Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats

Aileen F. Keating, Shannon M. Fernandez, Connie J. Mark-Kappeler, Nivedita Sen, I. Glenn Sipes, Patricia B Hoyer

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)743-751
Number of pages9
JournalBiology of Reproduction
Volume84
Issue number4
DOIs
StatePublished - Apr 1 2011

Fingerprint

Oocytes
Messenger RNA
Ovary
4-vinyl-1-cyclohexene dioxide
Staining and Labeling
Inbred F344 Rats
Receptor Protein-Tyrosine Kinases
Confocal Microscopy
Fluorescent Antibody Technique
Proteins
Western Blotting
Phosphorylation
Polymerase Chain Reaction
Forkhead Box Protein O3

Keywords

  • Follicle
  • Ovary
  • Toxicology

ASJC Scopus subject areas

  • Cell Biology

Cite this

Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats. / Keating, Aileen F.; Fernandez, Shannon M.; Mark-Kappeler, Connie J.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

In: Biology of Reproduction, Vol. 84, No. 4, 01.04.2011, p. 743-751.

Research output: Contribution to journalArticle

Keating, Aileen F. ; Fernandez, Shannon M. ; Mark-Kappeler, Connie J. ; Sen, Nivedita ; Sipes, I. Glenn ; Hoyer, Patricia B. / Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats. In: Biology of Reproduction. 2011 ; Vol. 84, No. 4. pp. 743-751.
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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.",
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