Role of Glutathione S-Transferase Class Mu During VCD-Induced Ovotoxicity in Neonatal Rat Ovaries.

Abstract

4-vinylcyclohexene diepoxide (VCD) is an occupational chemical that selectively destroys ovarian small pre-antral follicles in both rats and mice via apoptosis. Glutathione S-transferase (GST) enzymes catalyze glutathione (GSH) conjugation to xenobiotics. In cultured neonatal mouse ovaries exposed to VCD, mRNA encoding the GST isoform mu (GSTm) increased (d4) prior to the onset of follicle loss (d6). GST protein can also regulate cell signaling, and in extra-ovarian tissues GSTM prevents the action of apoptosis signal-regulating kinase 1 (ASK1) through formation of a protein complex. Upon xenobiotic exposure, this protein complex dissociates resulting in activation of pro-apoptotic signaling by ASK1. This study was designed to investigate an involvement of GSTm in VCD-induced ovotoxicity in rats. Post-natal day 4 (PND4) rat ovaries were incubated in control medium ± VCD (30 μM) for 2, 4, 6 or 8 d. Following culture, total RNA or protein were isolated (n=3; 10-20 ovaries per pool). GSTm mRNA and protein levels were determined using quantitative RT-PCR and Western blotting, respectively. To assess the presence of an GSTM:ASK1 protein complex, immunoprecipitation using an anti-ASK1 primary antibody was followed by Western blotting to measure GSTM protein level. There was no effect of VCD exposure on Gstm mRNA expression at any time point. Despite lack of a transcriptional effect GSTM protein was increased (P < 0.05) by VCD exposure after 6 and 8 d, relative to control. A GSTM:ASK1 protein complex was detected in the rat ovary. Relative to control, there was no effect of VCD on the GSTM:ASK1 protein complex after 4 d of exposure. GSTM-bound to ASK1 increased (P < 0.05) after 6 d of VCD exposure, when follicle loss is underway. Because there was no increase in the amount of GSTM protein unbound to ASK1, this indicates that the increase in total GSTM reflected an increase in the ASK1-bound form. These findings support that GSTM protein is involved in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. (Supported by ES016818 to AFK, ES09246 to PBH).