H. Krishnamurthy et al., Quantification of apoptotic testicular germ cells in normal and methoxyacetic acid-treated mice as determined by flow cytometry, J ANDROLOGY, 19(6), 1998, pp. 710-717
Several studies have reported the occurrence and significance of programmed
cell death (apoptosis) of testicular germ cells in mammals. In those studi
es, apoptotic germ cells were identified by morphological criteria or by in
situ end labeling (TUNEL) and were enumerated from histological sections b
y semi-quantitative and time-consuming techniques. In the present study, we
have established a flow cytometric technique for quantification of TUNEL-p
ositive 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 col
lected 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 genera
ted by the activation of endogenous endonuclease in the apoptotic germ cell
s were detected by the in situ labeling of the 3'-OH termini with biotinyla
ted dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL te
chnique). Histologically, TUNEL-positive germ cells were observed in contro
l 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 sperma
togonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes s
ynthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analy
sis of the percentages of TUNEL-positive cells within each population yield
ed 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 substa
ntial proportion of proliferating cells are undergoing apoptosis during nor
mal spermatogenesis. The overall incidence of apoptotic cells among all tes
ticular cells was around 2%. At 650 mg per kilogram of body weight, MAA ind
uced a fourfold to eightfold increase (P < 0.001) in the percentage of TUNE
L-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 ef
fect. These quantitative data demonstrate that 1) in the normal testis, it
is mainly proliferating cells that undergo apoptosis, and 2) MAA induces pr
imary spermatocyte loss by germ cell apoptosis.