Expression and redistribution of cellular Bad, Bax, and Bcl-xL protein is associated with VCD-induced ovotoxicity in rats

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25-100 mum) and large (100-250 mum) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 +/- 0.16; mitochondria, 2.20 +/- 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-x(L) protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 +/- 0.08; cytosol, 1.39 +/- 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day, 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-x(L) in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 +/- 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage I primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-x(L) protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-x(L) ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.