蛋白二硫键异构酶A3（PDIA3）参与卵母细胞成熟的机制研究

Oocyte maturation is a crucial process in ovarian follicle development and essential for further fertilization. Multiple proteins expressed differentially during this process are essential for oocyte maturation, fertilization and even embryonic development. As known, protein disulfide isomerases, considered as a chaperone that cooperated with numerous proteins, participate in lots of cell activities and are important for cell function. In our previous study, we found that protein disulfide isomerase A3 (PDIA3), localized in both membrane and cytoplasm of the oocyte, was increased expression during oocyte maturation. Moreover, reduce the expression of PDIA3 in oocyte by RNA interference will lead to the disturbance in oocyte maturation and display abnormal cell division and blocked mitochondria transportation. Notably, PDIA3, also named endoplasmic reticulum protein 57 (ERp57) or glucose regulated protein 58 (GRP58), was of assistance to protein folding and remodeling as well as the protein complexes assembling. Therefore, we presumed that PDIA3 might play multiple roles in oocyte maturation. A preliminary evidence also indicated that PDIA3 interacted with a cytoskeletal protein vimentin, which mainly involve in the maintenance of cell shape, cell division and organelles transportation. Based on these experiments, we supposed that PDIA3 in cytoplasm might participate in the maintenance of cell sharp, cell division and organelles transportation during oocyte maturation, depending on its regulation of disulfide formation as well as vimentin distribution. Thus, in this study, we will assess the effects of PDIA3 on oocyte maturation using the Pdia3 conditional knockout animal model, and then, improve the mechanism that PDIA3 effects on oocyte maturation. In conclusion, this applying project is focus on the function of PDIA3 in oocyte maturation and will further optimize the regulatory mechanisms in oocyte maturation. It will also contribute to improving the treatment of abnormal oocyte maturation induced by the dysfunction of these homologous proteins.

卵母细胞成熟过程中蛋白特异的时空表达为卵母细胞成熟和后续受精提供了重要保障，探索这些蛋白的作用机制有助于阐明卵母细胞成熟的奥秘，为辅助生殖技术改进提供理论依据。二硫键异构酶作为分子伴侣调节蛋白折叠及蛋白复合物的组装，参与了众多细胞活动。前期研究中我们发现二硫键异构酶PDIA3在卵母细胞成熟中表达升高，且抑制PDIA3表达可以阻碍卵母细胞成熟，并导致细胞异常分裂。进一步研究显示PDIA3还与细胞骨架蛋白vimentin相互作用，可能与维持细胞形态、细胞分裂及线粒体迁移有关。因此，本课题将通过条件基因敲除实验明确PDIA3在卵母细胞成熟中的作用，通过卵母细胞体外成熟、蛋白二硫键检测、化合物干预、蛋白组学分析等方法探索PDIA3通过调节蛋白二硫键形成、协同vimentin作用，进而参与卵母细胞成熟的作用机制。为了解卵母细胞成熟的调控机制，改进临床上卵母细胞成熟障碍的体外培养方法提供理论依据。

全球范围内，不孕不育率的急剧上升严重影响个体健康、家庭幸福和社会稳定。其中，卵巢因素造成的卵泡发育不良及卵子（卵母细胞）成熟异常是造成女性不孕的重要因素。卵母细胞成熟过程中蛋白特异的时空表达为卵母细胞成熟和后续受精提供了重要保障，探索这些蛋白的作用机制有助于阐明卵母细胞成熟的奥秘，为辅助生殖技术改进提供理论依据。二硫键异构酶具有独特的内质网表达特征，同时作为分子伴侣具有调节蛋白折叠及蛋白复合物的组装的功能，参与了众多细胞活动。其表达异常与细胞内质网应激密切相关。本课题通过比较卵母细胞成熟进程中二硫键异构酶PDIA3（protein disulfide isomerase A3）蛋白表达、体外实验和体内实验RNA干扰、卵母细胞特异的Pdia3基因敲除、蛋白相互作用分析等实验，探索了PDIA3在卵母细胞成熟中的作用。研究结果发现：PDIA3在卵母细胞成熟中表达升高，其分布类似细胞骨架样结构；通过RNAi技术抑制卵母细胞PDIA3表达可阻碍卵母细胞成熟，并导致细胞异常分裂；进一步，通过条件性基因敲除技术敲除卵母细胞Pdia3可造成雌性小鼠生育力下降、促排卵数量减少及卵细胞内质网应激升高；蛋白相互作用分析显示PDIA3可与细胞骨架蛋白Vimentin、透明带糖蛋白ZP1-3相互作用，可能与维持细胞形态、细胞分裂、线粒体迁移以及精子透明带结合等功能有关。本课题为全面了解卵母细胞成熟的调控机制，阐明PDIA3的生物学功能和以及卵母细胞成熟中蛋白二硫键形成的生物学意义奠定必要的基础。同时，也为改进临床上卵母细胞成熟障碍的体外培养方法提供必要的理论依据。此外，本课题构建的Pdia3条件性基因敲除小鼠模型结合不同的cre工具鼠，为后续研究PDIA3在子宫内膜容受性和附睾精子成熟中的功能指明了研究方向并奠定了研究基础。