Effects of methoxychlor in ovarian granulosa cell in vitro

Methoxychlor (MXC) has been shown to alter ovarian folliculogenesis through inhibition of granulosa cell steroidogenesis, which is required for normal reproductive physiology. The process of granulosa cell differentiation requires estrogen to augment follicle-stimulating hormone (FSH) activity and the ovulatory response to gonadotropins. This dissertation focuses on the effect of methoxychlor, which is suspected of acting through its metabolite, 2,2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on granulosa cell function. HPTE acts as an estrogen receptor α agonist and an estrogen receptor β antagonist. The well characterized immature rat granulosa cell model was used to generate samples for analysis of intracellular cAMP, protein, and gene expression. The transcriptional responses induced by MXC and HPTE were investigated using Affymetrix microarray Rat Genome 230 2.0. Alterations in guanine nucleotide binding protein-coupled receptors (GPCRs) signaling were suggested to be affected by MXC and HPTE. G-protein coupled receptors (e.g. FSH and LHR) are among the most common and successful drug targets produced by pharmaceutical companies and are critical to granulosa cell differentiation. Recent reports suggest that estrogen receptor β is required for FSH-induced granulosa cell differentiation, response to gonadotropins (i.e., LHR) in the pre-ovulatory follicle, and maximal cAMP production. MXC and HPTE reduced FSH-mediated LHR expression and cAMP levels. Our results using two estrogen receptor β antagonists, ICI 182,780 (fulvestrant or falsodex) and 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) support an association between estrogen receptor expression β and cAMP. However, co-treatment of granulosa cells with an estrogen receptor β agonist, diarylpropionitrile (DPN), did not reverse the effect of HPTE on cAMP. While there are clear differences in the morphology of FSH-stimulated granulosa cells treated with HPTE and MXC, as well as production of estradiol 17β (E2) when compared to FSH-alone, the use of dibutyryl cAMP (dbcAMP) was protective against the effects of MXC and HPTE on gene expression and steroidogenesis. Furthermore, MXC and HPTE suppressed estrogen signaling through inhibition of estrogen receptor expression and E2 production. We showed that cell viability was unaffected by MXC or HPTE at high doses, but MXC and HPTE did induce significant changes in gene expression and pathway activity.Ph. D.Includes bibliographical referencesby Craig Nicholas Harve