P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation

Abstract

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanisms underlying these radiosensitivity differences, p21(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21(Cip1/WAF1) is a cyclin-dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation. P21(Cip1/WAF1) expression was observed in the normal testis, using Western blotting analysis. After a dose of 4 Gy, but not after 0.3 Gy, an increase in p21(Cip1/WAF1) expression could be determined in whole testis lysates. To investigate which germ cells are involved in p21(Cip1/WAF1) protein expression, immunohistochemical analysis was performed on irradiated testis. In the normal testis a weak staining for p21(Cip1/WAF1) was found in pachytene spermatocytes in epithelial stage V up to step 5 spermatids. A dose of 4 Gy of X-irradiation resulted in a transient increase of p21(Cip1/WAF1) staining in these cells with a maximum at 6 h post irradiation, despite the fact that the irradiation did not induce an increase in the number of apoptotic spermatocytes. When a dose of 0.3 Gy was given, no increase in p21(Cip1/WAF1) staining was observed. Using the TUNEL technique, a 10-fold increase in apoptotic spermatogonia was found after a dose of 4 Gy. However, no staining for p21(Cip1/WAF1) was observed in spermatogonia, suggesting that these cells do not undergo a p21(Cip1/WAF1)-induced G1 arrest prior to DNA repair or apoptosis. These data imply that p21(Cip1/WAF1) is a factor which could be important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in spermatogonial cell cycle delay or apoptosis induction.