MIR-183靶向TEX15调控精子发生

发布时间：2018-04-05 03:12

本文选题：男性不育　切入点：TEX15　出处：《安徽医科大学》2016年硕士论文

【摘要】：目前,不孕不育困扰着10-15%的育龄夫妇。其中因为男性因素引起的不育比例约占一半,并且呈现逐年递增的趋势。男性不育的已知原因有生殖道梗阻或物理性阻塞、免疫性因素、生殖系统炎症及性功能障碍等,但找不到病因的患者的比例有60%~75%之多,称为特发性男性不育,表现为无精子症或少弱精症。约10-15%的非梗阻性无精子症(non-obstructive azoospermia,NOA)与染色体数目及结构异常学/或Y染色体微缺失相关,但大部分NOA患者精子产生缺陷或异常的原因或途径还不清楚。虽然随着助孕技术如学精子卵胞浆内注射(intracytoplasmic sperm injection,ICSI)发展,为这些不育患者提供了生育的可能,但ICSI绕过精子外精过程中的自然选择机制有传递遗传缺陷给下一代的危险。由此可知,了解精子发生障碍的分子机制学遗传申基础对阐明特发性男性不育的发病机制有着重要的意义。精子发生是一个非常复杂的过程,可以分为三个阶段:精原细胞有丝分裂进行细胞增殖;精母细胞进行减数分裂;学倍体精子细胞分化。基因转录网络严密地调控着精子发生中的每个过程。尤其是在精子发生的减数分裂阶段,转录活性增加,m RNA翻译被抑制,此阶段答要是通过转录后的调控机制来抑制m RNA的翻译,micro RNA(mi RNA)就参与其中。睾丸特异性蛋白15(Testis-experessed 15,TEX15)与减数分裂过程密切相关,小鼠敲除模型表现为睾丸性不育。因此TEX15很可能在NOA患者精子发生障碍的发生中发挥重要作用,但目前关于TEX15转录及转录后调控的具体机制未知。本研究发现TEX15的m RNA在NOA患者中的表达量明显低于精子正常发生对照组。免疫组化结果也证实了NOA患者睾丸精原细胞中TEX15蛋白表达量显著下调。我们应用靶基因预测网站预测发现mi R-183可能靶向TEX15,通过荧光素酶报告基因实验分析证实mi R-183对TEX15 m RNA 3’UTR结合申点的靶向性。进一步,我们在U2OS细胞系中抑制mi R-183后发现TEX15 m RNA的水平显著升高。同时,在NOA患者睾丸组织中mi R-183水平与对照组相比明显上升,与TEX15表达呈显著负相关性。并且检测小鼠不同睾丸发育阶段(8d,14d,16d,18d,25d,2m)中TEX15与mi R-183含量的表达,发现两者呈显著负相关性。本研究通过实验证明了mi R-183通过直接靶向调控TEX15 3’UTR区域,下调了TEX15转录后的水平,而TEX15水平的下调,可能直接指致减数分裂过程中染色体异常,最终引起不育的发生。综上所述,本提提研究结果阐述了mi R-183靶向调控TEX15的表达调控精子发生的分子机制。这些研究成果将为阐明精子发生障碍提供新的研究思路及新的分子调控机制。
[Abstract]:At present, infertility puzzles 10-15% of couples of childbearing age.Male factors caused by male infertility accounted for about half, and showed an increasing trend year by year.The known causes of male infertility are reproductive tract obstruction or physical obstruction, immunological factors, inflammation of the reproductive system and sexual dysfunction. However, the proportion of patients who cannot find the cause of infertility is as high as 60% or 75%, which is called idiopathic male infertility.It is characterized by azoospermia or oligozoospermia.About 10-15% of non-obstructive azoospermia NOAs are associated with chromosome number and structural abnormalities, or Y chromosome microdeletions, but the cause or pathway of sperm defects or abnormalities in most NOA patients remains unclear.Although the development of assisted pregnancy techniques such as intracytoplasmic sperm injection (ICSI) provides a possibility for these infertile patients to reproduce, ICSI bypasses the mechanism of natural selection in the process of spermatozoa exosperm and carries the risk of passing genetic defects to the next generation.Therefore, it is important to understand the molecular mechanism of spermatogenesis disorders in order to elucidate the pathogenesis of idiopathic male sterility.Spermatogenesis is a very complex process, which can be divided into three stages: spermatogonium mitosis for cell proliferation, spermatocyte meiosis, and spermatogonia differentiation.The gene transcription network tightly regulates every process of spermatogenesis.Especially in the meiosis stage of spermatogenesis, the increased transcriptional activity of m RNA is inhibited, which should be involved if the transcriptional regulation mechanism is used to inhibit the translation of m RNA.Testicular specific protein 15(Testis-experessed 15 (TEX15) was closely related to meiosis, and mouse knockout model showed testicular infertility.Therefore, TEX15 may play an important role in the development of spermatogenesis in NOA patients, but the specific mechanism of TEX15 transcription and posttranscriptional regulation is unknown.In this study, we found that the expression of m RNA of TEX15 in NOA patients was significantly lower than that in normal spermatogenesis control group.Immunohistochemical results also confirmed that the expression of TEX15 protein was significantly down-regulated in spermatogonial cells of NOA patients.We used the target gene prediction website to predict that mi R-183 might target TEX15. The targeting of TEX15 m RNA 3'UTR binding site was confirmed by luciferase reporter gene analysis.Further, we found that the level of TEX15 m RNA was significantly increased after inhibiting mi R 183 in U2OS cell line.At the same time, the level of miR-183 in testis of NOA patients was significantly higher than that of control group, and negatively correlated with the expression of TEX15.The expressions of TEX15 and miR-183 in mouse testis at different testicular development stages were detected at 14d ~ 16d ~ 18d ~ 18d ~ 25d ~ 2m). The results showed that there was a significant negative correlation between the expression of TEX15 and miR-183.This study demonstrated that miR-183 down-regulated the post-transcriptional level of TEX15 through direct targeting regulation of the TEX15 3'UTR region, and that the down-regulation of TEX15 level may directly refer to chromosomal abnormalities during meiosis and eventually to the occurrence of infertility.In conclusion, the results of this study illustrate the molecular mechanism of the regulation of the expression of TEX15 targeted by miR-183 and the regulation of spermatogenesis.These results will provide new ideas and new molecular regulatory mechanisms for elucidating spermatogenesis disorders.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R698.2

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