Investigation of mechanisms of follicle depletion and the ovarian protective response to ovotoxicant exposure

Abstract

<p>A female is born with a finite number of ovum-containing follicles, responsible for maintaining lifetime female fertility prior to menopause. Follicle quality and number can be compromised by ovotoxicant exposures; the chemotherapy drug cyclophosphamide (CPA) depletes ovarian follicles resulting in increased risks for infertility in female cancer survivors. In comparison, 7,12-dimethylbenz[a]anthracene (DMBA), liberated from burning of organic compounds, destroys all stage ovarian follicles. CPA and DMBA must be bioactivated to their active, ovotoxic, metabolites phosphoramide mustard (PM) and DMBA-1,2-epoxide, 3,4-diol, respectively. This dissertation investigated ovarian PM and DMBA exposures using a neonatal rat whole ovary culture method, to increase understanding for mechanisms driving PM- and DMBA-induced follicle depletion.</p> <p>Investigations of PM metabolism demonstrated that microsomal epoxide hydrolase (Ephx1) plays a role in detoxification of PM and while increased glutathione (GSH) levels lessened PM ovotoxicity, surprisingly, depleting (GSH) had no impact, rending the role of GSH in PM metabolism as inconclusive and requiring additional studies. Generation of an ovotoxic, volatile PM metabolite, presumably chloroethylaziridine (CEZ), was identified, independent of ovarian metabolism, suggesting that PM is not the only ovotoxic metabolite of CPA.</p> <p>Autophagy induction during PM-induced ovotoxicity was confirmed via identification of autophagosome formation by transmission electron microscopy and altered mRNA and protein levels of key autophagy genes. Pathway manipulations of PI3K and mTOR to inhibit or activate autophagy, respectively, revealed that PI3K inhibition had no effect on PM-induced ovotoxicity while, mTOR activation prevented PM-induced follicle depletion.</p> <p>Depletion of large preantral follicles along with altered mRNA levels of autophagy genes were also demonstrated with low-dose acute DMBA exposure. Induction of additional ovarian responses, including xenobiotic metabolism, oxidative stress and PI3K signaling were also observed in these experiments.</p> <p>Taken together, this dissertation supports that ovotoxicant exposure induces ovarian chemical biotransformation and autophagy in addition to a number of other protective responses. A thorough understanding of the detrimental effects of PM, CEZ and DMBA exposures are required before advancements towards preservation of the ovarian follicle reserve can progress.</p>