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

睾丸精小管固有层增厚是人类睾丸的独特形态特征，表明精子发生恶化。在此之前，我们发现增厚的固有层内层含有可被pna -凝集素识别的糖蛋白，并具有结合黄体酮的特性。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以确定产物。精子发生几乎正常的患者和精子发生恶化的患者的睾丸样本均质化和溶解。通过二维凝胶电泳对溶解蛋白进行分析。将印迹膜在含或不含黄体酮的溶液中预孵育，然后通过pna -凝集素亲和力筛选阳性斑点。这种筛选方法可以选择具有pna -凝集素亲和力和黄体酮结合亲和力的斑点。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固有层，因为仍然有几个斑点。另一方面，我们试图建立人睾丸永生化细胞系，研究细胞水平下糖蛋白的合成。将原代培养的人睾丸细胞与小鼠Sertoli细胞系（A31C4）共培养，可以维持人精原细胞和Sertoli细胞。成人睾丸支持细胞在共培养条件下具有增殖能力。人类和小鼠支持细胞的分离还需要进一步的步骤，但是我们相信这种共培养系统将有助于人类睾丸细胞细胞系的建立和体外诱导人类精原细胞的分化。

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.