Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection.
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 Ca(2)(+) 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 Ca(2)(+) 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 SCNT units induced Ca(2)(+) 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 blastocysts were higher in bovine nuclear transfer embryos 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.
Ross
Pablo J
PJ
Department of Animal Science, Michigan State University, East Lansing, Michigan 48824, USA.
Rodriguez
Ramon M
RM
Iager
Amy E
AE
Beyhan
Zeki
Z
Wang
Kai
K
Ragina
Neli P
NP
Yoon
Sook-Young
SY
Fissore
Rafael A
RA
Cibelli
Jose B
JB
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006657:Histones / Q000378:metabolism; D007267:Injections; D008745:Methylation; D053652:Nuclear Transfer Techniques; D018075:RNA, Complementary / Q000008:administration & dosage*; D010738:Type C Phospholipases / Q000235:genetics*
D006657:Histones; D018075:RNA, Complementary; C001020:N(6),N(6)-dimethyladenine; D003513:Cycloheximide; D010738:Type C Phospholipases; D000225:Adenine; D002118:Calcium