GDNF对人卵母细胞核成熟和胞浆成熟的旁分泌调节及机制研究

Traditionally, studies examining the control of oocyte maturation have focused mainly on endocrine regulation of the hypothalamic-pituitary-ovarian axis. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. Recent studies demonstrated that LH stimulated the production of ovarian factors, such as epidermal growth factor(EGF) like factors, insulin-like 3, brain-derived neurotrophic factor(BDNF), to promote oocyte nuclear and cytoplasmic maturation. We performed DNA microarray analysis of ovarian transcripts and identified glial cell line-derive neurotrophic factor (GDNF) as an ovarian factor stimulated by LH surge. Treatment of murine oocytes with GDNF enhanced first polar body extrusion and the GDNF action is likely mediated by its receptor GDNF family receptor alpha1(GFRα1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocyte. And the results of another study with porcine oocytes showed that GDNF promoted both oocyte nuclear and cytoplasmic maturation. In this study, we will investigate whether GDNF affect human oocyte nuclear and/or cytoplasmic maturation by in vitro maturation,in vitro fertilization ,embryo culture, quantitative RT-PCR,immunostaining and ELISA using immature human oocyte,follicular fluid, cumulus and granulosa cells. And we will further explore the mechanisms underlying GDNF promotion of oocyte maturation by animal experiments, including mouse oocyte IVM and siRNA mouse oocyte microinjection. Our hypothesis is that LH stimulates the production of GDNF, which promotes oocyte maturation through a two-compoent system, GFRα1/Ret or GFRα1/NCAM. MAPK, PI3-kinase or Fyn signaling may be involved in the process. This study underscores the importance of paracrine system for oocyte maturation and facilitates future formulation for the optimal culture conditions for in vitro maturation.

胶质细胞源性神经营养因子(GDNF)怎样通过旁分泌作用对哺乳动物卵母细胞成熟起调节作用这一科学问题尚未被阐明。我们在前期工作基础上，提出LH刺激颗粒细胞产生GDNF，与卵母细胞上的受体GFRα1结合，GDNF- GFRα1通过Ret或Fyn激活MAPK/PI3-kinase/Fyn等信号转导通路介导，促进卵母细胞核成熟和胞浆成熟这一科学假设。本项目拟以人卵母细胞和卵丘、颗粒细胞、卵泡液等为实验材料，采用人卵母细胞体外成熟、体外受精，胚胎培养，免疫荧光，定量PCR、ELISA等技术，揭示GDNF对人卵母细胞核成熟和胞浆成熟的调节作用。并采用小鼠卵母细胞体外成熟实验，siRNA小鼠卵母细胞内注射介导基因沉默等方法，阐明GDNF促进卵母细胞成熟的机制。本研究有助于阐明人卵母细胞成熟的旁分泌调节，更新过去认为卵母细胞的成熟主要受内分泌调节的观点，对设计合理的体外成熟培养液具有重大意义。

传统认为卵母细胞成熟主要受内分泌调节，但卵母细胞上几乎不表达黄体激素受体，且对直接的黄体激素也不敏感。因此，有报道认为黄体激素刺激颗粒细胞、卵丘细胞等产生旁分泌因子，促进了卵母细胞成熟。我们的前期研究发现在培养液中添加胶质细胞源性神经营养因子（glial cell-derived neurotrophic factor, GDNF）可促进小鼠卵母细胞第一极体的释放，且该作用可能是通过表达于小鼠卵母细胞上的GDNF的受体GDNF family receptor alpha1(GFRα1)和复合受体co-receptor ret proto-oncogene (Ret)介导的。本研究发现在培养液中添加GDNF能够促进生发泡期（germinal vesicle, GV）人卵母细胞成熟至MII期，但对生发泡破裂（germinal vesicle breakdown, GVBD）的发生无明显作用，证实GDNF能促进人卵母细胞核成熟。并且，我们发现GDNF促进人卵母细胞成熟的作用是通过miR-145-5p介导的，GDNF处理后的卵丘细胞中miR-145-5p水平显著下调。GFRα1和表皮生长因子受体（epidermal growth factor receptor，EGFR）是miR-145-5p的靶基因，且卵丘细胞转染miR-145-5p的inhibitor可显著上调GFRα1和EGFR的表达水平，提示GFRα1和EGFR也参与了GDNF诱导的卵母细胞成熟机制调控。