67 PREMATURE CHROMOSOME CONDENSATION IS NOT ESSENTIAL FOR BOVINE SOMATIC NUCLEAR REPROGRAMMING
L.Y. Sung A , F. Du A B , B.S. Jeong A , C.C. Chang A , J. Xu B , X.C. Tian A and X. Yang AA Department of Animal Science, Center for Regenerative Biology, University of Connecticut, Storrs, CT 05259, USA
B Evergen Biotechnologies, Inc., Storrs, CT 06269, USA. Email: lsung@canr.uconn.edu
Reproduction, Fertility and Development 17(2) 183-184 https://doi.org/10.1071/RDv17n2Ab67
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
The objective of this study was to determine the effect of donor nuclear exposure in MII oocyte cytoplasm on nuclear reprogramming events and subsequent development of cloned embryos in cattle. Somatic nuclear transfer (NT) was performed by electrofusion of the enucleated MII oocytes with cultured cumulus cells by ovum pickup. Donor cell-cytoplast pairs were fused by applying two direct current pulses at 2 kV/cm for 10 μs. Fused NT embryos were randomly divided in to Treatment A (immediate activation) and Treatment B (delayed activation, 4-h exposure in MII cytoplast before activation). In both treatment groups, the activation protocol was identical and consisted of incubation in cycloheximide (10 μg/mL) plus cytochalasin D (2.5 μg/mL) in M199 + 7.5% FBS for 1 h, followed by culture in cycloheximide (10 μg/mL) for an additional 4 h. Reconstructed embryos in both groups were subsequently cultured in CR1aa in 5% CO2, 5% O2, and 90% N2 at 39°C. Samples from both treatments were fixed at 0, 1, 2, 4, 6, 12, 18, 24, 30, 36, and 44 h after fusion. All fixed samples were double stained for tubulin and DNA, and observed with a laser-scanning confocal microscope for changes in nuclei and microtubules. The experiment was replicated three times. Cleavage rate and blastocyst rate were recorded and analyzed by Studen's t-test (SPSS 11.0, Chicago, IL). The staining revealed an absence of premature chromosome condensation (PCC) in all embryos in Treatment A. However, delayed activation (Treatment B) resulted in a high incidence of PCC, probably due to high levels of MPF in the MII cytoplasts. Chromosome condensation was observed in Treatment B at 4 h (82%, n = 17), 6 h (80%, n = 10), 12 h (36%, n = 25), 18 h (71%, n = 24), 24 h (50%, n = 16) and 30 h (6%, n = 18) after fusion. Subsequent culture results of these cloned embryos (Table 1) indicated that there were significantly higher cleavage rates and blastocyst development in Treatment A than in Treatment B. This study clearly demonstrated that PCC is not essential to support bovine cloned blastocyst development. Direct exposure of donor nuclei in a MII cytoplast enviroment for a very short time was sufficient for nuclear reprogramming.
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