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Title:Toxicity of TCDD in the mouse antral follicle
Author(s):Karman, Bethany
Director of Research:Flaws, Jodi A.
Doctoral Committee Chair(s):Flaws, Jodi A.
Doctoral Committee Member(s):Bahr, Janice M.; Bunick, David; Nardulli, Ann M.; Tischkau, Shelley Ann
Department / Program:Comparative Biosciences
Discipline:VMS - Comparative Biosciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
antral follicle
aryl hydrocarbon receptor
Estradiol 17-beta-dehydrogenase 1 (HSD17B1)
catechol-o-methyltransferase (COMT)
embryonic ovary
kinesin family member 11 (Kif11)
X-inactive specific transcript (Xist)
female germ cells
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Abstract:The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis, growth, and atresia of intact mouse antral follicles in vitro. Interestingly, TCDD has been shown to act mainly through binding to the aryl hydrocarbon receptor (AHR) in other tissues, and the AHR has been identified as an important factor in ovarian function. Thus, these studies were also designed to test the hypothesis that TCDD activates the AHR in antral follicles. Specifically, these studies were designed to test the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Mouse antral follicles were exposed for 96 hours to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture and to encompass environmentally relevant and pharmacological exposures (0.1-100nM). The results indicate that TCDD decreases progesterone (P), androstenedione (A4), testosterone (T), and estradiol (E2) levels in a dose response manner without altering growth of antral follicles. The 1nM dose of TCDD consistently lowered all four hormones measured. Thus, this dose was chosen to study more closely the mechanism of TCDD toxicity in the antral follicle. Interestingly, the addition of pregnenolone substrate (10μM) restores hormone levels to control levels, suggesting that TCDD has actions upstream of progesterone production. Thus, the next goal of these studies was to determine the mechanism by which TCDD inhibits steroidogenesis and whether this occurs in a time dependent manner. Specifically, experiments were designed to compare the effects of 48 and 96 hours TCDD exposure on hormone production/secretion, steroidogenic enzymes, atresia ratings, expression of members of the apoptotic pathway, and expression of the AHR and cytochrome P450, family 1, subfamily b, polypeptide 1 (Cyp1b1). TCDD exposure for 48 hours increased levels of A4, without changing HSD3B1 protein, HSD17B1 protein, estrone (E1), T or E2 levels. Further, TCDD did not alter atresia rating, but it did down-regulate the AHR protein compared to vehicle. TCDD exposure for 96 hours decreased transcript levels for Cyp11a1, Cyp17a1, Hsd17b1, and Cyp19a1, but increased Hsd3b1 transcript. TCDD exposure particularly lowered both Hsd17b1 transcript and HSD17B1 protein. However, TCDD exposure did not affect levels of E1 in the media. The down regulation of the AHR protein in TCDD exposed follicles persisted and Cyp1b1 transcripts were increased by 3-4 fold. Interestingly, levels of the proapototic factor, BCL2-associated X protein (Bax) were significantly reduced without affecting atresia ratings in antral follicles following TCDD exposure, suggesting an altered apoptotic pathway in exposed antral follicles. Finally, because Cyp1b1 is an important enzyme that is implicated in both biotransformation of TCDD and also in E2 catabolism to catecholestrogens and the highly reactive quinones, increased levels of Cyp1b1 could be an indication of a system under stress. Quinones are known to cause DNA damage, and if the DNA is left unrepaired, it can lead to mutations that propagate into daughter cells, leading to cancer. Besides its effects on E2 levels, chronic TCDD exposure causes promotion of ovarian tumors in rats and acute exposure is associated with an increased incidence of ovarian cancer in young woman. The enzyme catechol-o-methyltransferase (COMT) is regulated by E2 and is an important enzyme for inactivating the catecholestrogens. Thus, we hypothesized that TCDD exposure affects the levels of COMT. TCDD exposure reduced COMT protein levels, while there was no change in transcript levels for COMT after 96 hours exposure in antral follicles. Overall, these data indicate that TCDD exposure perturbs steroidogenesis by impairing the steroidogenic enzymes, and that it alters apoptotic pathways, and potentially decreases the ability to neutralize reactive metabolites in antral follicles. Finally, the activation and proteasomal degradation of the AHR in the antral follicle likely plays a central role in these processes, contributing to endocrine disruption, cancer promotion, and ovarian toxicity by TCDD.
Issue Date:2012-09-18
Rights Information:Copyright 2012 Bethany Karman
Date Available in IDEALS:2012-09-18
Date Deposited:2012-08

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