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Identification and function analysis of glycoprotein in lamina propria of human testis showing deteriorated spermatogenesis

人睾丸固有层糖蛋白的鉴定和功能分析显示精子发生恶化

基本信息

批准号：
17591706
负责人：
IWAMOTO Teruaki
金额：
$ 2.18万
依托单位：
St.Marianna University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591706/
关键词：
spermatogenesis lamina propria glycoprotein development-differentiation cell line

项目摘要

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 showed the property to bind progesterone. To clarify the role of lamina propria in spermatogenesis, we performed further analysis to identify products. 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 blotting membrane was pre-incubated in the solution with or without progesterone, and then the positive spots were screened by PNA-lectin affinity. This screening method could select the spots having both PNA-lectin affinity and progesterone binding affinity. However, we need to know the optimal condition for this screening method to determine the concerned glycoproteins in lamina propria because there are still several spots. As another projects, we tried to establish the immortalized cell line derived from human testis to investigate the synthesis of glycoproteins under cellular level. Co-culture of the primary cultured human testicular cells and mouse Sertoli cell lines (A31C4) allowed us to maintain the human spermatogonia and Sertoli cells. Furthermore, the Sertoli cells from adult human testis could proliferate under the co-culture condition. Further steps for separation of Sertoli cells derived from human and mouse are needed, however, we believe that this co-culture system will contribute to the establishment of cell line from human testicular cells and the induction of differentiation of human spermatogonia in vitro.

睾丸曲细精管固有层增厚是人类睾丸独特的形态特征，显示精子发生恶化。此前，我们发现增厚的固有层内层含有PNA-凝集素识别的糖蛋白，并显示出结合黄体酮的特性。为了阐明固有层在精子发生中的作用，我们进行了进一步的分析来鉴定产物。将来自显示几乎正常精子发生的患者和显示恶化精子发生的患者的睾丸样本均质化并溶解。通过二维凝胶电泳分析溶解的蛋白质。将印迹膜在有或没有孕酮的溶液中预孵育，然后通过PNA-凝集素亲和力筛选阳性斑点。该筛选方法可以选择同时具有PNA-凝集素亲和力和孕酮结合亲和力的点。然而，我们需要知道这种筛选方法的最佳条件来确定固有层中的相关糖蛋白，因为仍然有几个点。作为另一个项目，我们试图建立源自人类睾丸的永生化细胞系，以研究细胞水平下糖蛋白的合成。原代培养的人类睾丸细胞和小鼠支持细胞系（A31C4）的共培养使我们能够维持人类精原细胞和支持细胞。此外，来自成人睾丸的支持细胞可以在共培养条件下增殖。需要进一步分离来自人和小鼠的支持细胞，然而，我们相信这种共培养系统将有助于从人睾丸细胞建立细胞系并在体外诱导人精原细胞分化。