【作者】 孔祥斌；

【导师】 熊承良；

【作者基本信息】 华中科技大学 ， 男科学， 2007， 硕士

【摘要】 EnaC广泛分布于肾脏、结肠、肺脏、大脑、卵巢、睾丸和胰腺等组织,其生理功能就是跨越紧密连接上皮单向转运钠离子。构成EnaC的亚基有α、β、γ和δ,其中α和δ直接参与通道空隙的形成,而α2βγ是广泛分布的EnaC组成方式[4]。肾脏和结肠上皮细胞的EnaC接受盐皮质激素的正向调节,对于维持血钠血钾水平稳定十分重要。通过对EnaC的调控,肾小管两侧液体的成分和体积就能够维持稳定。在呼吸道上皮组织,钠离子通过EnaC吸收,氯离子则通过囊性纤维病跨膜传导调节蛋白受体分泌。呼吸道表面钠离子和氯离子反向运输形成的渗透压驱动力,决定了呼吸道表面液体容积。生理状态下这两种离子的运输维持平衡,使呼吸道粘膜表面液体维持在适当厚度。这样既可以保证粘膜纤毛清除率,又不至于让渗出的液体堵塞呼吸道。但是在大脑、卵巢、睾丸和胰腺组织,只检测到ENaC-α和ENaC-δ亚基。这提示ENaC-α和ENaC-δ应该是以同源多聚体的形式分别表达于精子鞭毛中段和顶体。与α、β、γ构成的异聚体相比,这种离子通道具有不同的生理和药理学特性。在大鼠和人精子上,ENaC-α亚基局限分布在鞭毛中段,它在小鼠精子上也有相同的分布特征;而膜片钳检测发现ENaC对小鼠精子获能所需的超极化有抑制作用。综合以上分析,ENaC-α亚基构成的ENaC很可能参与精子活力的调节。EnaC家族在精子发生和活力调控中所起的作用,目前所知甚少。加之精子形态结构的特殊性,相应的研究手段也受到很多限制。因此,我们首先运用western-blot、免疫荧光检测ENaC-α在大鼠和人的睾丸、精子上的表达和定位。初步的结果支持我们的猜测,即ENaC-α很可能参与精子活力的调控。为了证实这种推测,继而使用计算机辅助精液分析检测ENaC-α的阻断剂EIPA(5-(N-ethyl-N-isopropyl)-amiloride hydrochloride)对精子活力的影响。回顾整个研究过程,我们在实验中研究了上皮细胞钠离子通道α亚基(ENaC-α)在大鼠和人的睾丸、精子上的表达及其与精子活力之间的关系,并初步探讨ENaC-α在精子活力调控中的可能作用机制。一、检测ENaC-α在大鼠和人的睾丸、精子上的表达本部分的主要实验目的是研究上皮细胞钠离子通道α亚基(ENaC-α)在大鼠和人的睾丸及精子上是否表达。鉴定一种蛋白质分子是否存在于某种组织或细胞,最经典的方法就是蛋白质免疫印迹技术(western-blot)。从组织或培养细胞中提取的蛋白质样品经过十二烷基硫酸钠-丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS PAGE)和转膜,固定于硝酸纤维素膜。通过特异一抗和相应二抗(碱性磷酸酶或者过氧化物酶标记)的应用,目的蛋白分子被高度特异性地“辨识”。加入含有发光底物的溶液后,二抗上结合的辣根过氧化物酶作用于底物发出荧光使医用胶片曝光,最终得到记录有实验结果的胶片。该实验方法灵敏度非常高,不但可以鉴定蛋白有无和分子量大小,还可以半定量检测目的蛋白在组织或培养细胞中的丰度。本实验的结果证实,上皮细胞钠离子通道α亚基(ENaC-α)在大鼠和人的睾丸及精子上都有表达。其分子量约为75KD。二、免疫荧光和免疫组化染色检测ENaC-α在大鼠和人的睾丸、精子上的定位通过上面实验我们证实上皮细胞钠离子通道α亚基(ENaC-α)在大鼠和人的睾丸及精子上都有表达。但是,它在睾丸中的表达分布特征是什么样的?这需要进一步的研究。为此,我们运用免疫组化染色技术分析其在睾丸的分布特征。试验结果显示,ENaC-α分布于大鼠睾丸的间质细胞、血管内皮细胞、支持细胞、Ap型精原细胞,精母细胞和精子细胞以及精子;在人睾丸中,其生精细胞系亦见明显阳性表达。结果说明,在人和大鼠的睾丸中,ENaC-α都有表达,而且其分布具有共同特征。后来运用免疫荧光技术再次分析其在大鼠睾丸的分布特征。试验结果基本与组化染色一致,且在大鼠睾丸组织切片见精子鞭毛上有强阳性表达。不过相比之下,免疫荧光对胞浆中的目的蛋白显示更清晰,而免疫组化染色似乎更易于辨识组织中各类细胞。随后运用免疫荧光技术分析其在大鼠和人精子分布特征。试验结果显示,ENaC-α分布于大鼠和人精子的鞭毛中段,这和前人在小鼠精子上的发现是一致的。三、体外精子活力实验观测ENaC-α对大鼠和人精子活力的影响。在大脑、卵巢、睾丸和胰腺组织,只检测到ENaC-α和ENaC-δ亚基。这提示ENaC-α和ENaC-δ应该是以同源多聚体的形式分别表达于精子鞭毛中段和顶体。与α、β、γ构成的异聚体相比,这种离子通道具有不同的生理特性,但是仍对盐酸阿米洛利及其衍生物敏感。在大鼠和人精子上,ENaC-α亚基局限分布在鞭毛中段,它在小鼠精子上也有相同的分布特征;而膜片钳检测发现ENaC对小鼠精子获能所需的超极化有抑制作用。可透膜的cAMP拟似物或促获能液体诱导精子获能时,发现EnaC的功能被抑制了。综合以上分析,ENaC-α亚基构成的ENaC很可能参与精子活力的调节。EnaC家族在精子发生和活力调控中所起的作用,目前所知甚少。加之精子形态结构的特殊性,相应的研究手段也受到很多限制。我们使用EnaC抑制剂EIPA,间接观测EnaC活性对精子活力的影响。发现在获能的孵育环境下,EnaC抑制剂EIPA可以促进精子的活力。无论正常人精子还是弱精子组,实验组精子活力均高于对照组;在大鼠精子组也有同样的发现。这个事实说明ENaC-α亚基构成的EnaC至少部分参与精子活力的调控。四、总结总结前人的发现和我们的实验结果,认为ENaC-α与雄性生殖系统关系密切,可能对精子活力有调控作用。在这里,我们第一次提出EnaC抑制剂EIPA有促进精子活力的作用,并间接证明ENaC-α有抑制精子活力的趋势。同时,我们从精子周围环境pH变化的角度探讨了ENaC-α抑制精子活力的可能机制。此外,针对ENaC-α有可能研制成为抗雄性生育药品从而在男性避孕中发挥重要作用,以及作为一种诊断项目用于男性不育症病因的查找和为人工辅助生殖技术提供优质精子。更多还原

