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

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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