Synaptotagmin1在小鼠受精过程中的作用和机制探讨

Synaptotagmin1 (Syt1) enrichs in the membrane of nerve and secretory vesicles, and bond to intracellular calcium under exogenous stimulation to induce exocytosis of neurotransmitters and secretory granules. Cortical response in fertilization or parthenogenetic activation is also based on the mechanism of calcium regulation. So far, functional study of Syt1 on reproduction is limited. Our previous research showed that in mouse oocytes ,Syt1 played an important role in spindle assembly, chromosome segregation, Meterphase-Anaphase transition and its first polar body release process in the first meiotic division.We also found that Syt1 was co-localized with cortical granules in mouse oocytes, all of which suggested Syt1 was correlated with some events, such as the process of cell division and cortical reaction or fertilization. So adopting mice as a model, we intends to explore the role and molecular mechanism of Syt1 in the cortical reaction and fertilization by technologies including immunofluorescence, immunoblot, specific oligonucleotides interference and the real-time dynamic observation in living cell imaging. This study is to provide a theoretical basis for clarifing the normal process of fertilization and embryonic development and the regulatory mechanism; additonally,to supply realistic guiding significance for clinical application of high-risk infertile population and healthy offspring of patients undergoing In-vitro fertilization-embryo transplantation(IVF-ET).

Synaptotagmin1(Syt1)在神经和分泌囊泡膜富集，在外源刺激作用下，结合胞内钙离子诱导神经递质和分泌颗粒的胞吐。卵母细胞皮质颗粒在受精或者孤雌激活时发生的皮质反应也是基于钙离子调控机理。目前为止，关于Syt1与生殖相关的功能研究很少。我们前期研究结果表明Syt1对小鼠卵母细胞纺锤体的组装、染色体分离、第一次减数分裂中后期转换及其第一极体释放过程都起着重要调控作用；同时预实验发现Syt1在小鼠卵母细胞中与皮质颗粒共定位。说明Syt1与细胞减数分裂过程和皮质反应或者受精等功能相关。因此本研究，拟以小鼠为模型，采用免疫荧光，免疫印迹，特异性寡核苷酸干扰，活细胞工作站实时动态观察等技术初步探讨Syt1在皮质反应胞吐和受精中的作用和分子机制。为阐明正常的受精和胚胎发育过程及其调控机理提供理论依据；对目前不孕症高发人群、IVF-ET病患的健康后代的临床应用提供现实指导意义。

Synaptotagmin1(Syt1)作为神经突触囊泡蛋白分子，在神经和内分泌细胞中发挥着重要的作用。结合胞内钙离子诱导神经递质和分泌颗粒的胞吐。卵母细胞皮质颗粒在受精或者孤雌激活时发生的皮质反应也是基于钙离子调控机理。所以本研究拟以小鼠为模型，分析了Syt1在小鼠卵母细胞皮质反应过程中的作用；Syt1对卵母细胞受精率的影响；以及初步探讨了Syt1在皮质反应胞吐和受精中作用的分子机制。实验结果表明：1. 免疫荧光分析Syt1在卵母细胞孤雌激活后的分布和表达变化与CGs是相似的；不过Syt1降调后的卵母细胞受精形成的受精卵中皮质颗粒的表达较对照组增加（此方面的机理尚需进一步实验验证和分析）；2. 活细胞成像系统分析了Syt1降调后显著影响了[Ca2+]i, 皮质颗粒胞吐和F-actin的结构变化，表明Syt1有可能通过F-actin结构蛋白分子作用而影响小鼠孤雌激活时发生的[Ca2+]i波和CGs胞吐过程；F-actin可能为Syt1作用于胞吐生理过程的靶标分子之一；3. WB和激光共聚焦结果显示Syt1降调后显著影响了小鼠卵母细胞体外受精时受精率；4. 同时用显微注射和活细胞动态成像的方法观察了卵母细胞孤雌激活时发生皮质反应时的皮质颗粒胞吐的时间和位置；并且首次分析出了MII期成熟卵母细胞孤雌激活后发生的[Ca2+]i和CGs胞吐的时间关系。.上述实验结果为阐明正常的受精和胚胎发育过程及其调控机理提供理论依据；对目前不孕症高发人群、IVF-ET病患的健康后代的临床应用提供现实指导意义。