15 EFFECTS OF DEMECOLCINE ON THE MEIOTIC CELL-CYCLE AND MICROTUBULAR KINETICS OF ACTIVATED BOVINE OOCYTES SUBMITTED TO CHEMICAL ENUCLEATION

Abstract

Enucleation in traditional nuclear transfer (NT) is an invasive procedure for which alternative protocols have been sought. The present study was designed to explore the time course effects of demecolcine, a microtubule-depolymerizing agent, in bovine-activated oocytes submitted to induced chemical enucleation. For that purpose, after 26 h of in vitro maturation, the oocytes were parthenogenetically activated (5 μM ionomycin for 5 min and 10 μg mL^–1 cycloheximide for 4 h) and treated with demecolcine (0.05 μg mL^–1 for 2 h) 2 h after activation. Three groups were established: control (untreated oocytes), activated (oocytes exposed to activation) and deme (oocytes activated and treated with demecolcine). Then the nuclear and microtubular dynamics of the oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 59, 537–545). In each one of 3 replicates, 15 to 30 oocytes were evaluated per group. Oocytes were classified according to microtubule (MT) patterns as follows: present (evident MT), reduced (MT with reduced density), or absent. The results in percentage were submitted to ANOVA and means were compared by Tukey test, with a significance level of 5%. Effects of activation were observed after 2 h, when higher rates of oocytes presenting second polar body (2nd PB) extrusion were observed in the groups activated and deme (49.3% in both groups) compared with control (1.7%). At the end of activation treatment (4 h), the activated group presented 81.8% of oocytes with 2nd PB extrusion, whereas it was observed only in 37.8% of oocytes in the deme group. Effects of demecolcine on the microtubules initiated after only 0.5 h of treatment, when an increase (P < 0.05) of oocytes with reduced MT was observed in the deme group (26%; control – 3%; activated – 0%). After 6 h of culture in demecolcine-free medium, the deme group displayed ∼50% of oocytes with reduced MT (control – 0%; activated – 39%) and absence of MT in 23% of oocytes, which was superior to other groups (control – 0%; activated – 2%). Therefore, we detected a reduction of MT density after exposition of activated oocytes to demecolcine. However, MT were not completely absent in most of the evaluated oocytes, as previously described for bovine oocytes submitted to chemically assisted enucleation (Saraiva et al. 2009 Cloning Stem Cells 11, 141–152; Meng et al. 2011 Cell. Reprogram., in press). Apparently, there was no immediate repolymerization of MT after culture in demecolcine-free medium and this could result in negative consequences for subsequent embryo development. Moreover, demecolcine impaired the second PB extrusion during the activation process, probably due to inhibition of spindle rotation caused by the MT-disrupting drug. Nonetheless, considering the higher cytoplasmatic volume obtained with chemical enucleation and the lesser extent of injuries suffered by recipient oocytes, further studies focusing on the potential of embryo development and the quality of embryos are advisable.

Financial support: FAPESP.