肉羊卵母细胞超低温冷冻保存及影响其冻后发育能力的机理研究

The cryopreservation of mammalian oocytes increasingly displayed its huge potential of application in animal resource protection. Although various of protocols for oocyte freezing have been developed, most of them were applied in mouse, cattle and human oocyte cryopreservation successfully. To date, cryopreservation of ovine oocytes has not yet made any breakthough. Sutiable protocols used in cryopreservation of ovine oocytes were quite limited. Therefor, it became urgent to establish a high efficient cryopreservation platform for ovine oocytes. In addition, study on the mechanisms of cryotolerance against cryopreservation of ovine oocytes was also restricted. In the present study, ovine germinal vesicle (GV) stage and metaphase II (MII) stage oocytes were vitrified with open pulled straw (OPS) method in order to investigate the effect of cryopreservation on their post-warming survival and developmental competence. On the basis of the optimization of freezing procedure, the ultrastructure, cytoskeletal structure and membrance proteins of ovine oocyte were investigated systematically so as to enhance the understanding of mechanisms on influencing viability of vitrified ovine oocytes, to furnish a refernce for ovine oocytes cryopreservation, and to estabilish a basis for further improvement of cryopreservation of ovine oocytes.

哺乳动物卵母细胞冷冻保存，在动物种质资源保护等方面已日益显示强大的应用潜力。诚然，关于卵母细胞的冷冻保存，也已形成众多的技术方案，这些方案大多在小鼠、牛和人卵母细胞上应用并获得良好的冷冻效果。迄今为止，肉羊卵母细胞的冷冻保存尚未取得突破性进展。因此，建立有效的肉羊卵母细胞冷冻保存技术平台依然是当前最为迫切的研究任务。此外，有关肉羊卵母细胞冷冻耐受性的机理研究仍然偏少。本研究采用OPS(open pulled straw)法对肉羊未成熟(GⅤ期)和体外成熟(MⅡ期)卵母细胞进行玻璃化冷冻保存，研究其冻后存活力和发育能力所受的影响；在优化冷冻程序的基础上，对肉羊卵母细胞的超微结构、细胞骨架结构和膜蛋白变化进行系统研究，以期加深对影响肉羊卵母细胞冻后发育能力的机理认识，为建立适合于肉羊卵母细胞冷冻保存的技术方案提供参考，为进一步完善肉羊卵母细胞的冷冻保存技术奠定基础。

本课题采用免疫荧光染色技术检测了不同发育期绵羊卵母细胞冻融前后细胞骨架的变化，CD81卵巢组织、睾丸和附睾组织、卵母细胞及精子上的分布，结果显示，冷冻后 GV 期和 MII 期卵母细胞的形态正常率显著低于冷冻前，在卵巢组织、卵母细胞、睾丸和附睾组织上均有表达，强表达于精子的顶体部，其余部位均未检测到CD81蛋白荧光信号；采用RT-PCR技术，检测到CD81基因mRNA水平表达量新鲜MII期卵母细胞显著高于冷冻MII、新鲜GV和冷冻GV(P<0.01)，冷冻MII显著高于新鲜GV和冷冻GV(P<0.01)，新鲜GV与冷冻GV差异不显著(P>0.05)；经IVF试验，检测到CD81单克隆抗体和超低温冷冻降低每卵黏附精子数和精子穿透率。 采用RT-PCR、免疫印迹、免疫荧光法检测GPx5基因在不同不同发育期绵羊附睾不同部位上的mRNA和蛋白水平表达及分布。结果显示，GPx5基因mRNA在绵羊不同发育期附睾中均表达，但在不同部位表达却有差异；GPx5蛋白在附睾管假复层上皮细胞内表达，但2月龄和5岁羊附睾尾部不表达。用ELISA法、RT-PCR法和免疫印迹法研究雄激素对GPx5的影响，结果表明，GPx5受雄激素调控；采用机械剪刀结合酶消化法。成功建立了绵羊附睾上皮细胞系；成功构建靶向绵羊GPx5基因shRNA重组干扰腺病毒，感染绵羊体外培养的附睾上皮细胞，能够有效抑制GPx5在附睾上皮细胞中mRNA水平上的表达。并检测到SOD活力及GSH和MDA含量随着GPx5的降低而显著升高（P<0.01），因此GPx5基因与抗氧化机制有关。