Correlation of developmental differences of nuclear transfer embryos cells to the methylation profiles of nuclear transfer donor cells in Swine.

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues.

Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts.

Early embryonic development in the pig requires DNA methylation remodeling of the maternal and paternal genomes. Aberrant remodeling, which can be exasperated by in vitro technologies, is detrimental to development and can result in physiological and anatomic abnormalities in the developing fetus and offspring. Here, we developed and validated a microarray based approach to characterize on a global scale the CpG methylation profiles of porcine gametes and blastocyst stage embryos.

Na(+)/Ca(2+) exchanger in porcine oocytes.

The presence of the Na(+)/Ca(2+) exchange mechanism was investigated in porcine oocytes. Immature and in vitro-matured oocytes were loaded with the Ca(2+)-sensitive fluorescent dye fura 2 and changes in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) were monitored after altering the Na(+) concentration gradient across the plasma membrane. Decreasing the extracellular Na(+) concentration induced an increase in [Ca(2+)](i) possibly by a Ca(2+) influx via the Na(+)/Ca(2+) exchanger.

Cyclin B1 levels in the porcine 4-cell stage embryo.

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor alpha-amanitin.

Capacitative calcium entry mechanism in porcine oocytes.

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry.