GV phase of yak oocytes, and it gradually increased during the maturation of GVBD, MⅠ and MⅡ stages, and reached the peak at MⅡ stage, which was extremely significantly higher than GV and GVBD stages (P<0.01). GPR50 protein was mainly expressed on the membrane at GV stage, and then dispersed to cytoplasm gradually with the development of maturation, and high brightness dispersion expression was found at MⅡ stage. The above results indicated that GPR50 gene might play an important role in the meiosis of yak oocytes, and provided for the study on the function and mechanism of GPR50 in yak oocytes.