Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades.
Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25-100 microm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 +/- 0.23; Day 15, 3.25 +/- 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.
Hu
X
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Departments of Physiology and Pharmacology and Toxicology, Southwest Environmental Health Sciences Center, The University of Arizona, Tucson, Arizona 85724, USA.
Christian
P J
PJ
Thompson
K E
KE
Sipes
I G
IG
Hoyer
P B
PB
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism*; D002458:Cell Fractionation; D003510:Cyclohexanes / Q000494:pharmacology*; D053138:Cyclohexenes; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000096:biosynthesis; D005260:Female; D005455:Fluorescent Antibody Technique; D007202:Indicators and Reagents; D008928:Mitochondria / Q000378:metabolism; D006080:Ovarian Follicle / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D014753:Vinyl Compounds / Q000494:pharmacology*
D003510:Cyclohexanes; D053138:Cyclohexenes; D004792:Enzyme Precursors; D007202:Indicators and Reagents; D014753:Vinyl Compounds; C012606:4-vinyl-1-cyclohexene dioxide; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases