合并胰岛素抵抗的多囊卵巢综合征对小鼠及人卵母细胞内质网分布及功能的影响

Polycystic ovary syndrome(PCOS) is a common complex and heterogenous endocrine disorder with anovulation. It is generally recognized that insulin resistance(IR) is a key component in the pathogenesis of the disorder. Failure of the ER's adaptive capacity results in activation of the unfolded protein response (UPR), which intersects with chronic metabolic diseases such as obesity, insulin resistance, and type 2 diabetes. Our preliminary results showed that the diabetic condition induced the ER distribution defects during mouse oocyte maturation and early embryo development suggesting that PCOS IR might adversely affects the ER distribution pattern and ER stress in oocytes. We found that chronic dihydrotestosterone (DHT) exposure in mice reproduced features of both PCOS and IR. In this project, we will use time-lapse live cell imaging confocal microscopy to investigate the dynamic changes of ER and the effects of PCOS IR on the ER's structural dynamics during oocyte maturation in PCOS IR model mice. And then, we will examined the ability of the oocyte to release Ca2+ in response to fertilization or inositol 1,4,5-trisphosphate (IP3). The second, we will collect oocytes and granulosa cells to examine the expression of ER stress markers before and after treatment with two chemical chaperones, phenyl butyric acid or tauro-ursodeoxycholic acid in the PCOS IR model mouse oocytes . The end, we will compare the ER distribution and ER stress in human oocytes with or without PCOS IR. If our hypothesis could be confirmed, it would make an important complement to the mechanism of PCOS IR. At the same time, we will offer important data to investigate the clinical treatment of PCOS.

多囊卵巢综合征是高度异质的排卵障碍性疾病，研究发现胰岛素抵抗可能是其重要发病原因之一，代谢性疾病与内质网应激失调密切相关。本课题前期研究表明:糖尿病影响小鼠卵母细胞和早期胚胎内质网的动态分布，用双氢睾酮喂养的小鼠基本具有人类合并胰岛素抵抗的多囊卵巢综合征（PCOS IR）的特征，提示PCOS IR卵母细胞可能存在内质网分布异常及功能障碍。拟进一步：1）活细胞工作站分析PCOS IR小鼠卵母细胞内质网的分布特征及钙离子振荡。2）检测模型小鼠卵巢组织及生殖相关细胞GRP78,P-eIF2, p-JNK等表达，明确其内质网应激情况。3）应用苯基丁酸等改善内质网应激的药物，比较用药前后模型小鼠内质网应激状态及PCOS IR症状改善情况。4）比较正常人和PCOS IR患者生殖相关细胞内质网的分布及应激。若假说成立，它将对PCOS IR发病机理作出重要补充，同时也为探讨PCOS临床治疗提供一条新思路。

未经控制的糖尿病妇女常面临不孕，流产、子代畸形等生育问题。研究发现糖尿病小鼠卵母细胞GV泡破裂延迟，细胞内代谢异常，且卵母细胞减数分裂过程中易发生纺锤体组装及染色体排列异常，非整倍体率增高。糖尿病小鼠2细胞胚胎发育成囊胚速度减慢，甚至有50%左右的胚胎不能发育到8细胞阶段。然而，糖尿病对小鼠的卵母细胞及早期胚胎内质网分布影响未见报导。在卵母细胞成熟过程中，卵胞浆为了成功受精及胚胎发育做了大量的准备工作。其中钙离子释放是减数分裂成功和早期胚胎发育的关键步骤之一。内质网是由膜小管组成的网状多功能细胞器，与维持钙离子释放系统正常工作密切相关。在正常卵母细胞成熟过程中，内质网分布的动态变化与受精，钙离子峰及InsP3变化有关。. 按文献制作糖尿病模型雌性小鼠。我们用活细胞工作站动态观察糖尿病小鼠卵母细胞在体外成熟过程，发现内质网呈大片状聚集，且通常不能完成减数分裂，并在短期内退化。用激光共聚焦检测体内发育各发育时期的卵母细胞内质网的分布状况，发现：GV期卵内织网呈云雾状和团块状分布，MI及MI前期，大部分糖尿病小鼠卵母细胞内质网呈均质和块状分布。MII期的则在赤道面呈围核分布或小颗粒状均质分布，观察不到典型的皮集区内质网集聚的现象。形态学的异常通常会导致功能的异常。我们发现有19%糖尿病小鼠MII期卵母细胞形态异常。接下来，我们用活细胞工作站检测了糖尿病小鼠受精卵及早胚胎体外发育过程中内质网分布的变化。发现正常小鼠受精卵和早期胚胎内质网呈围核分布，随着胚胎发育，在2细胞胚胎中内质网在胞浆中呈大片状聚集。然而在糖尿病小鼠受精卵及早期胚胎中内质网基本呈匀质状分布。为了更清晰地了解内质网在早期胚胎中的分布规律，我们用激光共聚焦检测了内质网在皮质面及赤道面的分布。我们的研究发现正常小鼠合子期内质网在赤道面呈围核分布和糖尿小鼠相比，正常小鼠胞浆内质网呈大片状聚集。然而，在糖尿病小鼠中约80%1细胞胚胎，40%2细胞胚胎内质网均呈均质分布。糖尿病小鼠2细胞胚胎内质网呈巨大团块状聚集占30%，且大部分在较短时间内停止发育。. 我们的研究结果表明糖尿病小鼠卵母细胞和早期胚胎的内质网分布发生了改变，这可能与其生育缺陷相关，并对临床上糖尿病妇女卵母细胞及早期胚胎质量评估有一定的意义。