230 ACTIVATION OF IN VITRO-MATURED BOVINE OOCYTES WITH IONOMYCIN AND ROSCOVITINE AFTER INTRACYTOPLASMIC SPERM INJECTION

Abstract

In all mammalian species studied so far, fertilization induces oocyte activation necessary for pronuclear formation, syngamy, and the beginning of embryonic cleavage. The aim of this experiment was to evaluate the effectiveness of a protocol for artificial activation for bovine oocytes using ionomycin and roscovitine either in combination with intracytoplasmic sperm injection (ICSI) or alone. In this study, ionomycin was used to facilitate the increase of intracellular calcium, due to the release of calcium from intracellular stores. This compound was used in conjuction with roscovitine, a specific cdc2 kinase inhibitor. The success of the treatment was compared with that of oocytes fertilized by IVF. Three replicates were carried out using bovine oocytes harvested from slaughterhouse ovaries. In vitro-matured oocytes were cultured in TCM-199 plus 10% FCS, pyruvate, estradiol, hCG, and gentamicin at 39°C in an atmosphere of 5% of CO₂ in air for 20 h. After in vitro maturation, oocytes were divided into 3 groups. For parthenogenetic activation, 100 oocytes were stripped of cumulus cells and placed in H-MEM plus 10% FCS and 5 µm ionomycin for 8 min, maintained in H-MEM plus 10% FCS, 66 mm roscovitine and 7.5 mg mL^–1 cytochalasin B for 6 h, and placed into culture. In the ICSI group, oocytes were denuded and transferred to 5-µL H-MEM plus 20% FCS drops. Only MII oocytes were microinjected. The sperm drop was prepared with a mixture of 4 µL polyvinylpyrrolidone (PVP) and 1 µL of the sperm suspension produced by Percoll gradient. For injection, a single normal mobile sperm was aspirated with the tail first. A single oocyte was fixed by holding the pipette to position the polar body at the 6 or 12 o'clock position. The injection pipette was pushed through the zona pellucida and the oolema and the spermatozoan was released into the cytoplasm. After ICSI, the oocytes were subjected to the same activation protocol described earlier and cultured. For IVF, sperm was prepared by swim-up and 100 oocytes were fertilized in Fert-Talp for 18 h (sperm concentration: 1 × 10⁶). All oocytes were cultured in HTF:BME plus 0.6% BSA, 10% FCS, 0.01% myoinositol, and gentamycin at 39°C in an atmosphere of 5% of CO₂ in air for 72 h. Cleavage was evaluated visually and the embryos were stained with Hoechst 33342 for estimation of nuclei numbers. The data were analyzed by ANOVA, followed by the Tukey test (P < 0.05). The results showed a cleavage rate of 76% for the IVF group, 57% for the ICSI group, and 51% for the parthenogenic group. The artificial activation proposed was efficient in inducing oocyte activation and cleavage; however, the rates obtained were significantly lower then the ones observed after IVF. Injection of a viable sperm into the oocyte through ICSI did not improve the cleavage rate after activation. This result indicates that the membrane fusion and/or sperm interaction with the oocyte during fertilization is important for the physiological modifications that result in oocyte cleavage in bovine.