半滑舌鳎(Cynoglossus semilaevis)新型膜孕激素受体基因(mPRL)在卵母细胞成熟过程中的表达特征

Expression Characterization of the Novel Membrane Progestin Receptor (mPR-Like) Gene During the Oocyte Maturation of Half-Smooth Tongue Sole (Cynoglossus semilaevis)

关键词:  半滑舌鳎  新型膜孕激素受体  卵母细胞成熟  mRNA表达  蛋白表达

Key words:  Cynoglossus semilaevis  mPR-Like  Oocyte maturation  mRNA expression  Protein expression

摘要:

为进一步提高半滑舌鳎(Cynoglossus semilaevis)人工育苗技术的水平，对半滑舌鳎新型膜孕激素受体(mPR-like, mPRL)基因的表达特征和作用机制进行了研究。应用实时定量PCR方法分析mPRL mRNA在卵子形成过程中的时序表达，发现半滑舌鳎mPRL mRNA相对表达量的最高值出现在性成熟阶段卵巢的Ⅴ时相卵母细胞。原位杂交分析mPRL mRNA在繁殖相关组织的细胞学定位，发现mPRL mRNA分布在半滑舌鳎性成熟阶段卵巢的卵母细胞膜上；在脑的神经元和垂体内分散的细胞中，mPRL mRNA阳性信号较强。制备半滑舌鳎mPRL的多克隆抗体，采用Western blotting方法检测半滑舌鳎mPRL蛋白在不同组织的表达特征，发现mPRL蛋白的表达量在卵巢、脑、垂体中相对较高，在肝脏、头肾、肾脏中表达量相对较少。免疫组化结果显示，半滑舌鳎mPRL蛋白在卵巢、脑和垂体的细胞学定位与mPRL mRNA定位一致。应用实时定量PCR和Western blotting方法检测促性腺激素调控下半滑舌鳎不同时相卵母细胞mPRL基因mRNA和蛋白的表达变化，结果显示，促性腺激素对半滑舌鳎卵母细胞中mPRL基因mRNA和蛋白表达都有一定的上调作用，特别是对Ⅴ时相卵母细胞中mPRL mRNA和蛋白的表达量提升明显；发现表达量与促性腺激素调控作用具有剂量依存关系。半滑舌鳎mPRL在繁殖相关组织的表达特征表明其通过脑–垂体–卵巢轴参与繁殖调控，同时也揭示了mPRL介导卵母细胞成熟机制。

Abstract:

This research focuses on the expression characteristics and mechanism of the novel membrane progestin receptor-like (mPR-Like, mPRL) gene of half-smooth tongue sole (Cynoglossus semilaevis) to improve the technique of artificial breeding. In the present study, using the quantitative real-time PCR (qRT-PCR) assays, the mPRL mRNA expression levels were measured in the isolated oocytes during different stages of oogenesis. With the oocyte development, the mPRL transcript levels of oocytes significantly increased from stage Ⅱ to stage Ⅴ and reached the peak at stage Ⅴ. The spatial expression of mPRL mRNA in the ovary, pituitary and brain of C. semilaevis was demonstrated using in situ hybridization. Results revealed that the mPRL expression was observed on the membrane of oocytes, and the positive signals were also observed in the scattered cells throughout the pituitary and in the brain neurons. Western blotting analysis identified the immunoactive protein that bands in the ovary, brain, pituitary, liver, head kidney and kidney of C. semilaevis. However, the expression levels of the protein in the ovary, brain and pituitary were higher than other tissues. Immunohistochemistry detection revealed that the cellular localization of mPRL protein was similar with that of mRNA in C. semilaevis. The signals were observed in the scattered cells of pituitary and the brain neurons. The intensity of the positive signals was found in the membrane of oocyte. After incubation in vitro in the presence or absence of hCG, the mPRL mRNA and protein levels were measured in the oocytes at different developmental stages using qRT-PCR and western blotting analysis. In response to the hCG treatment, the mPRL mRNA and protein expression increased in a step-wise manner during follicle development, with the highest level detected in the stage V of oocytes. The mPRL expression characteristics at both transcript and protein levels implied that mPRL was involved in regulating the reproduction of female C. semilaevis through the brain-pituitary-ovary axis endocrine system. Moreover, the evidence supports that mPRL has a functional role in the oocyte maturation in C. semilaevis by acting as a mediator of progesterone.