NO介导的ERs-nNOS-GnRH通路对GnRH合成释放的影响机理

The main cause of reproductive disturbance in replacement gilts were generally known as sexual development lagged behind body development before their puberty. We have found that diet supplemented with N-carbamylglutamate (NCG) in replacement gilts could improve reproductive organ development, bring puberty and age suitanble for the first mating ahead, increase level of Arg, NO and E2 in the circulation systems and up-regulate the expression of puberty onset related genes in hypothalamus including Kiss-1, nNOS, ERα, etc. A hypothesis was put forward to explain why NCG can improve sexual development in replacement gilts. NO, one of the metabolites of Arg, could media the information of E2 to regulate GnRH synthesis and release through ERs-nNOS-GnRH pathway to improve sexual development. GT1-7 cell line, an immortalized GnRH neuron cells isloated from the hypothalamus of transgenic mice, was used to build a model of GnRH positively and negatively regulated by E2 in vitro, firstly. Then, NO donor and/or antagonist, as well as nNOS antagonist were added to the culture media of GT1-7 cell to evulate the effet of NO on GnRH synthesis and release. Moreover, qPCR, western blot, immunofluorescence were used to detect the mRNA and protein expression of genes in the hypothetic pathway, as well as interaction between proteins related with this pathway. All our researches were conducted to reveal the mechanism of NO regulation GnRH activity and to lay a foundation for NCG application on sexual development of young female livestock.

性发育落后于身体发育是造成后备母猪繁殖障碍的主要原因。我们研究发现，在后备母猪日粮中添加精氨酸内源激活剂—N-氨甲酰谷氨酸（NCG），可促进生殖器官发育，提前初情期和初配适龄，显著提高血液中Arg、NO和E2的浓度，并上调下丘脑初情期启动相关基因Kiss-1、nNOS、ERα等的表达。推测NCG是通过NO（Arg代谢产物之一）发挥作用，即NO介导E2通过ERs-nNOS-GnRH通路调控GnRH的合成释放进而促进动物性发育。本研究拟使用小鼠GnRH永生的细胞系GT1-7细胞，首选体外构建E2正负调控GnRH模型，进而通过添加NO供体/拮抗剂、nNOS拮抗剂研究NO对GnRH合成释放的影响，并利用qPCR、Western blot、免疫荧光等技术研究不同处理条件下该通路中相关基因、蛋白的表达及其相互作用，揭示NO调控GnRH合成释放的机理，为NCG在促进幼龄雌性家畜发育中的应用奠定基础。

本项目针对规模化养猪生产中后备母猪性发育滞后和繁殖障碍频发的生产实践，基于NCG添加能促进后备母猪性发育的研究结果，开展NCG影响SD大鼠性发育和产仔数的研究，并利用小鼠下丘脑GnRH神经元GT1-7细胞系建立体外动物性发育细胞模型，评价E2、NCG、kisspeptin-10（K10）等单独或组合对GT1-7细胞增殖、GnRH分泌及初情期相关基因表达的影响，验证NCG通过一氧化氮（NO）介导的ERs-nNOS-kisspeptin-GnRH通路影响GnRH分泌。结果表明：65日龄开始添加NCG（0.05%）可提前大鼠初情期3.5 d以上，可显著提高第一胎产（活）仔数，但对第二胎繁殖生产性能无显著影响，进一步证实了NCG促进性发育的效果；建立了体外E2促进和抑制GT1-7细胞GnRH分泌的模型（100 pM处理12 h促进和1 µM处理24 h抑制GnRH分泌，1 nM雌激素受体抑制剂能抑制E2对GnRH的刺激作用）；NCG（1 mM）处理12 h能显著促进GT1-7细胞增殖，并通过下调nNOS和GnRH基因表达抑制GnRH分泌；K10（100 pM）和NCG（1 mM）组合培养对GT1-7细胞增殖具有协同促进作用，K-10单独处理可促进GnRH分泌，组合处理后K10可补偿NCG对GnRH分泌的抑制，即在一定浓度K10存在的条件下，NCG能够呈剂量依赖性促进GnRH分泌。本研究初步揭示了NCG体内外促进GnRH分泌和性发育的分子机制。另外，NCG（1 mM）对能够调控猪小卵泡（1-5 mm）和大卵泡（>5 mm）颗粒细胞体外增殖和类固醇激素合成，其中大卵泡颗粒细胞对NCG更为敏感，表现在NCG能促进大卵泡颗粒细胞增殖并抑制P4和E2合成，NCG可抑制小卵泡颗粒细胞P4合成，但对E2合成和细胞增殖无显著影响。提示NCG直接影响猪的卵泡发育，其分子机理有待进一步研究。