Calyculin-A improves chromosome condensation for cytogenetic analysis of blastomeres from bovine and murine eight-cell stage embryos

Abstract

This study evaluated the serine/threonine phosphatase inhibitor calyculin-A for rapid, efficient induction of premature chromosome condensation (PCC) in blastomeres obtained from Day 3 bovine and Day 2 murine eight-cell stage embryos, and its potential for use in cytogenetic analysis. Experiment 1 tested calyculin-A duration (0, 60, 120, and 180min) to induce PCC in bovine blastomeres. More blastomeres that underwent PCC had chromosomes suitable for cytogenetic analysis if treated for 120 or 180min (P<0.005). Experiment 2 compared doses of calyculin-A (0, 10, 50, and 100nM) on bovine blastomeres; calyculin-A (50nM, 120min) induced PCC suitable for cytogenetic analysis in the greatest number of blastomeres when compared to other doses (52.5%; P<0.005). Effects of calyculin-A (50nM) on murine blastomeres at durations of 0, 60, 90, and 120min to induce PCC were tested in Experiment 3, with 90min inducing the highest frequency of condensed chromosomes suitable for cytogenetic analysis (34%; P<0.05). Finally, Experiment 4 evaluated calyculin-A treated bovine embryos under optimal conditions (50nM, 120min) for use in gender and cytogenetic analysis. Whole chromosome paint probes were successfully hybridized to chromosomes along with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) counterstaining, allowing detection of embryo gender (54% F:46% M) and ploidy of individual blastomeres within embryos (64% diploid:36% mixoploid embryos). In conclusion, we inferred that calyculin-A was useful for rapid induction of PCC, producing chromosome spreads suitable for cytogenetic analysis of blastomeres in G1 or G2/M phase of the cell cycle.