Expression of Rap1 during germ cell development in the rat and its functional implications in 2-methoxyacetic acid-induced spermatocyte apoptosis

Abstract

Objective: To investigate the expression pattern of Ras-related protein 1 (Rap1) during testicular development and to clarify whether its expression is developmentally regulated and whether this expression is involved in the process of germ cell apoptosis.

Materials and methods: The expression pattern of Rap1 in the adult rat testicle was compared with that in the developing rat testicle using immunoblotting and immunohistochemical analyses. After the adult rats were treated with methoxyacetic acid (MAA), which selectively depletes primary spermatocytes, we correlated Rap1 expression with apoptotic dynamics using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), double immunofluorescent staining, and coimmunoprecipitation assays.

Results: During testicular development, Rap1 was expressed in the nucleus of gonocytes and in the Golgi apparatus of spermatocytes. The expression pattern of Rap1 during spermatogenesis was also shown to be stage specific. After 12 hours of MAA treatment, we found that Rap1 was translocated into the nucleus of some spermatocytes and the overlapping rate of Rap1 and NF-κB was much greater than that of Rap1 and TUNEL staining. In addition, Rap1 protein could be co-immunoprecipitated with NF-κB protein.

Conclusion: In cooperation with NF-κB, Rap1 might be involved in the early stage of the apoptotic process occurring in the MAA-treated rat testicle. Additional characterization of this small guanosine triphosphatase in the apoptotic dynamics should provide information on the early diagnosis of MAA-induced male infertility.