Increasing numbers of couples are seeking assistance with fertility problems. A common cause of infertility, which affects ~10-15% of individuals, is anovulation resulting from menstrual cycle irregularities. Ovulation requires that ovarian follicular development and steroidogenesis are coordinated perfectly with precisely timed release of luteinizing hormone. This application presents the novel hypothesis that an ovarian circadian clock orchestrates the temporal synchronization of events that act through both endocrine (communication with the hypothalamic-pituitary axis) and paracrine (oocyte-follicle cell communication) mechanisms to assure successful ovulation. Dioxins, which are ubiquitously found in the environmental and within all organisms, exert significant deleterious effects on the female reproductive system. However, specific molecular mechanisms of dioxin effects within the ovary remain unclear. It is generally accepted that dioxin affects reproductive function through activation of the aryl hydrocarbon receptor (AhR) signal transduction cascade. AhR and its partner, the Aryl Hydrocarbon Nuclear Transporter (ARNT) are members of the bHLH-PAS (PER-ARNT-SIM) family of proteins, which also includes the genes known to regulate circadian rhythmicity. Like AhR:ARNT, the clock gene proteins CLOCK (CLK) and Brain/Muscle ARNT-like protein1 (BMAL1), form heterodimerizers and initiate transcription. Because members of the PAS domain family of proteins are notoriously promiscuous, it is not unreasonable to hypothesize that persistent activation of AhR may form a transcriptionally inert heterodimers with the clock gene, BMAL1, thereby disrupting timing mechanisms inherent to ovarian function. Immunohistochemistry will determine cell-specific localization of AhR, ARNT and the circadian clock genes in the rat ovary. Diurnal expression patterns of AhR/ARNT and clock genes across the estrous cycle will be examined using quantitative real-time PCR and Western blot analysis. Finally, alterations in the molecular rhythmicity of ovarian clock genes, AhR and ARNT will be examined after an acute cycle-disrupting exposure to dioxin-like compounds. Co-immunoprecipitation experiments will examine crosstalk among PAS domain family members. Our results will begin to elucidate the function of circadian clock genes in the ovary, as well as reveal novel mechanisms responsible for reproductive disruption in response to AhR activation ? ?
