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

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，增厚的固有层内层有糖蛋白识别的PNA-凝集素，并显示出属性结合孕酮。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，以确定产品。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。将印迹膜在含或不含孕酮的溶液中预孵育，然后通过PNA-凝集素亲和筛选阳性斑点。该筛选方法可筛选出同时具有PNA-凝集素亲和性和孕酮亲和性的斑点。然而，我们需要知道这种筛选方法的最佳条件，以确定有关的糖蛋白在固有层，因为仍然有几个斑点。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。将原代培养的人睾丸细胞与小鼠Sertoli细胞系（A31 C4）共培养，使我们能够保持人精原细胞和Sertoli细胞。成人睾丸支持细胞在共培养条件下可增殖。人和小鼠Sertoli细胞的分离尚需进一步的研究，但我们相信这种共培养体系将有助于人睾丸细胞系的建立和人精原细胞的体外诱导分化。

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.