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Bax, Caspase-2, and Caspase-3 Are Required for Ovarian Follicle Loss Caused by 4-Vinylcyclohexene Diepoxide Exposure of Female Mice in Vivo

Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School (Y.T., J.C., G.I.P., J.K.P., J.L.T.), Boston, Massachusetts 02114; Department of Environmental Health, Boston University Schools of Medicine and Public Health (J.J.S., D.H.S.), Boston, Massachusetts 02118; Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center (R.N.K.), New York, New York 10021; Department of Cell Biology, Harvard Medical School (J.Y.), and Departments of Pathology and Medicine, Dana-Farber Cancer Institute/Harvard Medical School (S.J.K.), Boston, Massachusetts 02115; and Section of Immunobiology, Yale University School of Medicine (R.A.F.), New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Dr. Jonathan L. Tilly, Massachusetts General Hospital, VBK113-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jtilly{at}partners.org.

The industrial chemical, 4-vinylcyclohexene diepoxide (VCD), kills oocytes within immature follicles in the ovaries of mice and rats and is considered a potential occupational health hazard. It has been reported that VCD-induced follicle loss occurs via a cell death process involving elevated expression of Bax, a proapoptotic Bcl-2 family member, and increased caspase-3-like activity. We have previously shown that oocytes lacking acid sphingomyelinase (ASMase; an enzyme that generates the proapoptotic stress sensor ceramide), the aromatic hydrocarbon receptor (Ahr), Bax, or caspase-2 are resistant to apoptosis induced by other chemical toxicants. Therefore, this study was designed to investigate the functional importance of ASMase, Ahr, Bax, and caspase-2 as well as the related executioner enzyme caspase-3 to VCD-induced ovotoxicity in mice using gene knockout technology. For each gene mutant mouse line, wild-type and homozygous-null female siblings derived from heterozygous matings were given once-daily ip injections of either vehicle (sesame oil) or VCD (80 mg/kg body weight) for 15 d (three or four mice per treatment group per genotype). Ovaries were collected 24 h after the final injection and analyzed for the total number of nonatretic primordial and primary follicles remaining per ovary. No differences in the extent of primordial or primary follicle destruction resulting from VCD exposure were observed in wild-type vs. ASMase- or Ahr-deficient mice. By contrast, the extent of VCD-induced primordial follicle depletion in Bax-deficient mice (45 ± 11%) was significantly (P < 0.05) lower than that in wild-type females (85 ± 2%). The extent of primary follicle loss in bax-null mice exposed to VCD (3 ± 22%) was also significantly (P < 0.05) lower than that in their wild-type sisters (86 ± 4%). In caspase-2-deficient mice, significantly (P < 0.05) fewer oocyte-containing primary follicles were destroyed by VCD (17 ± 19%) vs. wild-type controls (71 ± 6%); however, no significant difference in the extent of VCD-induced primordial follicle destruction was observed in caspase-2-null vs. wild-type females. Finally, in caspase-3-deficient mice, significantly (P < 0.05) fewer oocyte-containing primary follicles were destroyed by VCD (33 ± 3%) vs. wild-type controls (71 ± 2%); however, no significant difference in the extent of VCD-induced primordial follicle destruction was observed in caspase-3-null vs. wild-type females. We conclude that Bax, caspase-2, and caspase-3, but not ASMase or Ahr, are functionally important in VCD-induced follicle loss. However, as a loss of Bax, caspase-2, or caspase-3 function conveyed only partial protection from the ovotoxic effects of VCD, other cell death pathways that either function independently of Bax, caspase-2, and caspase-3 or are not apoptotic in nature are also involved.

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