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Proteome analysis of all proteins in the spermatic membrane using lectin, and relationship with spermatic functions

使用凝集素对精索膜中所有蛋白质进行蛋白质组分析，及其与精索功能的关系

基本信息

批准号：
15591777
负责人：
KUJI Naoaki
金额：
$ 2.24万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591777/
关键词：
human sperm proteome protein phosphorylation cryopreservation glycoprotein 質量分析アミノ酸配列解析

项目摘要

In this study, we tried to profile all the proteins expressed in the human sperm, analyzed the glycoproteins using lectin, and examined the effects of cryopreservation, association with infertility, and the relationship between spermatic functions and phosphorylation.We observed 560 and 318 independent spots, respectively, for proteins extracted from the human and mouse spermatic membranes, and were able to identify the proteins represented by 118 and 94 spots, respectively. The proteins identified in both mouse and human sperm were as follows : calreticulin and HSP90, the existence of which has already been confirmed by previous reports, as well as the nicotinic acetylcholine receptor, G-protein coupled receptor, ZFP35, and a cAMP response element binding protein. These findings are expected to contribute usefully to elucidation of spermatic functions in the future.In the mouse model, we examined the relationship between the signal transduction system via cAMP in the sperm and the acr … More osomal responses. We cultured sperm activated with a PKA stimulator, i.e., H8, in the presence of ^<32>P-ATP. This induced an acrosomal response, and a 45-kD protein with intensified phosphorylation was observed on autoradiography. We are currently analyzing this protein to determine its identity.Jn relation to the clinical aspect, we found that specimens from infertile males could be roughly divided into three groups according to the spot patterns appearing on the gel. At present, we are in the process of identifying the proteins present in markedly different quantities among the three groups, and analyzing the association of the expression pattern with the spermatic functions. Pattern comparison of the proteins extracted from the human spermatic membrane before and after freezing revealed four spots exhibiting obvious decrease in quantity after the cryopreservation. We did not succeed in our attempt to detect glycoproteins by lectin staining. In the future, we may need to adopt a different approach to stain the transcribed membrane with lectin, such as developing the extracted solution after treatment in the lectin column. Less

在这项研究中，我们试图分析人类精子中表达的所有蛋白质，用凝集素分析糖蛋白，并检查冷冻保存的效果，与不育的关系，以及精子功能和磷酸化之间的关系。我们观察到560和318个独立的斑点，分别从人类和小鼠精子膜提取的蛋白质，分别鉴定出118和94个蛋白质点。在小鼠和人类精子中鉴定的蛋白质如下：钙网蛋白和HSP 90，其存在已经被先前的报道证实，以及烟碱乙酰胆碱受体、G蛋白偶联受体、ZFP 35和cAMP反应元件结合蛋白。在小鼠模型中，我们研究了精子中cAMP信号转导系统与acr的关系，并探讨了精子中cAMP信号转导系统与acr的关系。 ...更多信息体细胞反应我们培养了用PKA刺激剂激活的精子，H8，在13 P-ATP的存在下<32>。这诱导了顶体反应，并观察到一个45 kD的蛋白与增强磷酸化放射自显影。我们目前正在分析这种蛋白质，以确定其身份。在临床方面，我们发现，不育男性的标本可以大致分为三组，根据出现在凝胶上的斑点图案。目前，我们正在鉴定这三组中存在明显不同数量的蛋白质，并分析表达模式与精子功能的关系。冷冻前后人精子膜蛋白质图谱比较显示，冻存后有4个蛋白质点数量明显减少。我们试图用凝集素染色法检测糖蛋白，但没有成功。将来，我们可能需要采用不同的方法用凝集素染色转录膜，例如在凝集素柱中处理后显影提取液。少