HDAC3在LH诱导卵母细胞成熟中的作用机制研究

The significance of epigenetic modification has been proved to be pivotal for gamete genesis and early embryo development, however, it is rarely reported regarding to the influence of the epigenetic modifications on oocyte maturation through its functioning in ovarian somatic cells. LH is known to promote oocyte maturation through up regulating EGF like factors, such as AREG in granulosa cells. Our pre-research has implied that LH surge down regulates the level of histone deacetylase 3 (HDAC3) in granulosa cells, which in turn releases the inhibitory factors that preventing the expression of molecules like AREG that are vital for oocyte maturation. Based on these findings, we plan to work out the detailed mechanisms of LH induced HDAC3 down regulation in mice by following assays based on conditional Hdac3 knockout mice and follicle in vitro maturation (IVM) models: (1) Which signal cascade in granulosa cells is activated by LH receptor in down regulating Hdac3 expression? (2) How does HDAC3 been degraded in response to LH surge? (3) Is there any relationship between down regulation of HDAC3 and luteinization of granulosa cells? (4) Does inhibiting HDAC3 contribute to improve the developmental potential of oocyte maturated in vitro? The findings will not only contribute to better understanding the mechanism of oocyte maturation, but also help to explore the possibility of improving oocyte maturation through modulating the epigenetic modification of granulosa cells.

表观遗传修饰对配子发生和早期胚胎发育至关重要，但其是否也通过调节卵泡体细胞的功能影响卵母细胞成熟及其质量鲜有报道。促黄体素（LH）可通过上调颗粒细胞中表皮样生长因子（AREG等）的表达促进卵母细胞成熟。我们的前期研究首次发现LH可能通过其受体下调颗粒细胞内组蛋白去乙酰化酶3（HDAC3）的水平，进而解除细胞对AREG等分子表达的抑制实现卵母细胞成熟。本项目在前期基础上，拟利用Hdac3条件性敲除鼠和卵泡体外成熟培养（IVM）模型，研究（1）LH受体通过哪条信号级联下调了细胞内HDAC3的表达；（2）LH诱导条件下细胞内HDAC3蛋白的降解途径及机制；（3）颗粒细胞内HDAC3水平的下调是否有助于促进其黄体化；（4）抑制HDAC3能否提高IVM体系中培养的卵母细胞的发育潜能。研究成果不仅将为深入揭示卵母细胞成熟的分子机制做出贡献，更有助于探索通过表观遗传调控实现促卵泡卵母细胞成熟的目标。

雌性哺乳动物能在性成熟后周期性地排出健康成熟的卵母细胞是其繁衍所必需的。已有研究证明，EGF样生长因子（如AREG等）是颗粒细胞响应LH作用而产生的，对促进卵丘细胞扩展和卵母细胞减数分裂恢复有重要作用的关键分子，但其具体产生机制目前不详。本课题利用双向电泳对小鼠卵巢中LH特异性响应蛋白进行了筛查，结果发现组蛋白去乙酰化酶3（HDAC3）被LH显著下调。流调结果也显示，多囊卵巢综合症患者卵巢颗粒细胞中HDAC3也呈高水平表达，且其卵发育能力下降也与HDAC3高水平显著正相关，故怀疑HDAC3可能是颗粒细胞中响应LH诱导效应的关键分子之一。我们的卵泡体外培养模型研究结果表明，与广谱性HDAC抑制剂相比，添加HDAC3特异性抑制剂 （HDACi 4b）可通过诱导AREG的表达进而促进卵母细胞成熟。ChIP等实验发现，HDAC3可直接结合于AREG启动子（-343到-150）区，通过改变该区域组蛋白H3K14的乙酰化修饰，改变转录因子（如SP1）对DNA的结合能力进而调控基因转录。进一步地，利用壁层颗粒细胞为饲养层与小鼠卵丘卵母细胞复合体 (COCs) 共培养发现，HDACi 4b处理的颗粒细胞可显著促进COCs中卵母细胞的成熟及发育能力。体外受精及早期胚胎体外培养证明，经HDACi 4b诱导成熟的卵母细胞的囊胚发育能力及其质量均显著高于对照组，胚胎移植后可得到健康子代。临床前研究发现，在人颗粒细胞中HDAC3也同样通过调控组蛋白H3K14的乙酰化水平及转录因子SP1与DNA的结合，调控AREG表达。综上，本研究发现在生理情况下，LH通过下调颗粒细胞内HDAC3的表达，上调其染色质组蛋白H3K14的乙酰化修饰，进而活化了染色质结构，促进了SP1等转录因子与AREG启动子区域的结合，进而起始其表达，最终诱导卵母细胞成熟。在体外，HDAC3抑制剂可模拟LH的效果，有助于提高卵的体外成熟及发育潜能，故有望用于对LH响应性不足患者卵母细胞的体外成熟治疗。本研究为理解促性腺激素诱导卵母细胞成熟的分子机制提供了新思路，也为促进临床卵母细胞体外成熟技术的提高提供了新方法。