Redox-induced apoptosis of human oocytes in resting follicles in vitro.

Abstract

Background: The age-related depletion of the resting follicle (RF) stock occurs as a result of two processes: atresia and entry in growth phase. Due to difficulties in obtaining sufficient numbers of RFs for study, little is known about the apoptotic mechanisms for RF atresia. The present study was designed to investigate the effects of oxidative stress on the apoptosis of RF oocytes.

Methods: RFs isolated from human adult ovaries were cultured in vitro, treated with H2O2 at various concentrations (50 microM, 100 microM, 1.0 mM) for 1 hour, and observed for up to 48 hours. The oxidant-induced apoptosis of oocytes were observed by detection of DNA fragments, mitochondria membrane potential (MMP), and cytochrome c release.

Results: Based on nuclear morphology and TUNEL (terminal deoxynucleotidyl transferase-mediated dDTP nick end-labeling), oocyte apoptosis was observed in the RFs treated with 50 microM H2O2 with rates of 35% and 43% at 24 and 48 h after treatment, respectively. But intensive oxidative stress (1 mM H2O2) caused mainly necrosis as measured by quantifying propidium iodide (PI)-positive oocytes (44% within 12 hours), with lower level of apoptosis (17%) being observed at 24 hours after treatment. RFs treated with 100 microM H2O2 showed both apoptosis with the similar rate observed at 50microM and necrosis (13% PI-positive oocytes). Although pre-incubation with cyclosporine A (CsA) could effectively prevent oxidant-induced MMP collapse, but failed to suppress apoptosis of oocytes in RFs.

Conclusions: Oocytes of RFs in adult ovaries retain their ability to undergo apoptosis under oxidative stress, which is both dose- and time-dependent.