松果体素激活自噬促进人卵巢颗粒细胞存活的分子机制研究

Previous studies indicated that granulosa cells from unfertilized oocytes of infertility patients showed a significantly higher incidence of apoptosis compared to those of fertilized oocytes, and embryo quality had a negative correlation with the incidence of granulosa cells apoptosis. The results of the animal experiment showed that melatonin suppresses apoptosis of granulosa cells via its receptors (MT1 and MT2). Induction of autophagy improves germ cells survival in the female murine, and facilitates embryos development. Our preliminary experimental results indicated that hamster ovarian cells autophagy can be induced by melatonin. Therefore，we speculate that “cultured human ovarian granulosa cell synthesize a certain amount of melatonin, and exogenous melatonin induces human granulosa cell autophagy through mTOR signaling pathways, inhibits human granulosa cell apoptosis, after binding to its receptor, and ultimately improves human granulosa cell survival, facilitates oocyte quality, and contributes to embryo development. ” To verify this hypothesis, firstly, at the cellular level, we investigate how melatonin prevents human granulosa cell apoptosis and ultimately promotes human granulosa cell survival by activating cell autophagy using primary cell culture、Western Blotting and Flow Cytometry. Additionally, at the molecular level, we explore how melatonin induces human granulosa cell autophagy through mTOR signaling pathway mediated by its receptor using SiRNA、Real-time PCR and Western Blotting. Lastly, we verify that melatonin improves human ovarian granulosa cell survival by activating cell autophagy using transgenic mouse model. This project aims to provide theoretical supports for clinical infertility application of melatonin and improvement of assisted reproductive technology (ART) outcome.

不孕症患者未受精卵子的颗粒细胞凋亡较受精卵子明显增加,胚胎质量与颗粒细胞凋亡率成负相关。动物实验结果表明，松果体素可通过膜受体抑制卵巢颗粒细胞凋亡；诱导自噬能促进小鼠生殖细胞存活，促进胚胎发育。本课题组预实验结果表明，松果体素可激活仓鼠卵巢细胞自噬。我们推测：松果体素与其受体结合后通过mTOR信号通路激活人颗粒细胞自噬，抑制其凋亡，促进其存活和继续发育，改善卵子质量、利于胚胎发育。为验证此假说，本课题①采用原代细胞培养、免疫印迹和流式细胞术等在细胞水平明确松果体素可通过激活自噬抑制颗粒细胞凋亡、促进其存活；②采用RNA干扰（SiRNA）、实时定量-PCR和免疫印迹等技术在分子水平研究松果体素作用的机制─在其受体介导下通过mTOR信号通路激活人颗粒细胞自噬；③利用转基因鼠模型验证松果体素通过激活自噬促进颗粒细胞存活、发育的机制，为将松果体素应用于临床、改善辅助生殖技术治疗结局提供理论依据。

不孕症患者未受精卵子的颗粒细胞凋亡较受精卵子明显增加,胚胎质量与颗粒细胞凋亡率成负相关。动物实验结果表明，松果体素可通过膜受体抑制卵巢颗粒细胞凋亡。本项目主要系统研究松果体素通过调控自噬，抑制颗粒细胞凋亡，促进其进一步发育，从而达到改善卵母细胞和胚胎质量的目的，并探讨可能的分子机制，为松果体素应用于临床疾病的防治策略提供新的依据。首先，我们发现培养的人卵巢颗粒细胞在体外能够合成松果体素，并表达松果体素合成的前体物质（5-羟色胺）和松果体素合成的两种关键酶（NAT和ASMT)。并且，松果体素促进了未成熟的人类卵母细胞的体外成熟，维持了人类卵母细胞的线粒体膜电位，并降低了过量的Ca2+水平。其次，我们构建了稳定表达EGFP-LC3的中国仓鼠卵巢细胞损伤模型，并利用该模型系统地研究了松果体素对卵巢细胞线粒体和卵巢储备功能的保护作用和分子机制。结果显示，10-9M松果体素不仅可以防止过度自噬诱导的抗苗勒氏管激素(AMH)表达的下降，而且可以挽救过度自噬诱导的线粒体表达和线粒体膜电位的损伤。此外，松果体素可以防止过度自噬诱导的核编码蛋白(SDHA和mitofilin)，以及线粒体动力相关蛋白（OPA1、MFN2和DRP1）表达的减少。松果体素还降低了线粒体氧化应激，增加了抗氧化酶超氧化物歧化酶2(SOD2)的表达，改善了过度自噬诱导的G2/M细胞周期阻滞。最后，松果体素抑制了过度自噬诱导的细胞外信号调节激酶(ERK)信号通路的激活。综上所述，松果体素能合成系统表达在体外培养的人卵巢颗粒细胞中，松果体素可能通过改善线粒体功能来降低人卵母细胞的氧化应激；松果体素通过下调ERK通路挽救了过度自噬诱导的线粒体和卵巢储备功能的损伤，以及线粒体ROS的产生和细胞周期阻滞。上述研究结果提示了使用松果体素改善线粒体功能、提高人类卵母细胞质量和卵巢颗粒细胞存活能力的潜在治疗策略。