ClpP介导线粒体功能对卵母细胞发育和成熟的作用及其调控机制

Low pregnancy rate and high miscarriage rate led from oocyte development and maturation disorder is still an unsolved problem in reproductive medicine. One of the important reason is dysfunction of mitochondria. Caseinolytic protease (ClpP) is an energy-dependent serine protease and plays a vital role in the protein quality control and functional regulation within mitochondria. Our previous study found ClpP decreased in both gene and protein level, accompanied with mitochondria morphological and distributional abnormality in oocytes of aging mice which have lower viability in vitro growth. Meanwhile, we confirmed that Clpp null mice had complete female infertility. According to above evidences we hypothesis that ClpP regulate oocyte development and maturation mediated by mitochondria. In the present study, we will mainly study the effect of ClpP in development and maturation of oocytes by regulating mitochondria function and to verify its mechanism in Clpp null mice by using follicle in vitro 3D culture system, oocyte/follicle intracytoplasmic mRNA injection technique and other technique in molecular biology. The present study will illuminate the molecular mechanism from the basic research point of view firstly, and moreover provides a theoretical instruction and a new strategy for the application of assisted reproductive techniques in oocyte development and maturation disorder.

卵母细胞发育和成熟障碍导致的低妊娠率和高流产率依然是生殖医学领域的重大难题。线粒体功能异常是卵母细胞发育和成熟障碍的重要原因之一。酪蛋白裂解酶ClpP是ATP依赖的蛋白水解酶，在线粒体蛋白质量控制和功能调节中起到非常重要的作用。既往研究表明Clpp基因敲除雌性小鼠生育能力丧失；本研究组前期工作发现高龄小鼠卵母细胞体外发育潜能下降，ClpP表达降低，伴随线粒体形态和分布异常；这强烈提示ClpP可能通过介导线粒体功能对卵母细胞发育成熟产生影响。本研究拟采用已建立的Clpp基因敲除小鼠模型，结合卵泡及卵母细胞mRNA胞浆注射和卵泡体外3D培养等关键技术，对线粒体功能变化进行研究，系统的阐述ClpP在卵母细胞发育和成熟过程中的作用，明确其通过介导线粒体功能调节卵母细胞发育和成熟的机制，从而进一步了解生殖生理,为线粒体功能异常导致的卵母细胞发育和成熟障碍的辅助生殖治疗提供科学依据。

项目基本完成预期研究目标。(1)明确了线粒体应激反应基因Clpp导致雌性不孕的原因是卵母细胞成熟障碍。研究者建立了Clpp敲除的雌性小鼠动物模型，通过体内实验和体外实验，均发现成熟卵母细胞数量显著降低；并且体内实验还发现，2细胞胚胎胚胎数量显著减少，无囊胚形成。（2）明确了Clpp导致卵母细胞成熟障碍的原因是线粒体功能异常，包括氧化应激水平增加，线粒体膜电位降低，ATP形成减少，线粒体动态受到干扰，线粒体DNA拷贝数异常增加等。（3）发现在衰老过程中，Clpp缺失可导致卵巢中早期卵泡数加速丢失，卵巢加速老化。（4）发现Clpp导致卵母细胞成熟障碍的机制之一是mTOR信号通路过度激活。在完成上述既定研究目标的同时，研究者进一步积极探索通过调节mTOR信号通路，改善卵巢老化的方法。目前研究表明，通过体内或体外使用mTOR通路抑制剂---雷帕霉素，可以改善Clpp敲除小鼠的卵母细胞成熟率，为临床卵巢功能减退的不孕症患者，尤其是由于线粒体功能异常导致的卵巢老化患者的生育力改善提供了新的治疗思路和理论依据。围绕上述工作的重要进展，本项目基金支持已发表如下文章：1）Aging Cell, 2018, May. 17(4): 0~e12784； 2) American Journal of Reproductive Immunology, 2017.May, 77(5): e12651；3）Fertility and Sterility, 2018, 110(7): 1387~1397；4）Human Reproduction. 2016, 31(4):763-773；5）Maturitas. 2016 Nov; 93:121-130.