77 BOVINE CYTOPLASTS PREPARED BY DEMECOLCINE-INDUCED ENUCLEATION OF ACTIVATED OOCYTES

Abstract

One of the most critical steps of the standard NT procedure is the removal of oocyte chromosomes for the production of enucleated cytoplasts. This process involves ultraviolet (UV) irradiation, which causes damage to the membrane and intracellular components of bovine oocytes. The objective of the present study was to evaluate the use of demecolcine, a microtubule depolymerizing agent, for chemical-induced enucleation of activated bovine oocytes. In the first experiment, oocytes obtained from cow ovaries collected in a slaughterhouse were IVM in TCM-199 medium supplemented with 10% FCS, FSH, hCG, estradiol, pyruvate, and amikacin for 26 h; then, they were artificially activated with 5 μM ionomycin for 5 min and 10 μg mL^-1 cycloheximide for 4 h. The following groups were established: control, C (no activation and no demecolcine); activated, A (exposure only to activating agents); and demecolcine-exposed oocytes, DEME (activation for 4 h and 0.05 μg mL^-1 demecolcine for 2 to 4 h after 0, 0.5, 1.0, 1.5, and 2.0 h of activation). The treatment DEME comprised the groups G1 (demecolcine from 0 to 2 h of activation; DEME 0-2 h); G2 (DEME 0-4 h); G3 (DEME 0.5-2.5 h); G4 (DEME 0.5-4 h); G5 (DEME 1-3 h); G6 (DEME 1-4 h); G7 (DEME 1.5-3.5 h); G8 (DEME 1.5-4 h); and G9 (DEME 2-4h). The oocytes were stained with 10 μg mL^-1 Hoechst 33342 for 15 min and enucleation rates (EN) were evaluated under epifluorescence microscope (330-385 nm).After EN evaluation, a second experiment was performed to verify the efficiency of demecolcine-induced enucleation in group G9, which showed the best result in the first experiment. After demecolcine treatment, the oocytes were incubated in HEPES-buffered SOF with 10% FCS and cytochalasin B for removal of the 2PB and minimal adjacent cytoplast under an inverted optical microscope. Traditional enucleation was performed on the control group without exposure of the oocytes to demecolcine. The same conditions were employed except that UV light was used to confirm enucleation. Samples of cytoplasts were stained with Hoechst for 15 min and analyzed for enucleation efficiency. Five and 3 replicates were evaluated, respectively, in experiments 1 and 2, and the results were analyzed by chi-square test in the statistical software Minitab^®, release 14.1 (Minitab Inc., State College, PA, USA). A level of 5% significance was used. Regarding enucleation rates, all treated groups were significantly different compared with the C (0/114) and A (0/130) groups. Considering the DEME groups, G8 (46/109; 42.2%) and G9 (61/113; 54.0%) presented superior rates (P < 0.05) to all other groups (26.8 to 36.3%), but they were similar despite the great tendency (P = 0.07) to difference. We observed high efficiency (P < 0.05) of demecolcine-induced enucleation (90/110; 81.8%) compared with a traditional technique (61/95; 64.2%). In conclusion, the present study shows that demecolcine-induced enucleation of activated oocytes enables good rates of enucleation and has greater efficiency than the traditional technique as well as avoiding UV irradiation of the cytoplast.

FAPESP 06/51481-5 and 07/55969-5