人类精子冷冻复苏后乌头酸水合酶的表达变化及其致损伤机制研究

发布时间：2018-05-31 11:31

本文选题：精子冷冻保存 + 蛋白降解　；参考：《南京医科大学》2014年硕士论文

【摘要】：据统计世界范围内大约有10%-20%的已婚夫妇遭遇生育困难,由于男性生殖能力异常所致的不育接近50%。并且随着我国经济迅速发展,有害的环境因素不断增加以及受不良生活习惯的影响,人类精液质量呈现下降趋势。人类精子库是指以治疗不育症以及预防遗传病等为目的,利用超低温冷冻技术,采集、检测、保存和提供精子的机构。人类精子库的建立为那些患有不可逆的无精子症、严重少、弱、畸形精子症及有遗传性疾病风险的患者提供了一种可供选择的方法。然而,人类精子在冷冻复苏后其活力、活率、获能、顶体反应等功能发生相应的减退,目前认为在冷冻复苏过程中,精子细胞内外的渗透压的改变是造成精子超微结构发生破坏的主要原因,但是超微结构的改变并不能直接解释其功能的损伤。由于蛋白质是细胞功能的重要执行者,了解精子冷冻复苏后蛋白质的改变对理解精子冷冻损伤机制具有重要的意义。因此,我们前期利用蛋白质组学的方法对我中心精子库9名健康志愿者的精液标本进行冷冻前后的差异蛋白质组学研究,发现了在三羧酸循环通路中的乌头酸水合酶(AC02)可能在人类精子冷冻损伤中发挥了重要作用。通过免疫印迹实验的验证证实了其在复苏精子中的表达下降,与蛋白质组中的结果一致,免疫荧光实验明确了其主要定位于精子中段线粒体鞘区域,通过文献复习及蛋白定位信息提示AC02可能参与了线粒体内三羧酸循环的过程,其冻后降解使得三羧酸循环释放ATP产量降低可能是导致精子复苏后活力下降的原因。我们进一步检测了冷冻前后精子内ATP含量的变化、线粒体膜电势的变化及三羧酸循环通路中AC02下游产物异柠檬酸含量的变化,发现冷冻复苏后ATP、异柠檬酸的含量显著下降,线粒体膜电势显著降低,差异均有统计学意义。综上所述,通过对差异蛋白AC02的一系列研究,我们提出了以下观点,AC02作为线粒体蛋白在精子冷冻复苏后发生了蛋白降解,使得下游产物异柠檬酸的产量下降,三羧酸循环受抑制导致了ATP的产生下降,复苏后的精子活力受损。通过对该机制的阐述,为研究人类精子冷冻损伤机制奠定了良好的理论基础,并为进一步全面地应用蛋白质组学技术筛选、分离、鉴定精子蛋白提供了系统、且详细的研究方法,同时为临床改进精子冷冻保护剂改善精子冻后活力提供了改进思路和作用靶点。
[Abstract]:According to statistics, about 10 to 20 percent of married couples worldwide experience fertility difficulties, and infertility due to abnormal male reproductive capacity is close to 50 percent. With the rapid development of Chinese economy, the increase of harmful environmental factors and the influence of bad living habits, the quality of human semen is declining. Human sperm bank is a kind of body which uses cryopreservation technology to collect detect preserve and provide sperm for the purpose of treating infertility and preventing genetic diseases. The establishment of human sperm bank provides an alternative method for those with irreversible azoospermia, severe, weak, deformed spermatozoa and risk of hereditary diseases. However, the motility, viability, capacitation, acrosome reaction and other functions of human spermatozoa decreased after cryopreservation. The change of osmotic pressure inside and outside of sperm cells is the main reason for the destruction of sperm ultrastructure, but the change of ultrastructure can not directly explain the damage of sperm function. Since protein is an important executor of cell function, it is important to understand the changes of protein after cryopreservation and resuscitation in order to understand the mechanism of cryopreservation injury. Therefore, we used proteomics to study the differential proteomics of semen samples from 9 healthy volunteers in our central sperm bank before and after freezing. It was found that aconitate hydratase AC02 in the tricarboxylic acid cycle pathway may play an important role in human sperm cryopreservation injury. The results of immunoblotting showed that the expression of the protein in resuscitation spermatozoa was decreased, which was consistent with that in proteome. Immunofluorescence assay showed that it was mainly located in the mitochondrial sheath region in the middle part of spermatozoa. The review of literatures and protein localization information suggested that AC02 might be involved in the process of tricarboxylic acid cycle in mitochondria. The degradation of tricarboxylic acid cycle resulted in the decrease of ATP production after resuscitation, which may be the reason for the decline of sperm motility after resuscitation. We further detected the changes of ATP content, mitochondrial membrane potential and isocitric acid content in the downstream of AC02 in the tricarboxylic acid cycle pathway before and after cryopreservation. It was found that the content of isocitric acid decreased significantly after cryopreservation. Mitochondrial membrane potential decreased significantly, and the differences were statistically significant. To sum up, through a series of studies on differential protein AC02, we put forward the following point of view: as a mitochondrial protein, the protein degradation occurred after cryopreservation and resuscitation of spermatozoa, which resulted in a decrease in the yield of isocitric acid, the downstream product. Inhibition of the tricarboxylic acid cycle resulted in a decrease in ATP production and impaired sperm motility after resuscitation. Through the elaboration of the mechanism, it lays a good theoretical foundation for the study of the mechanism of human sperm freezing injury, and provides a system for the further application of proteomics technology in screening, isolation and identification of sperm proteins. The detailed research methods also provide an improved idea and target for clinical improvement of spermatozoa cryopreservation protection agent for improving sperm viability after freezing.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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