γ-氨基丁酸及其A型受体α5亚单位在小鼠不同成熟度卵母细胞周围的卵丘颗粒细胞中的表达

Expression ofγ-aminobutyric acid and its type A receptor α5 subunits in mouse cumulus cells around oocytes at different mature stage

目的探讨γ-氨基丁酸(γ-aminobutyric acid,GABA)及其A型受体α5亚单位(GABRA5)在小鼠不同成熟度卵母细胞周围的卵丘颗粒细胞中的表达情况。方法在性成熟前小鼠给予孕马血清促性腺激素(PMSG)以刺激卵泡生长,并予以人绒毛膜促性腺激素(HCG)诱导排卵,于给药后不同时间取出卵巢和输卵管,并收集不同时期的颗粒细胞。釆用实时荧光定量聚合酶链反应检测GABRA5mRNA在生发泡期(GV)和减数第2次分裂中期(MⅡ)卵母细胞周围卵丘颗粒细胞中的表达;用Western blotting和免疫荧光技术检测GABRA5蛋白在GV期和MⅡ期卵母细胞周围卵丘颗粒细胞中的表达;用免疫组织化学方法检测GABA在GV期和MⅡ期卵母细胞周围卵丘颗粒细胞中的分布。结果 (1)GABRA5mRNA在GV期和MⅡ期卵母细胞周围的卵丘颗粒细胞中均有表达,且MⅡ期卵母细胞周围的卵丘颗粒细胞上的表达水平高于其在GV期卵母细胞时卵丘颗粒细胞中表达水平(P<0.05)。(2)GABRA5蛋白在GV期和MⅡ期卵母细胞周围卵丘颗粒细胞的细胞膜和细胞质中均有表达,且其表达量随卵泡发育呈上升趋势(P<0.05)。(3)免疫组化染色结果显示,GABA均定位于在GV期和MⅡ期卵母细胞周围卵丘颗粒细胞的胞质中。结论 GABA及其A型受体α5亚单位均表达于小鼠不同成熟度卵母细胞周围的卵丘颗粒细胞中,其中A型受体α5亚单位的表达与卵母细胞的成熟度相关,其表达量随卵母细胞成熟而升高,提示其可能参与小鼠卵泡发育和成熟的过程。

Objective： To investigated the expression of γ-aminobutyric acid（GABA）and its type A receptor a5 subunits（GABRA5）in cumulus ceils of mouse. Methods： Immature female Kunming mice were treated with pregnant mare serum gonadotropin （PMSG）to induce follicular development and HCG to promote ovulation. RT-PCR,western blotting and immunofluorescence technique were used to detect the expression of GABRA5 in cumulus cells isolated from oocytes at germinal vesicle（GV）or meiosis metaphase（M Ⅱ ）stage. The expression and localization of GABA protein in the GV and metaphase Ⅱ （M Ⅱ ） cumulus cells around oocytes were evaluated by immunohistochemical analysis. Results： The expression of GABRA5 in cumuluS cells derived from mouse oocytes at M 1I stage was significantly higher than that at GV stage（P〈0.05）. The GABRA5 protein was expressed in the cumulus ceils from mouse oocytes at GV or M Ⅱ stage, and the expression was increasing along with the follicle development（P（0.05）. The results of immunohistochemistry showed that GABA was located in the cytoplasm of the cumulus ceils from mouse oocytes at GV or M Ⅱ stage. Conclusions. The expression of γ-aminobutyric acid and its type A receptor a5 subunits in cumulus ceils of mouse indicates that GABA and its type A receptor a5 subunits may participate in regulation of ovarian follicular development and oocyte maturation.