Age-related decrease in active caspase-3 expression in germ cells of human testes

Abstract

OBJECTIVE: Advancing paternal age has been implicated in a broad range of abnormal reproductive and genetic outcomes, including diminished semen quality, increased DNA damage and reduced fertility. Suggested mechanisms include defective sperm chromatin packaging, disordered apoptosis, and oxidative stress. To clarify whether a deregulation of apoptosis could be involved in the increased incidence of sperm DNA damage in aged men, we examined age-related changes in the expression of active caspase-3, -8 and -9. We further assessed the effect of ageing on the expression of poly(ADP-ribose)polymerase-1 (PARP-1). DESIGN: Prospective comparative study. MATERIALS AND METHODS: Testicular tissue (orchidectomy/testicular biopsies) was obtained from 15 patients (mean age 60.2 years) with advanced prostate cancer and from 9 patients (mean age 38.4 years) with obstructive azoospermia (OA) showing normal spermatogenesis (controls). Immunohistochemical (IHC) and Western blot analyses were used for detection of testicular expression of caspase-3 (17/19 kDa). The number of caspase positive cells (IHC) was expressed relative to the total number of germ cells in over 20 seminiferous tubules (staining index). Cleaved caspases -8 (41/43kDa), -9 (37kDa) and PARP-1 (89kDa) were detected by Western blot. RESULTS: Immunolocalization of caspase-3 was detected in spermatogonias, primary spermatocytes and early spermatids. A significant decrease in the staining index was observed in testis sections of aged patients (8.1±1.7/18.0±5.0). In this group, the number of round spermatids positive for caspase-3 was remarkably decreased as compared with controls (3.0±0.6/15.1±5.6). In OA controls, immunostaining for caspase-3 was occasionally observed in Sertoli cells whereas this stain was higher (but not significant) in the aged group. Western blot analysis confirmed that active caspase-3 decrease (p< 0.001) in the testes of aged men. There were no significant changes in the levels of caspase-8 (extrinsic pathway of apoptosis) between both group of patients, whereas the expression of activated caspasa-9 (intrinsic apoptotic pathway) showed an overall decrease (p=0.054) in the aged group. Cleaved PARP also declined significantly (p<0.001) with age. CONCLUSIONS: Advancing male age is associated with a significant decrease in the expression levels of caspase-3 and PARP-1. In this context, down-regulation of two key proteins involved in apoptosis might be associated with increased frequencies of sperm DNA damage in spermatozoa.