Molecular and cellular biological mechanism of the deteriorated spermatogenesis : analysis of glycoprotein in basal membrane of seminiferous tubules.

睾丸曲细精管固有层增厚是人类睾丸生精功能下降的独特形态学特征。以前，我们发现，内层增厚的固有层的糖蛋白识别的PNA-凝集素，包括孕酮检测抗孕酮抗体。但是，没有关于固有层内层产品的其他信息。为了阐明固有层在精子发生中的作用，我们需要鉴定在内层增加的产物。结合免疫组织化学对睾丸细胞和固有层的信息，我们建立了一种筛选PNA-凝集素识别的糖蛋白的方法。将来自显示几乎正常的精子发生的患者和显示恶化的精子发生的患者的睾丸样品均质化并溶解。通过2-D凝胶电泳分析溶解的蛋白。PNA-凝集素亲和印迹法检测阳性斑点， ...更多信息 用IV型胶原和波形蛋白V9抗体免疫印迹法筛选阴性斑点。其中一个景点是M. W。LC-MS/MS和MASCOT检索结果表明，该蛋白分子量为43 kDa，等电点为5-6，是一种新的白蛋白样蛋白。然而，这种筛选方法不足以确定固有层中相关的糖蛋白，需要额外的信息。在对增厚的固有层的结合物质进行研究后，增厚的固有层显示出结合孕酮的性质，并且该糖蛋白也可以结合孕酮。我们继续用新的方法分析糖蛋白，包括糖蛋白性质的额外步骤。另一个研究项目是建立人睾丸组织来源的永生化细胞系，从细胞水平研究糖蛋白的合成。首先，选择生长较好的原代培养睾丸细胞，然后使用EGFP作为标记物检查转染效率。我们发现抗感染病毒载体是最适合我们需要的。少

Thickened lamina propria in seminiferous tubules of the testis is unique morphological feature of human testis showing deteriorated spermatogenesis. Previously, we showed that inner layer of thickened lamina propria had glycoproteins recognized by PNA-lectin and included progesterone detected by antiprogesterone antibody. However, there was no other information about the product of the inner layer of lamina propria. To clarify the role of lamina propria in spermatogenesis, we need to identify products which are increased in the inner layer. Combining information about the cells and lamina propria in testis by immunohistochemistry, we developed a screening method of isolating glycoproteins recognized by PNA-lectin. Testicular samples from a patient showing almost normal spermatogenesis and a patient showing deteriorated spermatogenesis were homogenized and solubilized. The solubilized proteins were analyzed by 2-D gel electrophoresis. The positive spots by PNA-lectin affinity blotting a … More nd negative spots by collagen IV and vimentin V9 Ab western blotting were screened. One of the spots was M.W. 43kDa with a pI of 5-6 and was thought to be a novel albumin like protein from the results by LC-MS/MS and MASCOT search. However, this screening method was insufficient to determine the concerned glycoproteins in lamina propria and additional informations were needed. After investigation of binding substances to the thickened lamina propria, thickened lamina propria showed the property to bind progesterone and this glycoprotein also could bind to progesterone. We continues the analysis of the glycoproteins by new method contained the additional steps for properties of glycoproteins. As another projects, we tried to establish the immortalized cell line derived from human testis to investigate the synthesis of glycoproteins under cellular level. First, primary cultured testicular cells showing better growth were selected and then examined the efficiency of transfection using EGFP as a marker. We found that anti-infectous virus vector is optimum for our needs. Less