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Mechanism of methoxychlor toxicity in mouse ovarian antral follicles

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Title: Mechanism of methoxychlor toxicity in mouse ovarian antral follicles
Author(s): Basavarajappa, Mallikarjuna
Advisor(s): Flaws, Jodi A.
Contributor(s): Hofmann, Marie-Claude; Dirikolu, Levent; Jeffery, Elizabeth H.; Nowak, Romana A.
Department / Program: Comparative Biosciences
Discipline: VMS - Comparative Biosciences
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Doctoral
Subject(s): methoxychlor antral follicles ovary mouse aryl hydrocarbon receptor atresia growth
Abstract: 1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor; MXC) is an organochlorine pesticide used against pests and insects that attack crops, gardens, vegetables, pets, and livestock. MXC targets the ovary and its exposure has adverse effects on reproductive function in adult female mice causing persistent estrus, reduced fertility, and ovarian atrophy. MXC reduces fertility by increasing atresia of antral follicles and by decreasing numbers of healthy antral follicles in adult female mice. Further, MXC inhibits growth and induces atresia of mouse antral follicles in vitro. Little is known, however, about the mechanisms by which MXC causes slow growth and atresia of antral follicles. Hence, this dissertation work was designed to help us to better understand the mechanism of action of MXC in antral follicles by examining the effects of MXC on steroid levels, Bcl2 factors and caspase activity, and by determining if MXC exerts toxicity through an AHR pathway. Since MXC is known to target antral follicles, the major producer of sex steroids in the ovary, the present study first tested the hypothesis that MXC decreases estradiol (E2) levels by altering steroidogenic and metabolic enzymes in the antral follicles. Given that MXC induces atresia after long term exposure, in part, by increasing the pro-apoptotic factor Bax and decreasing the anti-apoptotic factor Bcl2 in antral follicles, the present study also tested the hypothesis that MXC induces atresia at early time points and alters other pro-apoptotic (Bok and Casp3) and anti-apoptotic factors (Bcl-xL) in addition to Bcl2 and Bax. In addition, the present study tested the hypothesis that MXC alters caspase activity in the follicles. Several studies indicate that many chemicals act through the aryl hydrocarbon receptor (AHR) pathway and one study has shown that MXC binds to the AHR in liver cells. Hence, the present work also tested the hypothesis that MXC binds to the AHR to inhibit follicle growth and induce atresia of antral follicles. To test these hypotheses, antral follicles were isolated from ovaries of female wild-type (WT) or AHR knock-out (AHRKO) mice and cultured with either vehicle (dimethylsulfoxide; DMSO) or MXC. Follicle growth was measured every 24 h for 96 or 168 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes, the E2 metabolic enzyme Cyp1b1, and Bcl2 related factors were measured using qPCR. Caspase activity and atresia were also measured in follicles. In granulosa cells, MXC binding to AHR was also measured using transfection experiments. The results indicate that MXC decreases most of steroidogenic enzymes, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels. The results also indicate that, at 24 h, MXC increases Bax levels and does not affect Bcl2 levels. This increases the Bax/Bcl2 ratio, which in turn may increase the mitochondrial permeability leading to activation of caspase activities. Thus, these early changes in Bax expression and caspase may induce onset of morphological atresia beginning at 48 h. Further, the results indicate MXC binds to the AHR in granulosa cells and that MXC (10, 100 μg/ml) significantly inhibits follicular growth in WT antral follicles by 168 h. The results also indicate that MXC (10,100μg/ml) significantly induces atresia in WT antral follicles. Conversely, MXC (10 μg/ml) did not significantly inhibit growth or induce atresia in AHRKO follicles. These data indicate that AHR deletion protects antral follicles against MXC-induced growth inhibition and atresia. Thus, this study suggests that MXC acts through the AHR pathway to inhibit follicle growth and induce atresia in antral follicles of the ovary. Collectively, this dissertation work has shown that MXC exerts toxicity in antral follicles by inhibiting steroidogenesis and inducing apoptosis, Further, this work shows that MXC may exert toxicity via an AHR pathway.
Issue Date: 2012-02-01
Genre: thesis
URI: http://hdl.handle.net/2142/29409
Rights Information: Copyright 2011 Mallikarjuna Basavarajappa
Date Available in IDEALS: 2012-02-01
2014-02-01
Date Deposited: 2011-12
 

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