Morin´s (C15Hi007; 2- (2,4-dihidroxifenil)- 3,5,7-trihidroxi-4H-l-benzopiran-4-ona) effect on porcine oocyte in vitro maturation

GHERSA, J; LORENZO, MS.; TEPLITZ, G.; LOMBARDO, DM.

Ciudad Autónoma de Buenos Aires

Otro; IV REUNIÓN CONJUNTA DE SOCIEDADES DE BIOLOGÍA DE LA REPÚBLICA ARGENTINA; 2020

Sociedad Argentina de Biología

Porcine in vitro maturation (IVM) efficiency is modulated by intra and extracellular redox balance. Morin is a flavonoid from the flavone family which eliminate free radicals and protect DNA damage caused by free radicals. Recent studies have shown that Morin´s effect on cellular cultures is dose dependent. High doses of Morin stimulate oxygen reactive species (ROS) and promote apoptosis, whilst it has an antioxidant effect at low doses. The aim of this study was to evaluate the effect of adding different concentrations of Morin to the maturation medium on nuclear maturation and oocyte viability. We obtained pig cumulus oocyte complexes (COC) through follicular aspiration from slaughterhouse ovaries at 30-37°C. COCs were washed in PBS with 10% porcine follicular fluid (pFF). Groups of 50 COC per well were cultivated in 500 μL of medium at 39°C in a humidified atmosphere with 5% of CO2. The control group was matured in base medium (medium 199 Sigma® supplemented with 10% pFF, 2 mercaptoethanol, sodium pyruvate and antibiotics). For each treatment base medium was added with 100 μM of Morin (treatment 1), 50 μM (treatment 2), 10 μM (treatment 3) and 5 μM (treatment 4). The IVM procedure was done in 2 stages of 22 h. IVM medium was changed between stages and only the first one was supplemented with hMG and dAMPc. To evaluate oocyte viability, after IVM COC were denuded with hyaluronidase and were stained with Tripan Blue 0,16 % and observed under stereoscopic microscope. For nuclear maturation, oocytes were fixated with 4 % paraformaldehyde and stained with Hoechst 33342, then mounted on slides to be observed under fluorescent microscope. Statistical analysis was performed with a logistic regression in R with GLM function from Stats package version 2.6.2. None of the treatments influenced oocyte viability and treatment 1 negatively affected nuclear maturation rate (p value < 0.05). Thus, treatment 1 will be discarded for future determinations as it had a toxic effect. To conclude on the effects of adding Morin to IVM medium, oocyte redox balance must be analyzed.