FKBP12.6对FK506所致成年雄性小鼠不育的保护作用及其机制

发布时间：2018-05-26 16:33

本文选题：雄性不育 + FK506　；参考：《南昌大学》2017年硕士论文

【摘要】：研究背景与目的根据WHO的标准定义,不育是指夫妻同居一年以上,在未采取主动避孕而致女方不能怀孕的情况。近年来,随着工业化进程的加速,人们的生活方式也在发生改变,如过重的心里压力、不健康的饮食、恶化的环境等因素均可导致夫妻不孕不育,这一现象已引起社会的高度关注。目前,男性不育约占整个人类不孕不育的40%,但其发病原因及其机制尚不清楚。研究表明,FK506通过抑制钙调磷酸酶(calcineurin)活性,可引起未成熟精子的中端僵化,从而导致雄性小鼠不育。FKBP12.6是FK506结合蛋白家族中的一个重要成员,研究表明,该蛋白可与FK506结合,即可通过直接抑制calcineurin活性发挥免疫抑制作用,同时又可通过调节钙离子释放通道(ryanodine receptor 2,RyR2)而调控calcineurin活性。因此,本课题借助FKBP12.6敲除小鼠,旨在研究FKBP12.6在FK506所致雄鼠不育过程中的作用及其机制,从而为治疗男性不育提供新的靶点。方法与结果1.敲除FKBP12.6基因可明显缓解FK506引起的成年雄性小鼠不育:为了确定FKBP12.6在FK506所致雄鼠不育中的作用,我们首先将成年野生型(WT)和FKBP12.6基因敲除(FKBP12.6KO)雄性小鼠皮下注射FK506,结果显示,给予WT小鼠FK506后,精子发生中端僵化,受精比率急剧下降,而FKBP12.6KO鼠给药后与WT相比精子中端僵化明显减少,受精情况显著改善。结果表明FKBP12.6缺失在FK506所致雄鼠不育过程中起重要保护作用。2.FKBP12.6在睾丸组织内质网高表达:睾丸是精子发生的场所,为了明确FKBP12.6或FKBP12在雄性成年鼠睾丸中对精子发生发育的影响,我们分别提取雄性小鼠的睾丸总蛋白和微粒体(microsome,主要为内质网蛋白),并检测其表达情况。Western blot检测,结果显示:虽然FKBP12在睾丸中的表达较FKBP12.6更为丰富,但FKBP12.6在内质网上的相对表达量显著高于FKBP12。表明FKBP12.6在睾丸钙离子调控中可能起重要作用。3.FKBP12.6抑制睾丸特异性calcieneurin(PPP3CC/PPP3R2)活性:有研究表明,calcineurin的PPP3CC/PPP3R2亚基在睾丸组织特异性表达。为了进一步明确FKBP12.6和FKBP12对睾丸calcineurin活性的影响,我们首先采用体外原核细胞系统表达和纯化小鼠GST-FKBP12.6和GST-FKBP12融合蛋白,并通过亲和层析实验(pull down)观察FKBP12.6和FKBP12蛋白与睾丸组织裂解液或体外纯化的PPP3CC/PPP3R2蛋白的特异性结合。结果显示FKBP12.6与睾丸PPP3CC/PPP3R2蛋白的结合能力显著高于FKBP12,提示FKBP12.6在FK506抑制睾丸calcineurin活性过程中可能起主要作用。结论:我们的研究表明,FKBP12.6缺失可明显减轻给予FK506引起的雄性成年鼠不育,其机制可能与其在睾丸组织上表达缺失,解除了FK506对成年雄性鼠睾丸calcineurin活性的抑制,进而促进精子的正常发育有关。
[Abstract]:Background & objective according to the standard definition of WHO, infertility refers to couples cohabiting for more than one year and failing to conceive because they have not taken active contraception. In recent years, with the acceleration of industrialization, people's way of life is also changing, such as excessive psychological stress, unhealthy diet, deterioration of the environment and other factors can lead to infertility couples, This phenomenon has aroused the high attention of the society. At present, male sterility accounts for about 40% of infertility, but its pathogenesis and mechanism are not clear. It has been shown that FK506 can induce ossification of immature spermatozoa by inhibiting calcineurin activity, which leads to male sterility. FKBP12.6 is an important member of FK506 binding protein family. It has been shown that this protein can bind to FK506. The activity of calcineurin can be regulated by direct inhibition of calcineurin activity and regulation of ryanodine receptor 2 RyR2). Therefore, the purpose of this study was to study the role of FKBP12.6 in the process of male infertility induced by FK506 and its mechanism with the help of FKBP12.6 knockout mice, so as to provide a new target for the treatment of male sterility. Methods and results 1. Knockout of FKBP12.6 gene significantly alleviated male sterility induced by FK506 in adult male mice. In order to determine the role of FKBP12.6 in FK506 induced male sterility, we first injected FK506 subcutaneously into adult wild-type male mice and FKBP12.6 gene knockout FKBP12.6KOO male mice. After administration of FK506 in WT mice, the middle end of spermatogenesis became ossified, and the fertilization rate decreased sharply, while the ossification of sperm in FKBP12.6KO mice decreased significantly compared with WT, and the fertilization situation was improved significantly. The results show that FKBP12.6 deletion plays an important protective role in the male infertility induced by FK506. 2. FKBP12.6 is highly expressed in the endoplasmic reticulum of testis. Testis are the place where spermatogenesis occurs. In order to clarify the effect of FKBP12.6 or FKBP12 on spermatogenesis and development in the testis of adult male rats, FKBP12.6 was found in the testis of male mice. We extracted total testicular protein and microsomal microsome from male mice, mainly endoplasmic reticulum protein, and detected their expression. Western blot analysis showed that, although the expression of FKBP12 in testis was more abundant than that of FKBP12.6, However, the relative expression of FKBP12.6 in endoplasmic reticulum was significantly higher than that in FKBP12. It is suggested that FKBP12.6 may play an important role in the regulation of calcium ion in testis. 3. FKBP12.6 inhibits the activity of testicular specific calcieneurin PPP3CC / PPP3R2. Some studies have shown that the PPP3CC/PPP3R2 subunit of calcineurin is specifically expressed in testis. In order to further clarify the effect of FKBP12.6 and FKBP12 on testicular calcineurin activity, we first expressed and purified mouse GST-FKBP12.6 and GST-FKBP12 fusion protein by prokaryotic cell system in vitro. The specific binding of FKBP12.6 and FKBP12 proteins to testicular tissue lysate or purified PPP3CC/PPP3R2 protein in vitro was observed by affinity chromatography. The results showed that the binding ability of FKBP12.6 to testicular PPP3CC/PPP3R2 protein was significantly higher than that of FKBP12, suggesting that FKBP12.6 might play a major role in the inhibition of testicular calcineurin activity by FK506. Conclusion: our study shows that FKBP12.6 deletion can significantly alleviate the male infertility induced by FK506, and its mechanism may be similar to the loss of expression in testis, thus relieving the inhibition of calcineurin activity in testis of adult male rats by FK506. And then promote the normal development of sperm related.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.2

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