The Mouse Fetal Ovary Has Greater Sensitivity Than the Fetal Testis to Benzo[a]pyrene-Induced Germ Cell Death

Abstract

The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary.

Keywords: apoptosis; benzo[a]pyrene; germ cells; ovary; polycyclic aromatic hydrocarbon; testis.

FIG. 1

Benzo[a]pyrene (BaP) induces germ cell loss in mouse fetal ovaries. Total number of germ cells was counted as described in Materials and Methods section in 13.5 dpc ovaries cultured for 0, 6, 24, or 48 h with 0, 50, 500, or 1000 ng/ml respectively, BaP in 0.005% DMSO. A, Mean ± SEM total number of germ cells per fetal ovary. Representative images of germ cells in control (B) and BaP-treated (C) ovaries at 48 h identified by TRA98 immunofluorescence. *P = .025, effect of concentration by linear regression. n = 3 − 5/group. Scale bars, 100 µm. UC, uncultured (0 h).

FIG. 2

Benzo[a]pyrene (BaP) exposure activates caspases-3 and -9, and increases germ cell expression of BAX protein in fetal ovaries. Germ cell apoptosis was assessed by cleaved caspase-3, -9, and BAX immunostaining in 13.5 dpc ovaries cultured for 0, 6, or 24 with 0, 50, 500, or 1000 ng/ml, respectively, BaP in 0.005% DMSO. A, Mean ± SEM percentage of cleaved caspase-3 positive germ cells at 6 and 24 h (*P = .007 effect of concentration by linear regression, **P = .018 vs control). B–C, Representative images of cleaved caspase-3 immunostaining in fetal ovaries at 24 h. D, Mean ± SEM percentage of cleaved caspase-9 positive germ cells at 6 and 24 h (*P = .007 effect of concentration by linear regression, **P < .05 vs control and 50 ng/ml BaP). E and F, Representative images of cleaved caspase-9 immunostaining in fetal ovaries at 24 h. G, Mean ± SEM percentage of BAX-positive germ cells at 6 and 24h (*P = .036 effect of concentration by linear regression, **P = .024 vs control). H and I, Representative images of fetal ovaries identified by BAX immunostaining at 6 h. Arrows point to caspase-positive germ cells (C and F) or BAX-positive germ cells (I). n = 3–4/group. Scale bars, 15 µm. UC, uncultured (0 h).

FIG. 3

Benzo[a]pyrene (BaP)-mediated germ cell death in fetal ovaries requires caspase activation. Germ cells were counted as described in Materials and Methods section in 13.5 dpc ovaries cultured for 48 h with 0.005% DMSO alone, 50 μM of z-VAD-fmk pretreatment followed by control media, 1000 ng/ml of BaP alone, or z-VAD-fmk pretreatment followed by BaP. A, Mean ± SEM total number of germ cells per ovary. Representative images of germ cells identified by TRA98 immunodetection in fetal ovaries cultured with z-VAD-fmk (B); 1000 ng/ml BaP (C); z-VAD-fmk and 1000 ng/ml BaP cotreatment (D). n = 6–7/group. Scale bars, 100 µm. *P < .05 compared with vehicle and z-VAD-fmk control. UC, uncultured (0 h).

FIG. 4

Benzo[a]pyrene (BaP) does not affect germ cell proliferation in mouse fetal ovaries. Germ cell proliferation was assessed by Ki67 immunostaing in the same ovaries as for Figure 3. A, Mean ± SEM percentage of Ki67 positive germ cells at 0 and 48 h. Representative images of germ cells identified by Ki67 immunodetection in fetal ovaries: 0 h uncultured (B); 0.005% DMSO (C); z-VAD-fmk (D); 1000 ng/ml BaP (E); z-VAD-fmk and 1000 ng/ml BaP cotreatment (F). n = 4–5/group. Ki67 positive cells are dark brown in online version. Arrows point to examples of meiotic germ cells typical of the leptotene stage.

FIG. 5

Benzo[a]pyrene (BaP) does not deplete germ cells in cultured 13.5 dpc mouse fetal testes. A, Germ cells were counted as described in Materials and Methods section in 13.5 dpc testes cultured for 0, 6, 24, or 48 h with 0, 50, 500, or 1000 ng/ml, respectively, BaP in 0.005% DMSO. Mean ± SEM total number of germ cells identified by TRA98 immunodetection. B, Mean ± SEM percentage of apoptotic germ cells identified by immunohistochemical staining of cleaved caspase-3. n = 3–5/group. UC, uncultured (0 h).