【Abstract】 Mammalian spermatozoa are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. Capacitation is associated with changes in membrane properties, enzyme activities and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. Hernandez and his colleague found the localization of ENaC-αto the flagellar mid piece and of ENaC-δto the acrosome in mouse sperm (Hernandez-Gonzalez EO et al., 2006). By the methods of single cell fluorescence analysis and electrophysiology, they found that the two EnaCs participate in the regulation of the capacitation-associated hyperpolarization through producing a depolarization that was potently inhibited by amiloride and its analog EIPA, high pH, and by the incubation of mouse sperm under capacitating conditions.Our study investigated the regional distribution of epithelial Na+ channel (ENaC) alpha-subunit in the testis and sperm of rat and human. ENaC-αprotein was found on immunoblots of sperm and testis membrane preparations and indicated localization to the spermatocyte, spermatid, and sperm by Immunostaining. Taken together, these data indicate that ENaC-αis expressed in spermatocyte and distributed to the flagellar mid piece of sperm finally. The dynamic distribution changes of ENaC-αin spermatogenesis also suggest that ENaC-αis likely to participate in the regulation of sperm motility by someway. Sperm motility assays showed a significant difference between samples with EIPA and those without in human sperm. 1. Western-blot detecting ENaC-αsubunit in the testis and sperm of rat and humanThe main goal of this portion is to study the expression of ENaC-αprotein in the testis and sperm of rat and human.Western-blot is the classical method used in the assessment of a protein in tissue or cultivated cells. Proteins of the samples are obtained from tissue or cultivated cells, subjected to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Electrophoretic transfer of proteins to nitrocellulose filter and immunodetection of the goal protein are carried out by special antibody and the second antibody(alkaline phosphatase labelled). Using the ECL kit, immunoblots were developed with the film.We confirmed the presence of ENaC-αin the testis and sperm of rat and human by western blot analysis using polyclonal antibodies against the ENaC-αsubunit. Antibody detected bands with a Mr of ~75 KDa in testis and sperm.2. Indirect immunofluorescence and immunohistochemistry detecting ENaC-αdistribution in the testis and sperm of rat and humanAs discribed above, ENaC-αsubunit is expressed in the testis and sperm of rat and human. Indirect immunofluorescence and immunohistochemistry detecting ENaC-αdistribution in the testis and sperm of rat and human were applied for finding the distribution of ENaC-αin the testis and sperm.As a