| 摘要: |
| 在牙鲆(Paralichthys olivaceus)繁殖周期，新型膜孕激素受体基因(mPR-Like, mPRL)在脑–垂体–性腺轴的表达变化规律与性腺发育成熟密切相关。为进一步研究牙鲆mPRL作用机制，本研究对其mRNA和蛋白的表达特征进行了分析。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，发现牙鲆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在繁殖相关组织中的细胞学定位，发现mPRL mRNA主要分布在牙鲆性成熟阶段卵巢的卵母细胞膜上；在脑组织的神经元附近区域mPRL mRNA阳性信号也较强。制备牙鲆mPRL的多克隆抗体，采用Western blotting方法检测牙鲆mPRL蛋白在不同组织的表达特性，发现mPRL蛋白表达量在卵巢和脑组织中相对较高，在肝脏、头肾、肾脏中表达量相对较少。免疫组化结果显示，牙鲆mPRL蛋白在卵巢和脑组织的细胞学定位与mPRL mRNA定位一致。牙鲆mPRL在繁殖相关组织的表达特征说明其参与卵母细胞成熟的过程，并通过内分泌方式参与牙鲆的繁殖调控。 |
| 关键词: 牙鲆 新型膜孕激素受体 mRNA表达 蛋白表达 |
| DOI：10.11758/yykxjz.20151213002 |
| 分类号: |
| 基金项目:国家自然科学基金项目(31201982)、国家鲆鲽类产业技术体系(CARS-50)和山东省优秀中青年科学家科研奖励基金项目(BS2013SW042)共同资助 |
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| Expression Characterization of the Novel Membrane Progestin Receptor (mPR-Like) in Sexual Maturation of Female Japanese Flounder (Paralichthys olivaceus) |
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SHI Bao1,2,3,4, LIU Xuezhou1,2,3,4, XU Tao5, LI Xiaoni1,2,3, XU Yongjiang1,2,3,4, ZHANG Jinyong1,2,3
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1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture;2.Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology;3.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;4.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071;5.Shandong Province Fisheries Technology Extension Station, Jinan 250013
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| Abstract: |
| This research examined the expression characteristics and mechanism of the novel membrane progestin receptor-like (mPRL) in Japanese flounder Paralichthys olivaceus. In the present study, the temporal changes of mPRL mRNA expression level in isolated oocytes at various oogenesis stages were measured using quantitative real-time PCR (qRT-PCR). The mPRL transcript levels significantly increased with the oocyte development, with the peak level at stage Ⅴ. However, the transcript level decreased significantly at stage Ⅵ. These expression characteristics of mPRL showed that mPRL is a good marker for studying the oocyte maturation. The spatial expression of P. olivaceus mPRL mRNA in the ovary and brain was demonstrated using in situ hybridization. Results revealed that mPRL transcript was localized at the membrane of oocytes, and the positive signals were also observed in the brain neurons of P. olivaceus. Specifically, high-titer rabbit polyclonal antibody was generated against synthetic peptides derived from the C-terminal domain of P. olivaceus mPRL amino acid. Immunobloting analysis identified the immunoactive protein that bands in the ovary, brain, head kidney, kidney and liver of P. olivaceus. However, the expression levels of protein in ovary and brain were higher than other tissues. The protein was localized in the major reproductive associated organs including the ovary and brain using immunohistochemistry. Moreover, the cellular localization of mPRL protein was similar with that of mRNA in P. olivaceus. The intensive signals were observed at the edge of pituitary and brain, and the intensity of positive signals was found on the membrane of oocytes. This is to date the first study that examined the tissue specific localization of mPRL protein and transcript in the reproductive organs of P. olivaceus. The mPRL expression characteristics implied that mPRL regulates the reproduction of P. olivaceus through the endocrine system. Furthermore, the evidence supports a functional role of mPRL in the oocyte maturation of P. olivaceus, possibly, by acting as a mediator of progesterone. |
| Key words: Paralichthys olivaceus mPR-Like mRNA expression Protein expression |
