【作者】 王勇；

【导师】 桂耀庭；

【作者基本信息】 汕头大学 ， 生理学， 2009， 硕士

【摘要】 精子从附睾中射出后需要经过一段距离的运动才能与卵细胞相遇而受精,因此,精子活力对于正常受精是十分必要的。弱精子症(asthenospermia)或精子活动力低下是导致男性不育的主要原因之一。导致精子活力低下的原因很多,包括精子尾部结构的异常和功能的缺失。因此,通过对正常精子鞭毛功能和精子活动力的分子机制的理解,可以为弱精子症提供一条可行性的治疗途径。Ropporin为一种精子鞭毛相关蛋白,高表达于睾丸组织中,并特异性的定位于精子鞭毛的主段和尾段上,在细胞粘连、胞质分裂和细胞运动方面有着重要的作用。同时,Ropporin是鸟苷三磷酸酶(GTPase)Rho结合蛋白和Rhophilin的精子特异性结合蛋白。在体外, Ropporin、Rhophilin和GTP-Rho可形成一种三元络合物,通过Rho信号通路调控精子运动。作为一种AKAP结合蛋白,该蛋白还可与AKAPs相互结合,参与精子运动。ODF1(outer-dense fiber 1)作为外周致密纤维(ODFs)家族成员之一,为精子尾部重要的组成部分,它包绕着轴丝,由9根纵行的柱状结构组成,每一根致密纤维与其相对的轴丝二联微管平行,其外面被纤维鞘包裹着,主要定位于精子尾部的中段和主段,在精子的形态结构和活动力方面发挥着重要的作用。本研究采用RT-PCR和实时荧光定量RT-PCR检测了Ropporin和ODF1 mRNA在正常生育男性和弱精子症患者精液精子中的表达差异,发现Ropporin和ODF1在弱精子症患者精子中的表达明显下降。另外,免疫细胞化学方法检测到Ropporin特异性地定位于精子鞭毛的主段和尾段上,与正常生育男性精子相比,Ropporin蛋白在弱精子症患者精子中的表达明显下降。Western-Blot结果显示Ropporin和ODF1蛋白在正常人精液精子中的表达量均高于弱精子症患者精液精子。总之,本研究证实了Ropporin和ODF1在人精液精子中的表达情况,并且,与正常生育男性精液精子相比,Ropporin和ODF1在弱精子症患者精液精子中的表达显著下降。Ropporin和ODF1的表达降低可能是导致精子活动力低下的原因之一。更多还原

【Abstract】 Spermatozoa,ejaculated from the epididymis, travel a long distance to meet and fertilize the oocytes, so sperm motility is a requisite for normal fertilization. Asthenozoospermia, or low sperm motility, is a common cause of human male infertility, and it may be caused by sperm structural or functional deficiencies. So the understanding of the mechanisms that contribute to normal mammalian flagellar fuction and sperm motility will provide a feasibility treatment for asthenospermia.Ropporin, a sperm flagellum associated-protein, which is highly expressed in testis and localized in the principal piece and end piece of sperm flagella. It plays an important role in cell adhesion, cytokinesis and cell motility. Meanwhile, Ropporin is GTPase Rho binding protein and also a sperm-specific binding protein of rhophilin. In vitro, Ropporin, Rhophilin and GTP-Rho can make a ternary complex regulate sperm motility. As an AKAP binding protein, it can regulate the sperm motility through the interaction with AKAPs. Outer-dense fiber 1(ODF1), as a member of ODF family, is an important ingredient of sperm tail. And as a sperm tail-specfic cytoskeletal structures, ODF1 consists of nine fibres which surround the axoneme on its outer side accompanying the tubuli doublets in the middle and principal piece of the sperm tail. Each dense fiber axis relative to its two-wire parallel microtubules, and its outside wrapped by fibrous sheath. ODF1 is localized in the connecting piece and principal piece of sperm flagella, and may play an important role in the formation of sperm structure.In the present study, we demonstrated the differential expression of Ropporin and ODF1 between fertile men and asthenospermia men through semi-quantitative RT-PCR and Real-time RT-PCR. Furthermore, we found that the expression of Ropporin and ODF1 mRNA were significantly lower in spermatozoa from asthenospermia men than that from fertile men. To further confirm the results from semi-quantitate RT-PCR and real-time RT-PCR, immunocytochemistry was used to investigate the expression of Ropporin protein in human ejaculated spermatozoa. The results showed that Ropporin was localized in the principal piece and end piece of sperm flagella, and the expression of Ropporin was significantly decreased in the patients with asthenospermia, which was similar to the results from semi-quantitative RT-PCR and real-time RT-PCR. Western blot showed that the expression of Ropporin and ODF1 protein were significantly lower in spermatozoa from asthenospermia men than that from fertile men.In summary, our study demonstrated that Ropporin and ODF1 were expressed in human ejaculated spermatozoa and their expression were significantly lower in asthenospermia men than in normozoospermic men. The decreased expression of Ropporin and ODF1 may be one possible reason for low sperm motility.更多还原