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特有的形态特征，表现为生精功能减退。以前，我们发现增厚的固有层内层有PNA-凝集素识别的糖蛋白，并包括抗孕酮抗体检测到的孕酮。然而，没有关于固有层内层产物的其他信息。为了阐明固有层在精子发生中的作用，我们需要确定内层增加的产物。结合免疫组织化学中睾丸细胞和固有层的信息，我们建立了一种分离PNA-凝集素识别的糖蛋白的筛选方法。取自一名精子发生几乎正常的患者和一名精子发生恶化的患者的睾丸样本被匀浆和溶解。用双向凝胶电泳法对溶解的蛋白质进行分析。核酸-凝集素亲和印迹法检测…阳性斑点用IV型胶原蛋白和波形蛋白V9抗体免疫印迹筛选出更多的ND阴性斑点。其中一个斑点为43 kDa，等电点为5-6，经LC-MS/MS和吉祥物搜索，确定为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的相关糖蛋白，需要额外的信息。通过对增厚的固有层结合物质的研究，发现增厚的固有层具有与黄体酮结合的特性，该糖蛋白也能与黄体酮结合。我们继续用新的方法分析糖蛋白，新方法包含了额外的糖蛋白性质的步骤。作为另一个项目，我们试图建立人睾丸永生化细胞系，在细胞水平上研究糖蛋白的合成。首先，选择生长良好的原代培养的睾丸细胞，然后以绿色荧光蛋白为标记检测其转染率。我们发现抗感染病毒载体最适合我们的需要。较少

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