Quantification of apoptotic testicular germ cells in normal and methoxyacetic acid-treated mice as determined by flow cytometry

Abstract

Several studies have reported the occurrence and significance of programmed cell death (apoptosis) of testicular germ cells in mammals. In those studies, apoptotic germ cells were identified by morphological criteria or by in situ end labeling (TUNEL) and were enumerated from histological sections by semi-quantitative and time-consuming techniques. In the present study, we have established a flow cytometric technique for quantification of TUNEL-positive cells in the mouse testis. Groups of five adult mice each received 0, 650, or 1300 mg/kg (IP) of methoxyacetic acid (MAA), and testes were collected 24 hours later. MAA is known to induce germ cell apoptosis in rodent testes. MAA induced a significant (P < 0.01) dose-dependent decline in the percentage of pachytene spermatocytes (4C cells). DNA strand breaks generated by the activation of endogenous endonuclease in the apoptotic germ cells were detected by the in situ labeling of the 3'-OH termini with biotinylated dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL technique). Histologically, TUNEL-positive germ cells were observed in control testes, and the number of these cells was visibly increased following MAA exposure. As determined by flow cytometry, four cell populations contained TUNEL-positive cells: 1C cells (round spermatids), 2C cells (mainly spermatogonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes synthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analysis of the percentages of TUNEL-positive cells within each population yielded values of 1.57 ± 0.23% for 1C cells, 1.65 ± 0.27% for 2C cells, 6.26 ± 1.03% for S-ph cells, and 3.24 ± 0.39% for 4C cells. Hence, a substantial proportion of proliferating cells are undergoing apoptosis during normal spermatogenesis. The overall incidence of apoptotic cells among all testicular cells was around 2%. At 650 mg per kilogram of body weight, MAA induced a fourfold to eightfold increase (P < 0.001)in the percentage of TUNEL-positive cells, compared with saline-treated controls, and, overall, 17% of testicular cells were apoptotic. This effect of MAA was most pronounced for S-ph and 4C cells, with 25-30% of cells being affected in each of those populations. At 1300 mg per kilogram of body weight, MAA had no further effect. These quantitative data demonstrate that 1) in the normal testis, it is mainly proliferating cells that undergo apoptosis, and 2) MAA induces primary spermatocyte loss by germ cell apoptosis.