DIMETHYLTHIOUREA MODIFIES GSH CONCENTRATION AND MALE PRONUCLEAR FORMATION IN PIG OOCYTES.

GHERSA, J; LORENZO, MS.; LUCHETTI, CG.; TEPLITZ, G.; CRUZANS, PR.; LOMBARDO, DM.

Ciudad Autónoma de Buenos Aires

Jornada; IX Jornadas de Jóvenes Investigadores; 2019

Facultad de Ciencias Veterinarias UBA

Successful in vitro embryo production is determined, amongst other factors, by sustaining a balanced redox state during oocyte maturation. In vitro matured oocytes are subject to higher oxygen levels (20%) than those in vivo (7%). Manipulation of oocytes also contributes to high levels of reactive oxygen species (ROS) production and resulting in oxidative stress. It is known that sperm chromatin decondensation and later male pronuclear (MPN) formation is related to oxidative stress and glutathione (GSH) levels in oocytes during in vitro maturation (IVM). We previously reported that the antioxidant dimethylthiourea (DMTU) reduces ROS level in oocytes and enhances sperm penetration after in vitro fertilization (IVF) without increasing polispermy. This study aimed to evaluate the effects of different concentrations of DMTU on matured oocyte GSH concentrations and MPN formation. We obtained cumulus-oocyte-complexes (COC) through follicular aspiration from slaughterhouse ovaries. Three treatments were applied during aspiration and further search: PBS + 10% porcine follicular fluid (control group); or supplemented with 1mM of DMTU (1mM treatment); or 10mM of DMTU (10mM treatment). Oocytes were matured in vitro in wells containing supplemented Medium 199, under 5% of CO2 in humidified air at 39ºC, during 44 h. COC were denuded with hyaluronidase and oocytes (30 per tube, 8 tubes per treatment) were homogenized in PBS on ice and centrifuged at 4500 x g for 10 minutes. The supernatants were used to measure oocyte protein levels and total GSH. Bradford technique was used to assess protein levels. Total GSH was measured through colorimetric assay adapted to 96 well microplates, and it was relativized to the protein level. IVF was performed with fresh boar semen (1x106 sperm/mL) in 100 μL-drops of modified Medium 199, under 5% of CO2 in humidified air at 39°C, during 6 h. After IVF, presumptive zygotes were washed and cultured for additional 12 h and MPN formation was evaluated through Hoechst stain (n control= 77; 1 mM treatment= 96, and 10 mM treatment= 87). There was a significant decrease in GSH levels in the 10 mM treatment in comparison with control and 1 mM treatment (p< 0.05). MPN formation was significantly less in the 10 mM treatment than those in the 1mM treatment and control (p< 0.05). We concluded that the addition of 10 mM of DMTU during aspiration and searching does not represent a benefit as it reduces MPN formation and GSH concentrations. Considering our previous reports, the use of 1 mM of DMTU needs to be further analyzed to determine its effects on embryo development