25 COMPARISON BETWEEN CHEMICALLY ASSISTED, CHEMICALLY INDUCED AND MECHANICAL ENUCLEATION OF MOUSE OOCYTES

Abstract

Chemically-assisted (AE) and chemically induced (IE) enucleation using demecolcine (DEM) or nocodazole (NOC) have proven to be technically simple procedures to prepare developmentally competent cytoplasts for nuclear transfer (NT) in different species. In this study, we analyzed AE and IE in mouse oocytes in terms of enucleation efficiency, amount of cytoplasmic volume removed and distribution of spindle-associated γ-tubulin after enucleation, and spindle morphology after cytoplast reconstruction by NT. Results were compared to the standard mechanical enucleation (ME) method. Outbred CD-1 and hybrid B6CBAF1 oocytes were collected at 13 to 16 h post-hCG. In AE experiments, oocytes were treated with either 0.4 μg mL–1 DEM or 0.3 μg mL–1 NOC in KSOM for 30 min. Protrusions induced in CD-1 (92.2%, n = 695) and B6CBAF1 (83.3%, n = 370) oocytes were aspirated by piezo-actuated micromanipulation, in H-KSOM with 2.5 μg mL–1 cytochalasin B and 0.05 m sucrose. In IE experiments, oocytes were preactivated with 7% ethanol for 5 min and treated with DEM or NOC in calcium-free KSOM containing 10 mm strontium. At 90 min postactivation (p.a.), completely- and partially-extruded second polar bodies (PBs) were mechanically aspirated. Enucleation efficiencies were higher than 90% both for AE (90.8%, n = 509 CD-1; 90.4%, n = 260 B6CBAF1) and IE methods (90.3%, n = 167 CD-1; 92.9%, n = 197 B6CBAF1), though they were significantly lower than those obtained for ME in nontreated CD-1 (98.4%; n = 126) or B6CBAF1 (100%, n = 498) oocytes. The amount of cytoplasmic volume removed in CD-1 oocytes was smaller in AE than in ME (2.1%, n = 35 and 3.9%, n = 30, respectively). In B6CBAF1 oocytes, used to compare IE (5.4%, n = 60) and ME (4.9%, n = 41), no differences were found. Volumes were calculated using the CellA software on images of cytoplasts and karyoplasts taken after enucleation. Even though both AE and IE methods avoided the removal of the oocyte spindle microtubules, spindle-associated γ-tubulin was eliminated from the cytoplasts generated by all 3 enucleation procedures, as confirmed by immunofluorescence analysis of the cytoplasts and the complementary karyoplasts produced. Finally, spindle morphology was examined in enucleated oocytes reconstructed by NT with a cumulus cell nucleus. Cytoplasts prepared by NOC-AE or NOC-IE displayed morphologically normal bipolar spindles by 2 h post-NT or 18 to 20 h post-activation (hpa), respectively, similar to cytoplasts prepared by ME. However, when DEM was used, microtubule repolymerization was slower and bipolar spindles could not be observed until 4 h post-NT (AE) or 22 to 24 hpa (IE). In conclusion, although enucleation rates are slightly higher for ME, AE and IE protocols allow oocyte enucleation without removal of the meiotic spindle, and a very small cytoplasm volume is eliminated during AE. Treatments with NOC and DEM are reversible, and cytoplasts produced by AE and IE can form morphologically normal spindles after NT, similar to those of cytoplasts produced by ME. MEC BIO 2006-11792; 2005-SGR00437; Portuguese FCT.