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(311) CO-LOCALIZATION OF ESTROGEN RECEPTOR (ER) , ER, ARYL HYDROCARBON RECEPTOR, AND HEAT SHOCK PROTEIN 90 IN RAT OVARIAN SMALL PREANTRAL FOLLICLES.
Thompson, Kary1, Christian, Patricia2, Sipes, I. Glenn3, Hoyer, Patricia2, 1 Reproductive Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC2 Department of Physiology, University of Arizona, Tucson, AZ3 Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ
ABSTRACT- The occupational chemical, 4-vinylcyclohexene diepoxide (VCD), induces the selective loss of small pre-antral (primordial and primary) follicles in the ovaries of rats and mice. The specific mechanism of this loss is unclear. However, in rats, the pathways affected by VCD have been shown to involve the estrogen receptor (ER), aryl hydrocarbon receptor (AhR), and heat shock protein 90 (Hsp90), a protein that chaperones both ER and AhR. It is unknown whether these proteins interact in mediating the effects of VCD. This study was designed to examine Hsp90 in small pre-antral follicles and compare its localization to the nuclear receptors ER, ER, and AhR. F344 rat ovaries (d42) were fixed, sectioned, and stained with either anti-ER, -ER, -AhR, or -Hsp90 antibodies. Protein recognized by antibody was visualized by confocal microscopy with Cy-5 and cell nuclei (DNA) were visualized with YOYO-1. Intensity of Hsp90 protein staining was quantified using Scion Image. Hsp90 protein was observed primarily in the oocyte nucleus and cytoplasm in all sizes of follicles. Relative to controls, there was a 47.6% +/- 4.5% decrease (p<0.05) in Hsp90 staining in primary follicles from rats dosed daily with VCD (80 mg/kg, i.p., 15d). ER protein was localized in oocyte nuclei in all sizes of follicles in the ovary. ER protein was seen in oocyte and granulosa nuclei in all sizes of follicles, but was also found in the cytoplasm of oocytes and granulosa cells specifically in primordial and primary follicles. The AhR was visualized in granulosa cell nuclei in all sizes of ovarian follicles, however it was also found in the oocyte nucleus of primordial and primary follicles. Taken together, all four of these proteins are expressed in VCD-targeted primordial and primary follicles, and the oocyte nucleus is the only potential site of direct interaction for these proteins in the rat ovary. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy. This work was completed at the University of Arizona with funding from the NIEHS (ES 09246 & Center Grant ES 06694).
KEY WORDS: 4-vinylcyclohexene diepoxide, estrogen receptor, aryl hydrocarbon receptor, heat shock protein 90