AMH-INH-GnIH共表达质粒转染和免疫对牛卵泡与胚胎发育及性比率的调控研究

AMH (anti-Mullerian hormone) and INH (inhibin) are secreted by ovaries and are able to inhibit the development of both small and large follicles respectively. GnIH (gonadotrophin inhibiting hormone) is secreted by hypothalamus and is also able to inhibit folliculogenesis via inhibition of secretion of follicle stimulating hormone at pituitary. Moreover, AMH acts on sex-differentiation by inhibition of the development of Müllerian duct. Results in our previous researches indicated that genetic immunizations against both INH and GnIH eukaryotic coexpression plasmid (pIG, each gene was previously fused with hepatitis B surface antigen gene) promoted folliculogenesis and ovulation as well as litter size, which were greater than those obtained by immunization against INH eukaryotic expression plasmid fused with hepatitis B surface antigen gene (pI). Four novel coexpression plasmids, namely pA, pAI, pAG, pAIG are constructed by two A peptide methods on the basis of previously constructed fusion expression plasmids of pI and a coexpresion plasmid of pIG in this research. Six plasmids are used for both transfection of grannular cells from bovine ovaries in different development stages and immunization of mice. Results of both in vivo and in vitro, in both competitive inhibition or overexpression and immunonetralization, are harvested to study the effects and mechanism of 3 kinds hormones and their combinations on folliculogenesis, ovulation, embryo development and sex ratio in genetic immunization, competitive inhibition or overexpression. These fundamental results will be useful to develop new techniques such as induction of estrus ovulation, superovulation and twinning by hormone genetic immunization to improve animal fertility.

卵巢分泌的AMH（抗苗勒管素）和INH（抑制素）分别对小卵泡和大卵泡发育有抑制作用，下丘脑分泌的GnIH（促性腺激素释放激素）通过抑制垂体促性腺激素的分泌而抑制卵泡发育。此外，AMH还可抑制苗勒管发育，影响性别发生和发育。我们前期研究证明，用融合乙肝表面抗原的INH-GnIH共表达质粒免疫动物，可促进卵泡发育，增加排卵率和产仔数，且其效果比单用INH融合表达质粒好。本项目在现有2种真核表达质粒基础上，采用自剪切2A 肽策略，构建4种新型表达质粒、形成6种质粒组合，分别用于转染不同时期的牛卵泡颗粒细胞和免疫小鼠，从体内外、过表达或竞争性抑制和免疫中和内源性激素等方面，分析3种激素及其两两组合与联合对卵泡发育、排卵、胚胎发育与性别比率的影响，阐明3种激素在卵泡发育、排卵及胚胎发育和性别发生调控中的作用及其机制，为申请转基因生物安全评定、开发发情排卵调控和人工孪生的激素基因免疫新技术奠定基础。

本项目采用分子生物学技术并结合2A肽策略，成功构建并鉴定了4种新型真核表达质粒。分别将荷载单表达、双表达和三表达质粒的减毒沙门氏菌免疫小鼠后，发现p-AMH、p-INH、p-RFRP、AMH-INH-2A、AMH-RFRP-2A、INH-RFRP-2A、AMH-INH-RFRP-2A组均分别产生了较高的抗体水平，其中AMH-RFRP-2A、INH-RFRP-2A、AMH-INH-RFRP-2A组的产仔数均显著高于对照组。结果还发现：含AMH组母：公比例高于不含AMH组，但差异不显著；AMH-INH-2A 组以及 AMH-INH-RFRP-2A 组雌激素和孕酮水平显著高于其他实验组以及对照组。安全性评估结果显示：整体实验组和对照组的体重差异不显著；3周仔鼠整体实验组和对照组脏器指数正常、石蜡切片无异常，显示所构建疫苗对于母鼠和仔鼠是安全的。此外，研究还发现AMH处理组的次级卵泡数（64.67±4.58）显著高于对照组（52±2.65）。项目的实施揭示了AMH-GnIH-INH三种负向调控激素间的关系，有助于提高动物繁殖力。