Enhanced mitotic arrest and chromosome resolution for cytogenetic analysis in the eastern mosquitofish, Gambusia holbrooki

Abstract

Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24-96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (μm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species.

Keywords: Chromosome elongation; Ethidium bromide; Metaphase resolution; Mitotic index; Sex dimorphism.