Adição de paraoxonase-1 durante a maturação oocitária e seu efeito sobre a produção in vitro de embriões bovinos

Abstract

Some studies suggest that the enzyme paraoxonase-1 (PON1) can exert antioxidant effects on cell membranes, thereby improving the competence of the oocyte, particularly by reducing oxidative stress. Based on this, the aim of the current study was to evaluate the effect of different levels of human recombinant PON1 (rPON1) in the maturation medium, on embryo development and gene expression in bovine oocytes and embryos. The study was performed in 8 repetitions, using 1600 cumulus oocyte complexes (COCs) collected from bovine ovaries at local slaughterhouses and divided randomly into four groups of 50 COCs each containing 0.0; 0.02; 0.04 and 0.08 mg/mL rPON1 in the IVM medium. Maturation occurred in the incubator at 5% CO2 and 39 °C for 24 hours. After the sperm selection using the mini Percoll®, IVF was performed with a sperm concentration of 1x106/ml. After 18 hours, presumptive zygotes were cultured for 7 days using IVC medium with the addition of 10% FBS under controlled conditions (5% CO2, 5% O2 and 90% N2) at 39˚C. In addition, denuded oocytes and cumulus cells (600 COCs, 3 repetitions) and D7 embryos (8 repetitions) were collected for RNA extraction and gene expression analysis (Bax, Bcl-2 and MnSOD). PON1 activity was evaluated at 0 and 24 hours of maturation by spectrophotometry. The average PON1 activity in the IVM medium was 2.15±0.38; 15.51±1.48; 30.24±2.96 and 57.94±5.03 U/mL according to the addition of 0.0; 0.02; 0.04 and 0.08 mg/mL of rPON1 in the IVM medium. There was no difference (P>0.05) in cleavage rates among treatments: 81.7%; 85.1%; 81.0%. 82.5% respectively. However, the addition of rPON1 to the IVM medium improved embryo development linearly (P<0.0001), resulting in D7 embryo production of 22.1%, 29.2%, 31.7% and 33.8%, respectively. Moreover, this effect was supported by the positive correlation between the level rPON1 in the IVM medium and the D7 blastocyst rate (r=0.35; P=0.04). There was no difference in gene expression in oocytes, cumulus cells and embryos. Finally, the addition of rPON1 in the IVM medium had a positive effect on the IVP of bovine embryos, indicating that PON1 improves embryo rate.