ANTIOXIDANT SUPPLEMENT AS PREVENTION OF POSTOVULATORY IN VITRO OOCYTE AGING

WETTEN P.A.; KLINSKY LAHOZ O.G.; MICHAUT M.A.

Congreso; LVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB) and XVI Annual Meeting of the Argentinean Society for General Microbiology (SAMIGE); 2021

After ovulation, mature oocytes only have a short optimal time span for fertilization to take place. If not fertilized in time, these oocytes will undergo a time-dependent quality degradation process called postovulatory aging. In assisted reproduction technologies (ART), oocytes are inevitably subjected to postovulatory aging. Although quite significant technical progress has been made to improve ART technologies, poor oocyte quality is the key factor closely associated with ART failure. Understanding the underlying mechanisms in the oocyte aging process and finding chemicals that can reverse postovulatory aging are two of the most important research topics today. In a previous work, we showed that ROS levels increased during in vitro mouse oocyte aging. In addition, we also observed that: 1) cortical granules density (CGD) decreased, indicating a premature exocytosis, and 2) the localization of alpha-SNAP and NSF, two proteins involved in membrane fusion during cortical granule exocytosis, were altered in postovulatory in vitro aged oocytes. Dithiothreitol (DTT) is a dithiol with two end sulfhydryl groups that works as an antioxidant. We hypothesized that DTT treatment might prevent the aforementioned alterations during in vitro mouse oocyte aging. Mature oocytes were obtained from hormonally stimulated CF-1 female mice of 8-12 weeks of age. For achieving in vitro aging, oocytes were collected 16 h post hCG (time 0 h, control oocytes) and in vitro cultured by 4 or 8 h (aged oocytes) in presence or absence of DTT. ROS levels were measured using the fluorescent indicator DCF-DA. When in vitro oocyte aging was performed in presence of DTT, ROS levels diminished significantly to similar levels observed in control oocytes. To determine the effect of DTT on the premature cortical granules exocytosis, mouse oocytes were aged in presence of DTT, fixed, and stained with fluorescent LCA to label cortical granules. Results showed that aged oocytes incubated with DTT had a CGD akin to control oocytes. Likewise, immunofluorescence indirect analysis of the localization of alpha-SNAP and NSF, showed that in aged oocytes treated with DTT both proteins had a similar distribution pattern to control oocytes. Altogether, these results suggest that the addition of antioxidants to culture medium might be useful to avoid the alterations produced by the postovulatory in vitro oocyte aging and, in consequence, improve the ART rate success.