108 CALYCULIN-A INDUCES CHROMOSOME CONDENSATION FOR CYTOGENETIC ANALYSIS IN BOVINE AND MURINE BLASTOMERES

Abstract

Cytogenetic studies of preimplantation embryos have traditionally used interphase fluorescence in situ hybridization (FISH) to examine chromosome copy number, chromosomal rearrangements, or sex determination. Comprehensive analysis of chromosomes is hindered by the low mitotic index of blastomeres, combined with the technical limitations of interphase FISH. Efforts to overcome these limitations include inducing premature chromosome condensation (PCC) for metaphase FISH, by fusing blastomeres with metaphase II-stage oocytes, or with time-consuming incubations with okadaic acid or vinblastine. Our objective was to evaluate Calyculin-A (CA) as an alternative to induce PCC in blastomeres. In vitro-produced bovine 8-cell embryos and frozen–thawed mouse 8-cell embryos were cultured with CA and examined for changes in morphology before fixation. Colcemid (0.1 mg mL^–1), the standard for cytogenetic analysis, and vehicle served as positive and negative controls. Blastomeres were washed in hypotonic solution, loaded onto slides, and fixed in methanol:acetic acid (3:1). The degree of chromatin condensation and quality of chromosome spreads were determined by 42,6-diamidino-2-phenylindole staining and visualization on an epifluorescence microscope (100×). Experiment 1 (1 rep, 136 cells) tested dose of CA on bovine blastomeres at 50, 100, 150, 250, 500, and 750 nm. Experiments 2 and 3 tested culture duration of CA (50 nm) at 60, 120, and 180 min in bovine blastomeres and 60, 90, and 120 min in mouse blastomeres (2 reps each, 132 and 207 cells, respectively). Data were analyzed by chi-square with significance deemed P < 0.05. Cell lysis and blebbing was observed in bovine blastomeres treated with greater than 150 nm CA. Duration of CA treatment affected the frequency of bovine and mouse blastomeres undergoing PCC. Fewer bovine blastomeres treated for 60 min underwent chromatin condensation compared to blastomeres treated for at least 120 min with 50 nm CA (25% v. 100%; P < 0.05). In mice, the frequency of blastomeres undergoing PCC was lower (43%) for 50 nm CA at 60 min than at 90 and 120 min (90 and 100%, respectively; P < 0.05). Chromatin condensation suitable for cytogenetic analysis was 20, 34, and 20%, respectively, but did not differ (P > 0.10). Further examination (Exp. 4, 5 reps, 293 cells) comparing the degree of condensation revealed 55% of the total bovine blastomeres treated with 50 nm CA for 120 min were suitable for cytogenetic analysis, as compared to 30% treated with colcemid for 16 h (P < 0.05). Bovine and mouse blastomeres treated with vehicle had lower PCC than all other treatments averaging 2 and 7%, respectively (P < 0.05). These results suggest that CA can rapidly induce PCC in blastomeres from pre-implantation bovine and murine embryos, but the degree of chromatin condensation may not always be suitable for detailed cytogenetic analysis from a single blastomere.