title: Enhanced expression of bax in small preantral follicles during 4-vinylcyclohexene diepoxide-induced ovotoxicity in the rat.
abstract: 4-Vinylcyclohexene diepoxide (VCD) destroys small preantral (25-100 microns) ovarian follicles after repeated dosing in mice and rats. A previous study determined this follicular destruction is via apoptosis (physiological cell death). The purposes of this study were to examine the effects of VCD on amounts of mRNA for several genes that might be involved in this ovotoxic response and to determine the specificity of this response for small preantral follicles. The genes of interest were bax, a cell death gene; three forms of the antioxidant enzyme, superoxide dismutase (mitochondrial manganese-containing or MnSOD, cytosolic copper/zinc-containing or Cu/ZnSOD, and secreted or secSOD); and microsomal epoxide hydrolase (mEH), involved in detoxification of VCD. Female Fischer 344 rats were administered daily doses (10 days) of vehicle control (sesame oil) or VCD (80 mg/kg, ip). Four hours after the last injection, livers and ovaries were removed. Small (25-100 microns) and large (100-250 microns) preantral follicles were separated from the ovaries by gentle dissociation and collected by mouth pipeting. Total RNA was extracted from all tissues, reverse transcribed into first-strand cDNA, and amplified by polymerase chain reaction using oligonucleotide primers specific for each gene. Relative levels of mRNA were visualized by agarose gel electrophoresis and autoradiography and quantified by densitometric analysis. Coamplification of ribosomal protein L19 (constitutively expressed in ovarian tissue) was used for normalization in each sample. Increased levels of mRNA for bax (172 +/- 20% of control, p < 0.05), MnSOD (248 +/- 70% of control, p < 0.05), and mEH (352 +/- 120% of control, p < 0.05) were measured in 25- to 100-microns follicles collected from VCD-treated compared with control rats. Unlike 25- to 100-microns follicles (the targets of ovotoxicity), in 100- to 250-microns follicles (nontargets) there were no changes (p > 0.05) in mRNA levels for bax or MnSOD in VCD-treated rats; however, mRNA levels for mEH were significantly decreased (79 +/- 4% of control, p < 0.05), compared with control. No changes in levels of mRNA for mEH were observed in liver from VCD-treated rats relative to control. Additionally, in liver VCD caused a significant decrease in mRNA levels for bax (31 +/- 5% of control, p < 0.05) and Cu-ZnSOD (56 +/- 17% of control, p < 0.05). In summary, dosing of rats with VCD enhanced expression of mRNA encoding several genes that might respond during the induction of ovotoxicity. The selective increase in bax in the population of follicles destroyed by repeated dosing with VCD may reflect their susceptibility to apoptosis.
authors.affiliation: Department of Physiology, University of Arizona, Tucson 85724, USA.
authors.forename: L N
authors.initials: LN
authors.lastname: Springer
authors.forename: J L
authors.initials: JL
authors.lastname: Tilly
authors.forename: I G
authors.initials: IG
authors.lastname: Sipes
authors.forename: P B
authors.initials: PB
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002273:Carcinogens / Q000008:administration & dosage / Q000633:toxicity*; D003510:Cyclohexanes / Q000008:administration & dosage / Q000633:toxicity*; D053138:Cyclohexenes; D004851:Epoxide Hydrolases / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D007274:Injections, Intraperitoneal; D008969:Molecular Sequence Data; D006080:Ovarian Follicle / Q000187:drug effects*; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011916:Rats, Inbred F344; D013482:Superoxide Dismutase / Q000378:metabolism; D014753:Vinyl Compounds / Q000008:administration & dosage / Q000633:toxicity*; D051028:bcl-2-Associated X Protein
chemical_list: C494828:Bax protein, rat; D002273:Carcinogens; D003510:Cyclohexanes; D053138:Cyclohexenes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014753:Vinyl Compounds; D051028:bcl-2-Associated X Protein; C012606:4-vinyl-1-cyclohexene dioxide; D013482:Superoxide Dismutase; D004851:Epoxide Hydrolases