A simplified approach for oocyte enucleation in mammalian cloning.
Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.
Faculty of Veterinary Medicine, University of Teramo , Teramo, Italy .
Domenico
D
Iuso
Marta
M
Czernik
Federica
F
Zacchini
Grazyna
G
Ptak
Pasqualino
P
Loi
D000818:Animals; D001755:Blastocyst; D019976:Cloning, Organism; D003470:Culture Media; D003703:Demecolcine / Q000008:administration & dosage; D005260:Female; D009865:Oocytes / Q000648:ultrastructure*; D012756:Sheep
D003470:Culture Media; D003703:Demecolcine