result, we found that that ENaC-αexpress in leydig cell, vascular endothelial cell(VEC), Ap type spermatogenous cell, spermatocyte, spermatid and sperm. The sample of human testis is not good enough for use in Indirect immunofluorescence and immunohistochemistry, no sperm is found. But the positive staining was found in spermatocyte of the human sample.ENaC-αdistribution character in the sperm of rat and human were detected too. ENaC-αwas locallized on the midpiece of flagella of the sperm of rat and human.3. Assessment of sperm motilityMammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. Capacitation is associated with changes in membrane properties, enzyme activities and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. Hernandez and his colleague found the localization of ENaC-αto the flagellar mid piece and of ENaC-δto the acrosome in mouse sperm (Hernandez-Gonzalez EO et al., 2006). By the methods of single cell fluorescence analysis and electrophysiology, they found that the two EnaCs participate in the regulation of the capacitation-associated hyperpolarization through producing a depolarization that was potently inhibited by amiloride and its analog EIPA, high pH, and by the incubation of mouse sperm under capacitating conditions.Our study investigated the regional distribution of epithelial Na+ channel (ENaC) alpha-subunit in the testis and sperm of rat and human. ENaC-αprotein was found on immunoblots of sperm and testis membrane preparations and indicated localization to the spermatocyte, spermatid, and sperm by Immunostaining. Taken together, these data indicate that ENaC-αis expressed in spermatocyte and distributed to the flagellar mid piece of sperm finally. The dynamic distribution changes of ENaC-αin spermatogenesis also suggest that ENaC-αis likely to participate in the regulation of sperm motility by someway. Sperm motility assays showed a significant difference between samples with EIPA and those without in human sperm.4. SummarySummarizing the findings of other researchers and ours, ENaC-αis closely relevant to the male fertility and may take part in the regulation of sperm mortility. Here, for the first time, we raise and confirm a new action of EIPA on promoting male fertility, which provide an indirect evidence that the open ENaC-αmay inhibits sperm motility by some way. Meanwhile, we further investigate the mechanism of ENaC-αinhibiting sperm motility in the aspect of pH change in the liquid environment surrounding sperm. The role of ENaC-αin regulation of sperm motility might also be the target of antifertility medicine for male contraception, and may provide a way to evaluate male sperm functions in clinics.更多还原