CYCLOPHILIN-A IS PRESENT IN RAT GERM-CELLS AND IS ASSOCIATED WITH SPERMATOCYTE APOPTOSIS

Recent investigations in our laboratory revealed divalent cation-depen dent endonuclease activity in testes from 2-methoxyethanol-treated rat s, which was able to cleave substrate DNA into a pattern of DNA fragme ntation consisting of similar to 180-200 base pairs. Further studies w ere undertaken to characterize the active nuclease. F344 rats were tre ated with 2-methoxyethanol, a glycol ether that causes the death of pa chytene spermatocytes in juvenile and adult rats. The active nuclease was found in nuclear extract from treated animals, but not controls. A radioactive gel nuclease assay, which detects degradation and loss of P-32-labeled DNA from a DNA-containing polyacrylamide gel, localized the nuclease activity to a band of approximate to 18 kDa. This activit y was dependent on calcium and was inhibited by both zinc and aurintri carboxylic acid. Amino acid sequence data showed that this protein was identical to cyclophilin A. Immunohistochemistry using antibodies aga inst cyclophilin A found specific staining in pachytene spermatocytes, spermatids, interstitial cells, and Sertoli cell nuclei. Cyclophilin A staining was present in both control and 2-methoxyethanol-treated ra t testes in a stage-dependent manner, with pachytene spermatocytes in stage-VIII-XIV seminiferous tubules most heavily stained. These data d emonstrate that rat testis germ cells contain relatively high levels o f cyclophilin A whose nuclease activity is associated with spermatocyt e apoptosis induced by 2-methoxyethanol.