The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. to regulate on d4-d8; whereas GSTp protein was increased (< 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control the amount of GSTp-bound JNK was increased (= 0.09) while unbound JNK was decreased (< 0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis. < 0.05) mRNA encoding GST classes pi (GSTp) and mu (GSTm) on d4 by 1.55- and 1.7-fold respectively. Yet this effect was reversed on d6 and d8. GSTp but not GSTm protein was elevated by 47% after 8d of VCD exposure. It was hypothesized that in mice despite early up-regulation of GST repeated VCD exposure eventually overwhelmed the induction of GST enzymes thereby reducing its detoxification capacity during the onset of ovotoxicity (Keating culture system has been developed using ovaries from PND4 rats (enriched in small pre-antral follicles) to examine effects of ovotoxicants without a metabolic influence from the liver (Devine ovary culture system. The formation of an ovarian GSTp and JNK-containing protein complex was also investigated. Materials and Methods Reagents VCD (mixture of isomers >99% purity) 2 30 acrylamide/0.8% bis-acrylamide ammonium persulfate glycerol N’ Mouse monoclonal to CD9.TB9a reacts with CD9 ( p24), a member of the tetraspan ( TM4SF ) family with 24 kDa MW, expressed on platelets and weakly on B-cells. It also expressed on eosinophils, basophils, endothelial and epithelial cells. CD9 antigen modulates cell adhesion, migration and platelet activation. GM1CD9 triggers platelet activation resulted in platelet aggregation, but it is blocked by anti-Fc receptor CD32. This clone is cross reactive with non-human primate. N’ N’ N’-Tetramethyl-ethylenediamine (TEMED) Tris base Tris HCl sodium chloride Tween-20 bovine serum albumin (BSA) ascorbic acid (Vitamin C) phosphatase inhibitor protease inhibitor and transferrin were purchased from Sigma-Aldrich Inc. (St Louis MO). Dulbecco’s Modified Eagle Medium: nutrient mixture F-12 (Ham) 1X (DMEM/Ham’s F12) albumax penicillin/streptomycin (5000U/ml 5000 μg/ml respectively) Hanks’ Balanced Salt Solution (without CaCl2 MgCl2 or MgSO4) custom designed primers and superscript III one-step RT-PCR Fasiglifam System were obtained from Invitrogen Co. (Carlsbad CA). Millicell-CM filter inserts anti-p-c-Jun and anti-GSTp ant ibodies were purchased from Millipore (Bedford MA). 48 well cell culture plates were obtained from Corning Inc. (Corning NY). RNeasy Mini kit QIAshredder kit RNeasy MinElute kit and Quantitect? SYBR Green PCR kit were purchased from Qiagen Inc. (Valencia CA). Anti-JNK antibody was purchased from Santa Cruz Biotechnology (Santa Cruz CA). Anti-ACTB antibody and agarose G beads were purchased from Santa Cruz Biotechnology (Santa Cruz CA). Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Pierce Biotechnology (Rockford IL). Animals A Fasiglifam breeding Fasiglifam colony was established from Fischer 344 (F344) rats that were originally purchased from Harlan Laboratories (Indianapolis IN) to use as a source of PND4 female rat pup ovaries for culture. All pregnant animals were housed singly in plastic cages and maintained in a controlled environment (22 ± 2°C; 12h light/ 12h dark cycles). Animals were provided a standard diet with access to food and water and allowed to give birth. All animal experiments were approved by the University of Arizona’s Institutional Animal Care and Use Committee. ovarian culture Ovaries from PND4 F344 rats were cultured as described by Devine rat ovary culture system (Keating culture ovaries treated with control or VCD (30 μM) were stored in RNAat ?80°C. Total RNA was isolated (n=3; 10 Fasiglifam ovaries per pool) using an RNeasy Mini kit. Briefly ovaries were lysed and homogenized using a motor pestle followed by applying the mixture onto a QIAshredder column. The QIAshredder column containing ovarian tissue sample was then centrifuged at 14 0 rpm for 2 min. The resulting flow-through was applied to an RNeasy mini column allowing RNA to bind to the filter cartridge. Following washing RNA was eluted from the filter and concentrated using an RNeasy MinElute kit. Briefly isolated RNA was applied to an RNeasy MinElute spin column and after washing RNA was eluted using 14 μL of RNase-free water. RNA concentration was determined using an ND-1000 Spectrophotometer (λ = 260/280nm; NanoDrop technologies Inc. Wilmington DE). First strand cDNA synthesis and real-time polymerase chain reaction (PCR) Total RNA (0.5 μg) was reverse transcribed into cDNA utilizing the Superscript III One-Step RT-PCR System. cDNA was diluted (1:25) in RNase-free water. Two microliters of diluted cDNA were amplified on a.