Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection

Pablo J. Ross, Ramon M. Rodriguez, Amy E. Iager, Zeki Beyhan, Kai Wang, Neli P. Ragina, Sook Young Yoon, Rafael A. Fissore, Jose B. Cibelli

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca2+ oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca2+ and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNTunits induced Ca2+ oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocystswere higher in bovine nuclear transferembryos activated using cycloheximide and 6-dimethylaminopurine(DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming.

Original languageEnglish
Pages (from-to)427-437
Number of pages11
JournalReproduction
Volume137
Issue number3
DOIs
StatePublished - 2009

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