PARENT SESSION
NUCLEAR TRANSFER, CLONING, AND REPRODUCTIVE TECHNOLOGIES
Harborside C
7:30 AM-10:00 AM

(346) ACTIVATED BOVINE CYTOPLASTS PRODUCED BY INDUCED ENUCLEATION SUPPORT DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS IN VITRO.

Fischer, Daniela¹, Ibanez, Elena¹, Cibelli, Jose², Albertini, David³, Overstrom, Eric^1,3, ¹ Department of Biomedical Sciences, North Grafton, MA² Advanced Cell Technology, Worcester, MA³ Department of Anatomy and Cellular Biology, Boston, MA

ABSTRACT- Poor efficiency of somatic cell nuclear transfer (NT) has been associated with the preparation of developmentally competent enucleated oocytes. Induced enucleation (IE) of mouse oocytes has been shown to support enhanced term development of cloned mice. The objective of this study was to characterize the kinetics and phenotypic progression of bovine oocytes subjected to IE, and to evaluate their competence to support NT embryo development in vitro. In vitro matured (26 h) oocytes were denuded, activated (5 mM ionomycin 5 min, then 10 mg/mL cycloheximide 5 h) and cultured for up to 5 h post-activation (pa). Oocyte enucleation was induced by demecolcine (0.4 mg/ml, DM) exposure at 30, 60, 90, 120 min post activation for various time periods (1 to 4.5 h). Activation rates and meiotic progression of control and DM treated oocytes (n=31-49/group) was evaluated at 5 h pa by immunofluorescence microscopy (microtubule Mab-FITC, microfilament Texas red-phalloidin and chromatin - H33258). DM treatment at 30 min pa resulted in low activation rates (10-16%) whereas DM exposure at 1, 1.5, or 2 h pa resulted in higher (79-100%) oocyte activation rates. Onset and duration of deme treatment significantly altered IE rates, which varied from 60-91% at 5 h pa. Maximum rates of IE were obtained when oocytes were exposed to DM between 1.5 and 5 h pa (91% IE at 5 h pa). DM treatments elicited a range of distinct oocyte spindle, chromatin, microfilament and PB phenotypes. Development of reconstructed IE embryos was evaluated by in vitro culture for 7 days. Mechanically isolated adult fibroblast nuclei were injected into IE cytoplasts between 1.5-3 h pa (n=106). Parthenogenetically activated and DM treated oocytes were cultured simultaneously for 7 days and served as controls. Control group cleavage and morula/ blastocyst rates were 49% (23/47) and 30% (7/23) respectively, whereas IE group rates were 48% (51/106) and 27% (14/51) respectively. These results demonstrate that the IE method can be used to produce enucleated bovine cytoplasts, and that IE cytoplasts are competent to support in vitro development. This technically simple approach may provide a more efficient method to prepare competent cytoplasts for use in nuclear transfer procedures. (Supported by Advanced Cell Technology and USDA #2001-35205-09966).

KEY WORDS: Induced Enucleation, Bovine, Nuclear Transfer, Demecolcine