输出小管的结构与功能与miR-34b/c-/-；miR-449-/-双敲小鼠不育的关系及临床意义

The efferent ductuli play an important role in regulating the testicular fluid homeostasis, which is critical for spermatogenesis as well as male fertility. Previous study showed that the miR-34b/c-/-; miR-449-/- double knock out (dKO) mice were sterile, with increased testicular fluid and atrophic seminiferous epithelium. It is not clear whether these two miRNA clusters, miR-34b/c ,miR-449, are essential for the structure and function of the efferent ductuli, which may induce infertility in the miR-34b/c-/-; miR-449-/- dKO mice . In order to investigate the relationship between the structure and function of the efferent ductuli and the infertility of the miR-34b/c-/-; miR-449 dKO mice, in this project we will use the miR-34b/c-/-; miR-449-/- dKO mouse as model to compare the structure of WT and dKO efferent ductuli, compare the differences of the transcriptome and proteotome between WT and dKO efferent ductuli, screen the targets of these two miRNA clusters, examine the interactions between the two miRNAs clusters and their targets in vitro using efferent ductuli epithelial cells culture system. In addition, this project will detect the expression levels of these two miRNA clusters in serum and seminal plasma samples of the primary cilia dyskinesia (PCD) and oligoasthenoteratozoospermia (OAT) patients to assess the importance of these two miRNA clusters for human male fertility. This research work may provide evidences for clarifying the mechanisms of dysspermatogenesis such as oligozoospermia, azoospermia, asthenozoospermia, teratozoospermia and it may bring new ideas for diagnosis and treatment of male infertility.

输出小管在调节睾丸液的动态平衡中起关键作用，从而维持正常的精子发生和雄性生育力。研究表明，miR-34b/c-/-, miR-449 -/-双敲小鼠雄性不育，睾丸液增多，生精上皮变薄萎缩。miR-34/449是否通过调控输出小管的结构和功能而影响雄性生育？尚未明了。本项目以双敲小鼠为模型，通过比较正常小鼠与双敲小鼠输出小管的结构差异、转录组学与蛋白组学变化，筛查miR-34/449的靶基因，并通过输出小管上皮细胞培养模型，验证miR-34/449与其靶基因在调控输出小管的纤毛生成与重吸收功能中的作用，以明确输出小管的结构功能与双敲小鼠不育的关系。通过检测临床上原发性纤毛功能障碍以及少弱畸精症患者血清和精浆中的miR-34b/c，miR-449表达水平来探讨这两个miRNA 簇对于男性生育力的重要性。为阐明精子发生障碍的机理提供实验依据，并可能为临床上不育的诊断与治疗带来新思路。

输出小管在调节睾丸液的动态平衡中起关键作用，从而维持正常的精子发生和雄性生育力。研究表明，miR-34b/c-/-, miR-449 -/-双敲小鼠雄性不育，睾丸液增多，生精上皮变薄萎缩。miR-34/449是否通过调控输出小管的结构和功能而影响雄性生育？尚未明了。本项目以双敲小鼠为模型，通过比较正常小鼠与双敲小鼠输出小管的结构差异、转录组学变化，筛查miR-34/449的靶基因，并通过原代输出小管上皮细胞培养和在体动物实验，探讨miR-34/449与其靶基因在调控输出小管纤毛生成中的作用，以明确输出小管的结构功能与双敲小鼠不育的关系。通过检测临床上少弱畸精症患者血清中的miR-34b/c，miR-449表达水平来探讨这两个miRNA 簇对男性生育力的重要性。我们发现： 1）miR-34/449高表达于小鼠输出小管、定位于小鼠输出小管纤毛细胞 ；2）敲除miR-34b/c、miR-449，输出小管上皮纤毛细胞数量减少，纤毛结构异常，这种现象从2周龄开始出现；3）纤毛细胞的数量减少和结构异常导致精子运送受阻，积聚于输出小管，无法进入附睾，输出小管压力增大，通过反压力使生精上皮萎缩，生精细胞减少，导致雄性双敲小鼠梗阻性不育；4）双敲小鼠输出小管上皮中细胞周期相关基因表达水平上调，使上皮细胞不能正常退出细胞周期而持续处于增殖状态，导致纤毛细胞分化障碍，纤毛细胞数量减少；5）抑制双敲小鼠周期蛋白依赖性激酶的异常活跃活性，可部分挽救双敲小鼠输出小管上皮细胞的纤毛发生；6）miR-34b/c，miR-449c在少弱畸精症患者血清中低表达。本研究探讨了miR-34b/c,miR-449在输出小管上皮纤毛细胞纤毛生成过程中的作用和机制，为输出小管梗阻性不育提供基础研究与临床诊断思路，并为化学小分子治疗这种不育提供了实验依据。