153. XENOBIOTICS; INFLUENCE ON OVARIAN FOLLICULAR DEVELOPMENT

Abstract

The mammalian female reproductive lifespan is largely defined by a finite pool of ovarian follicles established around the time of birth. It is now understood that certain synthetic chemical compounds, known as xenobiotics, can cause premature ovarian senescence through the destruction of small ovarian follicles. Although the ovotoxic effects of these chemicals are well documented, the exact molecular mechanisms behind their action are only just becoming understood. Recent evidence suggests that bioactivation of xenobiotics by Phase I detoxifying enzymes may lead to the generation of free oxygen radicals (ROS), which we suspect may perturb intracellular signalling pathways in primordial follicles. In this study we attempted to identify ovarian follicle signalling pathways activated by xenobiotic exposure using ovotoxic agents which target immature follicles. Neonatal ovaries obtained from 3/4-day old Swiss mice were exposed to either 4-Vinylcyclohexene (25µM), Methoxychlor (25µM) or Menadione (5µM) for 96hrs using our in vitro culture system. Total RNA was then collected and analysed using Affymetrix Mouse Genome 430 2.0 Arrays. Bioinformatic analysis identified between ~500–1000 genes with a two-fold significant difference in gene expression (p<0.05) for each xenobiotic compared to the control. Differentially expressed genes were analysed for pathways and molecular functions using Ingenuity Pathways Analysis (Ingenuity Systems). In agreement with the current literature, many of the genes belonged to toxic response pathways, such as; Xenobiotic metabolism (10); p53 (15) and Apoptosis (11) signalling. However, the vast majority of the differentially expressed genes belonged to canonical pathways implicated in follicular development, such as PI3K/AKT (18), Wnt/ b -catenin (21), and JAK/Stat (8) signalling. Further qPCR analysis has confirmed a substantial increase in the transcription factor Sox4 and cell cycle inhibitor Cdkn2a in 4-Vinylcyclohexene and Menadione treated ovaries respectively. These results suggest that xenobiotics which target primordial follicles may exert part of their ovotoxic effects by perturbing signalling pathways involved in follicular activation and development.