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

在这项研究中，我们试图对所有在人类精子中表达的蛋白质进行图谱分析，使用凝集素分析糖蛋白，并研究冷冻保存的效果、与不育的关联以及精子功能与磷酸化的关系。我们从人和小鼠精子膜中提取的蛋白质分别观察到560个和318个独立的点，并分别鉴定出118个和94个点所代表的蛋白质。在小鼠和人类精子中鉴定出的蛋白质有：钙网蛋白和HSP90，以及烟碱型乙酰胆碱受体、G蛋白偶联受体ZFP35和cAMP反应元件结合蛋白。在小鼠模型中，我们研究了精子中通过cAMP的信号转导系统与acr…的关系。更多的腔体反应。我们用PKA刺激物，即H8，在^&lt；32&gt；P-ATP存在下培养激活的精子。这引起了顶体反应，并在放射自显影中观察到了磷酸化增强的45-kD蛋白。我们目前正在分析这种蛋白质以确定其身份。联系临床方面，我们发现不育男性的样本可以根据凝胶上出现的斑点图案大致分为三组。目前，我们正在鉴定三组之间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的图谱比较显示，冷冻后有4个斑点的蛋白质数量明显减少。我们试图用凝集素染色来检测糖蛋白，但没有成功。未来，我们可能需要采用不同的方法来用凝集素对转录膜进行染色，例如将处理后的提取液在凝集素柱中显色。较少

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