title: Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats.
abstract: 4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.
authors.affiliation: Department of Physiology and Pharmacology, University of Arizona, Tucson, AZ, USA.
authors.forename: Aileen F
authors.initials: AF
authors.lastname: Keating
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Fernandez
authors.forename: Connie J
authors.initials: CJ
authors.lastname: Mark-Kappeler
authors.forename: Nivedita
authors.initials: N
authors.lastname: Sen
authors.forename: I Glenn
authors.initials: IG
authors.lastname: Sipes
authors.forename: Patricia B
authors.initials: PB
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D001483:Base Sequence; D053138:Cyclohexenes / Q000633:toxicity*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D010053:Ovary / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D015398:Signal Transduction / Q000187:drug effects; D014753:Vinyl Compounds / Q000633:toxicity*
chemical_list: D053138:Cyclohexenes; D017931:DNA Primers; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger; D014753:Vinyl Compounds; C499515:FOXO3 protein, rat; C012606:4-vinyl-1-cyclohexene dioxide; C494920:Akt1 protein, rat