Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins. Increased levels of these factors were not associated with mRNA polyadenylation or increased transcription rates of CCNB1 and MOS (Moloney murine sarcoma viral oncogene homolog) during CHX arrest. We found that treatment of CHX-arrested oocytes with the actin filament-stabilizing agent Jasplakinolide (Jasp) delayed GVBD following release from CHX arrest and that this was correlated with reduced maturation-promoting factor (MPF) activity. These results suggest that CCNB1 mRNAs released from actin filaments during CHX arrest increase CCNB1 transcripts available for translation after release from CHX arrest, leading to the precocious activation of MPF and accelerated meiotic progression.
Induced pluripotent stem cells (iPSCs) share similar characteristics with ESCs of indefinite in vitro growth, and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Since derivation of stable ESC lines from farm animals has not been possible, it is important to determine whether iPSCs can be used as substitutes to ESCs for generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were bFGF-and Activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities and failed to activate endogenous NANOG.Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Since cloning of farm animals is best achieved with diploid cells (G 1 /G 0 ), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPS-NT embryos was lower than with somatic cells (2% vs 10% blastocysts, p< 0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental foetal fibroblasts was similar to those generated by in-vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.
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