33 ENUCLEATION OF PRE-ACTIVATED MOUSE OOCYTES INDUCED BY DEMECOLCINE, NOCODAZOLE, AND VINBLASTINE

Abstract

Demecolcine-induced enucleation has been previously used to prepare developmentally competent enucleated mouse and bovine cytoplasts for nuclear transfer (Gasparrini et al. 2003 Biol. Reprod. 68, 1259–1266; Fischer-Russell et al. 2005 Mol. Reprod. Dev. 72, 161–170). The approach is technically simple, but the proportion of pre-activated oocytes that extrude all of the chromatin within the second polar body (PB) after exposure to demecolcine is relatively low, especially in the mouse (20%). This study was designed to explore the potential of other antimitotic drugs (nocodazole and vinblastine), besides demecolcine, to induce enucleation of mouse oocytes and to characterize the morphological progression of the treated oocytes after drug removal. Metaphase II (MII) oocytes were collected from cytochalasin D-1 (CD-1) females (6–12 weeks old) at 16 h post-hCG, activated in 7% ethanol (for a fast release from MII arrest) for 5 min and immediately treated for 15, 30, or 60 min with demecolcine (DEM, 0.4 µg mL^-1), nocodazole (NOC, 0.3 µg mL^-1), or vinblastine (VIN, 0.1 µg mL^-1), prepared in calcium-free KSOM containing 10 mM strontium. Then, the oocytes were cultured in drug-free medium for up to 2 h, 6 h, or 20 h post-activation (p.a.) and fixed in a microtubule stabilization buffer-extraction fixative. A triple-labelling protocol for microtubules, microfilaments, and chromatin was used to analyze oocytes (approximately 60 per treatment) by fluorescence microscopy. Results were statistically analyzed by chi-square. At 2 h p.a., the highest rates of enucleation were achieved when pre-activated oocytes were treated with VIN (63.8%) or NOC (41.9%) for 15 min or with DEM (66.1%) for 30 min. Although antimitotic treatments did not affect activation rates (91.8–100%), a significant proportion of DEM- (19.6%) and of VIN-treated (15.5%) oocytes failed to complete second PB extrusion when compared to control (0%) or NOC-treated (4.8%) oocytes. From the total of the enucleated oocytes, 11.5%, 24.3%, and 29.7% had an incomplete second PB extrusion in NOC, VIN, and DEM groups, respectively, and therefore were classified as partially enucleated. Further culture of oocytes after drug withdrawal resulted in 100% of activated oocytes having a completely extruded second PB in all groups by 6 h p.a. and resulted in a significant and similar decrease in enucleation rates for all treatments by 6 h (20.3–34.9%) and 20 h p.a. (10.2–16.1%). This decrease might be caused by the reintegration of the chromosomes into the oocyte after incomplete second PB extrusion, or by re-fusion of second PBs to enucleated oocytes. Thus, our results show that both VIN and NOC, in addition to DEM, can be successfully applied to produce enucleated mouse cytoplasts, omitting the potentially harmful step (staining and ultraviolet illumination) of the traditional enucleation method. However, removal of the second PB at 2 h p.a. is recommended in order to achieve an irreversible oocyte enucleation. It remains to be demonstrated if the cytoplasts prepared with VIN or NOC are as competent as those prepared by DEM to support embryo development to term after being reconstructed by nuclear transfer.