One percent of all women living today has or will develop premature ovarian failure (POF), defined as the precocious depletion of ovarian follicles before the age of 40. However, the mechanisms underlying POF remain poorly defined. Our previous work shows that normal ovarian aging is associated with increased ovarian oxidative stress and decreased antioxidant capacity. We have observed that Gclm -/- mice, genetically deficient in the modifier subunit of the rate-limiting enzyme in synthesis of the antioxidant glutathione (GSH), exhibit increased ovarian oxidative stress and accelerated ovarian aging. Gclm -/- mice show similar numbers of oocytes and ovarian follicles at birth and weaning compared to Gclm +/+ counterparts, but thereafter exhibit more rapid age-related declines in primordial and total follicle numbers. This makes the Gclm null mouse an ideal model to study premature ovarian failure.
The aims i n this proposal will identify the contribution of GSH to the proper maintenance of the primordial follicle pool and clarify effects of oxidative stress on the ovarian primordial follicle reserve, quiescent primordial to activated primary metabolism, and female fecundity. We will test our hypothesis that genetic deficiency in the antioxidant GSH causes prolonged oxidative stress that alters the redox equilibrium of the ovary, resulting in downstream effects that promote premature primordial follicle depletion by increasing primordial follicle activation and altering follicular metabolism and energetics in favor of accelerated ovarian aging. To identify GSH contribution to maintenance of the primordial follicle pool and fecundity, we propose the following specific aims: 1) Quantify primordial follicle activation and atresia in cultured neonatal ovaries of Gclm -/- and Gclm -/+ mice compared to Gclm+/+ mice, and assess phosphoinositide-3 kinase (PI3K) cellular signaling across genotypes. Neonatal ovary culture offers an effective model to investigate the response of primordial follicles to increased oxidative stress during normal culture conditions. [2) Perform Phasor FLIM at 740nm 2-photon excitation to characterize the levels of free-to-bound NADH during initial to early stages of follicular development and compare with immunolocalization of Silent mating type information regulation 2 homolog (Sirt1) and Forkhead Box O3a (Foxo3a) in stage matched follicles in both wildtype and Gclm +/+ vs Gclm -/- glutathione deficient mice.] 3) Establish if oocyte-specific and granulosa cell specific GSH depletion leads to premature ovarian failure and/or reduced fecundity, as observed in global Gclm null GSH deficient mice. In vivo requirements for GSH in maintenance of the primordial follicle pool will be identified utilizing tissue specific GSH depleted mouse models that have deletion in the catalytic subunit of the rate-limiting enzyme in GSH synthesis, Gclc. Mechanisms uncovered by the research performed in this proposal will provide new insights into POF etiology and contribute to future therapies and preventive measures.
There is a significant medical need to understand the mechanisms underlying premature ovarian failure (POF) so that we may progress towards designing appropriate preventive and therapeutic strategies. The aims in this proposal will identify the contribution of glutathione (GSH) to proper maintenance of the primordial follicle pool and clarify effects of oxidative stress on the primordial follicle reserve, follicular metabolsm, and accelerated ovarian aging. If better understood, mechanisms uncovered by the research performed in this proposal could provide new insights into POF etiology and contribute to future therapies and preventive measures.
| Cinco, Rachel; Digman, Michelle A; Gratton, Enrico et al. (2016) Spatial Characterization of Bioenergetics and Metabolism of Primordial to Preovulatory Follicles in Whole Ex Vivo Murine Ovary. Biol Reprod 95:129 |
