Racl在小鼠原始卵泡形成中的功能与机制

发布时间：2018-04-17 17:15

本文选题：Rac1 + 原始卵泡形成　；参考：《中国农业大学》2016年博士论文

【摘要】：哺乳动物出生前后形成的原始卵泡库是雌性动物一生中发育卵泡与成熟卵母细胞的唯一来源,其大小代表了雌性哺乳动物一生的生育能力。原始卵泡形成异常会导致合胞体破裂受阻、多卵子卵泡发生,从而缩短雌性动物的生育年限,降低卵母细胞的体外成熟率,最终影响生殖健康。原始卵泡的形成需要多种必要基因时空特异性地表达,但这些基因是如何协同调控的,至今仍不清楚。RhoGTPase家族的Racl分子是一个广泛存在的信号分子开关,具有多种细胞生物学功能。研究表明,Rac1参与了许多生殖相关事件,但在原始卵泡的形成过程中是否具有功能,至今仍未见到报道。相关研究表明Rac1可以通过转录因子STAT3调控诸多基因的转录。我们推测Racl可能在原始卵泡的形成过程中具有生物学功能,并且有可能调控原始卵泡形成必要基因的转录。本研究发现,在小鼠原始卵泡形成过程中,Racl表达于生殖细胞中,且表达量逐步上升。为了研究Racl在小鼠原始卵泡形成过程中是否具有功能,我们利用小鼠卵巢体外培养体系,通过向培养基中添加Rac分子特异性抑制剂NSC23766和特异性敲降Racl的方法,发现抑制Racl能够显著抑制原始卵泡的形成,卵巢皮质区出现了大量合胞体；在胎鼠卵巢中过表达Rac1则加速原始卵泡的形成；同时,体内注射Racl抑制剂,未破裂的合胞体可以持续到青春期形成多卵子卵泡。为了探索Racl是如何调控原始卵泡形成的,我们研究了Rac1与现有的原始卵泡形成必要基因之间的关系,结果表明Racl可以调控生殖细胞中.Jagged1、GDF9、BMP15以及转录因子Nobox的转录,并且影响前体颗粒细胞中Notch2的翻译。后续研究表明,在生殖细胞中,Rac1与STAT3直接互作促进其入核,直接激活原始卵泡形成必要基因Nobox、Jngged1、GDF9及BMP15的转录。受Racl调控的GDF9与BMP15可以通过旁分泌的方式作用于前体颗粒细胞,激活前体颗粒细胞中的mTORC1并进而调控Notch2的翻译。过表达和蛋白纯品添加实验也表明Jagged1、GDF9与BMP15不仅能够逆转由NSC23766引起的原始卵泡形成受抑制的表型,而且添加这3种蛋白纯品能够促进原始卵泡的形成。以上结果表明：在原始卵泡的形成过程中,Racl作为关键性的调控因子调节了原始卵泡形成过程中必要基因的表达,从而调控了原始卵泡的形成。
[Abstract]:The primordial follicle bank formed before and after birth in mammals is the only source of follicular and matured oocytes in female animals in their lifetime, and its size represents the fertility of female mammals in their lifetime.Abnormal primordial follicle formation will lead to the rupture of syncytial cells and the formation of multiple oocytes, thus shortening the reproductive life of female animals, reducing the rate of oocyte maturation in vitro, and ultimately affecting reproductive health.The formation of primordial follicles requires a variety of necessary genes to be specifically expressed in time and space, but how these genes are co-regulated remains unclear. The Racl molecule of the RhoGTPase family is a widespread signal molecule switch.It has many cell biological functions.Studies have shown that Rac1 is involved in many reproductive events, but whether it has function in the formation of primordial follicles has not been reported.Related studies have shown that Rac1 can regulate the transcription of many genes through the transcription factor STAT3.We speculate that Racl may have biological function in the process of primordial follicle formation and may regulate the transcription of genes necessary for primordial follicle formation.In this study, it was found that Racl was expressed in germ cells during primordial follicle formation in mice, and the expression level increased gradually.In order to study the function of Racl in the process of mouse primordial follicle formation, we used the mouse ovary culture system in vitro by adding Rac molecular specific inhibitor NSC23766 and specific knockout Racl to the culture medium.It was found that inhibition of Racl could significantly inhibit the formation of primordial follicles, and a large number of syncytial bodies appeared in the ovarian cortex; overexpression of Rac1 in fetal ovaries accelerated the formation of primordial follicles; at the same time, Racl inhibitor was injected in vivo.Unruptured syncytial can continue to form multiple oocyte follicles until puberty.In order to explore how Racl regulates primordial follicle formation, we studied the relationship between Rac1 and the necessary genes for primordial follicle formation. The results showed that Racl could regulate the transcription of. Jagged1, GDF9, BMP15 and Nobox.It also affects the translation of Notch2 in precursor granulosa cells.Further studies have shown that the direct interaction between rac1 and STAT3 in germ cells promotes the nucleation of the primordial follicles and activates the transcription of GDF9 and BMP15, which are essential genes for the formation of primordial follicles.GDF9 and BMP15 regulated by Racl can act on precursor granulosa cells through paracrine way, activate mTORC1 in precursor granulosa cells and then regulate the translation of Notch2.Overexpression and protein addition also showed that Jagged1GDF9 and BMP15 could not only reverse the inhibited phenotype of primordial follicles induced by NSC23766, but also promote the formation of primordial follicles.These results suggest that Racl, as a key regulatory factor, regulates the expression of necessary genes in the process of primordial follicle formation and thus regulates the formation of primordial follicles.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q492

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