GnIH对母猪生殖调控的研究

Regulation of GnIH on Reproduction of Female Pig

【目的】研究促性腺激素抑制激素(GnIH)mRNA在母猪组织器官中的分布,不同剂量GnIH对母猪卵巢类固醇激素合成和细胞增殖相关蛋白表达的影响,及GPR147与GnRH的形态学关系。从分子、细胞水平和形态学角度,探讨GnIH在下丘脑-垂体-性腺轴对母猪生殖调控的作用机制,丰富神经内分泌学的内容。【方法】以30日龄健康仔母猪为研究对象,采用半定量RT-PCR方法研究GnIH mRNA在母猪24个组织中的分布定位。采集青年母猪卵巢样品,分离卵巢颗粒细胞,研究不同剂量GnIH(10-6、10-8、10-10和10-12 mol·L-1)对体外培养的母猪卵巢颗粒细胞类固醇激素(雌二醇,孕酮)的合成、细胞增殖相关蛋白(cyclin B1,PCNA)表达的影响。取30日龄健康仔母猪下丘脑,制作脑片,采用荧光双标记,激光共聚焦显微镜观察拍照分析GPR147与GnRH的形态学关系。【结果】GnIH mRNA主要在母猪的中枢神经系统中表达,尤其是在间脑表达含量最高。在外周组织,如子宫、卵巢、垂体中也有一定量表达。不同剂量的GnIH作用于体外培养的卵巢颗粒细胞,其中10-6和10-10 mol·L-1 GnIH能极显著地抑制雌二醇的分泌(P<0.01),但不同剂量的GnIH对孕酮分泌的影响并不显著。GnIH能极显著地抑制卵巢增殖相关蛋白PCNA和cyclin B1的表达(P<0.01)。此外,激光共聚焦结果显示GPR147与GnRH在下丘脑室旁核及视前区存在密切联系。【结论】GnIH可在下丘脑水平通过其受体直接作用于GnRH神经元,影响GnRH的合成和分泌,进而在中枢水平调控母猪的生殖活动。在外周生殖器官中,GnIH可通过影响卵巢激素的分泌和细胞增殖相关蛋白的表达参与母猪的生殖调控。

[ Objective ] The expression of GnlH mRNA in female pig, the effect of GnlH on steroid hormone secretion and cell proliferation proteins expression in granulosa cells cultured in vitro, and morphological relationship between GPR147 and GnRH were investigated in this study. The purpose of all the studies was to explore the regulatory mechanism of GnlH to pig reproduction in vitro, at molecular and cell levels, and from cell morphology images. [Method] Twenty-four tissue and organ samples were collected from healthy female piglets aged 30 d, expression levels of GnlH mRNA were detected by semi-quantitative RT-PCR. The effect of GnlH on steroid hormone secretion and cell proliferation proteins expression in granulosa cells cultured in vitro was analyzed by the method of radioimmtmoassay and Western blotting. Hypothalamus tissues were also collected from healthy female piglets aged 30 d and used for preparation of histological sections. The morphological relationship between GnlH receptor and GnRH was revealed by immunohistochemistry and examined with confocal microscopy. [ Result] The result showed that GnlH mRNA mainly expressed in the central nervous system, especially in the diencephalon. After in vitro cultured, the granulosa cells were treated with 10-6, 10-8, 10-10 and 10-12 mol·L-1 GnlH. Compared with the control cells, 106and 10-10 mol·L-l doses of GnlHsignificantly reduced （P〈0.01） E2 secretion. All concentrations of GnlH could attenuate P4 secretion. GnlH significantly inhibited （P〈0.05） PCNA and cyclin B1 expression in cultured granulosa cells. Moreover, immunohistochemistry analysis revealed the close contact between immunoreactive fibers or cell bodies of GPR147 and GnRH in pig hypothalamus tissue. [ Conclusion ] These results demonstrated that GnlH not only plays a central regulatory role on GnRH secretion in the hypothalamus, but also peripherally regulates pig reproduction by affecting steroid hormone secretion and cell proliferation proteins expression in the ovary.