miR-10a在小鼠精子发生过程中的调控作用

发布时间：2018-01-03 18:46

本文关键词：miR-10a在小鼠精子发生过程中的调控作用　出处：《西北农林科技大学》2016年硕士论文　论文类型：学位论文

【摘要】：Mi RNA是一类长度约22个核苷酸,广泛分布和高度保守的非编码RNA。它能够与靶基因对应的3,-UTR结合,调节其表达水平。在哺乳动物睾丸中存在丰富的micro RNAs,一些mi RNA直接参与生殖细胞和支持细胞的功能,调控精子发生过程。本研究中我们利用Cre-flox P重组酶系统获得生殖细胞过表达mi R-10a的小鼠,试图揭示mi R-10a对小鼠精子发生过程的调控作用。1.生殖细胞中过表达mi R-10a(Mvh-Cre;mi R-10alsl-tetoff,mi R-10a+/-)小鼠表现为精子发生障碍。利用Mvh-Cre雄鼠与mi R-10alsl-tetoff雌鼠交配,经基因分析和q PCR检测,获得了生殖细胞过表达mi R-10a(mi R-10a+/-)的小鼠;mi R-10a+/-成年雄鼠睾丸显著变小,交配试验表现为雄性不育;睾丸组织H.E.染色结果显示,出生7天的mi R-10a+/-小鼠曲细精管内没有明显的异常,但在出生10天后,mi R-10a+/-小鼠曲细精管内细胞显著减少,成年睾丸曲细精管上皮空泡化并且精子发生受阻。2.生殖细胞过表达mi R-10a抑制靶基因Rad51的表达水平导致减数分裂阻滞。采用免疫荧光分析显示,生殖细胞过表达mi R-10a不影响SYCP1和SYCP3的表达定位;但是γ-H2AX不仅性染色体上,而且在常染色体定位,可能导致染色部位的断裂损伤修复受阻;进一步分析显示,mi R-10a+/-小鼠睾丸中Rad51表达水平显著下调。mi R-10a候选靶基因预测、通过双荧光素酶报告检测以及mi R-10a minics转染3T3、GC-1、Hela、293T细胞中显著下调Rad51的表达,确定Rad51是mi R-10a的一个靶基因。因此,生殖细胞过表达mi R-10a通过抑制靶基因Rad51的表达水平影响减数分裂期染色断裂损伤修复,导致减数分裂阻滞。3.生殖细胞过表达mi R-10a抑制精原干细胞分化。出生7天的mi R-10a+/-小鼠,其睾丸组织Mvh、GATA4、Stra8和PLZF双荧光染色显示,Mvh阳性细胞相对于GATA4阳性细胞明显减少,但是PLZF阳性细胞似乎没有明显变化,表明位于基底膜的精原干细胞数量不变,而分化的生殖细胞数量减少。4.生殖细胞过表达mi R-10a导致睾丸细胞极性紊乱可能影响生殖细胞分化。分别对出生第6、10、14和60天的mi R-10a+/-小鼠睾丸进行γ-actin和cdc42表达定位检测,结果显示,与野生型小鼠相比,出生第6天的mi R-10a+/-小鼠睾丸曲细精管中γ-actin表达分布没有明显差异;出生第10天后mi R-10a+/-小鼠睾丸曲细精管中γ-actin表达低表达;而从出生第6天开始,mi R-10a+/-小鼠睾丸曲细精管细胞极性相关蛋白cdc42在睾丸显著低表达,这显示mi R-10a+/-小鼠睾丸生殖细胞中γ-actin和cdc42表达受到抑制,这可能是导致mi R-10a+/-小鼠睾丸细胞极性紊乱影响生殖细胞分化进程。
[Abstract]:Mi RNA is a class of non-coding RNA with a length of about 22 nucleotides, which is widely distributed and highly conserved. It can bind to the 3H-UTR corresponding to the target gene. There are abundant micro RNAss in mammalian testis, and some mi RNA are directly involved in the functions of germ cells and Sertoli cells. In this study, we used Cre-flox P recombinant enzyme system to obtain overexpressed mi R-10a mouse germ cells. This paper attempts to reveal the regulatory effect of mi R-10a on mouse spermatogenesis. 1. Overexpression of mi R-10a Mvh-Creh-Crein in germ cells; Mi R-10alsl-tetoff. Mi R-10a / -) mice showed spermatogenesis disorder. Male Mvh-Cre mice were mated with MI R-10alsl-tetoff female mice. By gene analysis and Q PCR analysis, we obtained the mouse with overexpression of mi R-10aI mi R-10a / -; Mi R-10a /%-the testis of adult male rats were significantly smaller, and the mating test showed male sterility. The results of H.E. staining in testicular tissue showed that there was no obvious abnormality in the seminiferous tubules of the 7-day-old miR-10a / -mouse tubule, but 10 days after birth. The number of cells in the seminiferous tubule of the mouse was significantly decreased. Adult testicular seminiferous tubules vacuolated and spermatogenesis blocked. 2. Overexpression of germ cells mi. R-10a inhibits the expression of target gene Rad51 and results in meiotic arrest. Overexpression of mi R-10a in germ cells did not affect the expression localization of SYCP1 and SYCP3. But 纬 -H2AX not only on sex chromosome, but also on autosomal location, may lead to damage repair of staining site. Further analysis showed that the expression of Rad51 in testis of R10a /-mice was significantly down-regulated, and the prediction of candidate target genes was significantly down-regulated. The expression of Rad51 was significantly down-regulated by double-luciferase report detection and transfection of miR-10a minics into 3T3GC-1HelaF93T cells. Rad51 was identified as a target gene of mi R-10a. Overexpression of mi R-10a in germ cells affected the repair of meiotic staining damage by inhibiting the expression of target gene Rad51. The over expression of mi R-10a inhibited the differentiation of spermatogonial stem cells. Stra8 and PLZF double fluorescent staining showed that Mvh positive cells were significantly decreased compared with GATA4 positive cells, but PLZF positive cells did not appear to change significantly. The results showed that the number of spermatogonial stem cells located in basement membrane was unchanged. However, the number of differentiated germ cells decreased .4. overexpression of mi R-10a in germ cells may affect the differentiation of germ cells. The expression localization of 纬 -actin and cdc42 in the testis of 14 and 60 d mi R-10a /-mice were detected. The results showed that the expression of 纬 -actin and cdc42 were compared with those of wild type mice. There was no significant difference in the expression of 纬 -actin in testicular seminiferous tubule of MI R-10a / -mouse on the 6th day of birth. The expression of 纬 -actin was low in testicular seminiferous tubules 10 days after birth. From the 6th day after birth, the expression of MIR-10a / -mouse testicular tubule cell polarity related protein (cdc42) was significantly lower in the testis. This indicated that the expression of 纬 -actin and cdc42 in testicular germ cells of miR-10a /-mice was inhibited. This may lead to the disturbance of testicular cell polarity in mi R-10 a /-mice and influence the process of germ cell differentiation.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S814

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