糖皮质激素通过其受体介导的PI3K/Akt及凋亡途径影响始基卵泡池的效应及机制探讨

Loss of finite primordial follicle pool with age is the primary cause of ovarian aging. The reduced primordial pool leads to infertility, early menopause and premature ovarian failure, which harm for women's health and life. Until now, the effects and mechanism of ovarian microenvironment such as the level of glucocorticoid on the assembly and activation of primordial follicle remained unclear. In the former study, we found that the number of primordial follicles in the neonatal mouse ovary exposed to dexamethasone declined significantly through over activation. Therefore, we confer that glucocorticoid may affect the primordial follicle pool through PI3K/Akt and apoptosis pathway. Therefore, our study was aim to explore the effects of glucocorticoid on primordial follicle pool through in vivo animal experiment and in vitro neonatal mouse ovary culture model; to study the expression and location of glucocorticoid receptor through confocal laserscanning microscope(CLSM); Furthermore, to explore the mechanism that glucocorticoid reduced primordial follicle pool through premature activation of the primordial follicle pool by PI3K/Akt pathway activation, and the apoptosis by Bcl-2/Bax ratio decline. Finally, fully understanding the mechanism of glucocorticoid on the effects on primordial follicle pool will allow for the prevent and intervention strategies of reproductive endocrinological diseases such as premature ovarian failure.

始基卵泡池的不断耗竭是导致卵巢衰老的根本原因。其规模的缩小可导致不孕不育、过早绝经及卵巢早衰，严重影响了女性的身体健康及家庭幸福。目前卵巢微环境如糖皮质激素水平的变化对始基卵泡池形成及激活的影响尚不明确，相关分子机制亦不明了。我们前期研究发现糖皮质激素可过早激活始基卵泡导致其规模的缩小。由此我们推测糖皮质激素可通过其受体介导的PI3K/ Akt及凋亡途径影响始基卵泡池的规模。因此，本项目欲采用体内动物实验及我们建立的体外新生小鼠卵巢培养模型，探寻糖皮质激素影响始基卵泡池的效应；利用激光共聚焦扫描显微镜研究新生小鼠卵巢中糖皮质激素受体的定位及核转移效应；继而探索糖皮质激素通过其受体介导的PI3K/Akt通路过早激活始基卵泡池及下调Bcl-2/Bax比例促进卵母细胞凋亡的途径影响始基卵泡池的机制。最终阐明糖皮质激素影响始基卵泡池的效应及相关机制，为预防及治疗卵巢早衰等生殖内分泌疾病提供新策略。

始基卵泡池的不断耗竭是导致卵巢衰老的根本原因。其规模的缩小可导致不孕不育、过早绝经及卵巢早衰，严重影响了女性的身体健康及家庭幸福。目前卵巢微环境如糖皮质激素水平的变化对始基卵泡池形成及激活的影响尚不明确，相关分子机制亦不明了。本项目通过体内小鼠动物实验发现糖皮质激素可通过影响生殖巢破裂及始基卵泡的聚集从而影响始基卵泡池的形成，在女性人群中发现高浓度糖皮质激素可导致始基卵泡池的加速耗竭，糖皮质激素可直接或间接影响排卵或黄体功能继而影响卵巢功能导致卵巢衰老；同时，构建了改良的可供体外研究始基卵泡池形成与激活的新生小鼠卵巢培养模型，将新生小鼠卵巢放置于0.4um的Millicell-CM滤膜上，使其漂浮于400ul的培养基中，在α-MEM、0.1% Albumax, 0.1% 牛血清白蛋白, 0.05 mg/ml L-抗坏血酸, 0.23 mmol/l 丙酮酸钠, 50 μg/ml 胰岛素, 27.5 μg/ml 转铁蛋白, 5 U/ml 青霉素, 5 µg/ml 链霉素的体系下生长状态最佳，接近体内的卵巢生长变化，为卵巢相关实验的开展提供良好的实验平台与基础；明确了新生小鼠卵巢中糖皮质激素受体的定位，在颗粒细胞及卵泡膜细胞中发现GR mRNA表达并无明显异常，但是在蛋白水平有差异。并发现EGR1可通过NF-kB的途径导致颗粒细胞凋亡，上调EGR1可上调Caspase-3和9等促凋亡蛋白，而下调BCL2等抗凋亡蛋白，而这些蛋白均与线粒体途径相关，而死亡受体途径的caspase-8却无明显差异，因此，上调的EGR1可通过线粒体途径促颗粒细胞凋亡，并最终导致卵泡闭锁，从而消耗始基卵泡池而导致了卵巢衰老；本项目阐明了糖皮质激素影响始基卵泡池的效应及相关机制，为预防及治疗卵巢早衰等生殖内分泌疾病提供了新策略。