Detection of micronuclel in the germ cell line of male mice using multi-color fluorescence in-situ hybridization
Conference
·
OSTI ID:149610
The ability to detect genetic abnormalities in germ cell helps further our understanding of the effects of toxicants on congenital defects during birth and development. We are currently developing a method to detect one such genetic abnormality, the micronucleus in the male germ line. A micronucleus, a chromatin piece of whole chromosome separated from the main nucleus, is an indicator of a chromosomally abnormal gamete. Our goals are to detect and determine the type of micronuclei in round spermatids, the haploid precursors of sperm, and to investigate the effects of exposure of mice to germinal mutagens. To make microscope slide preparations of spermatid micronuclei, seminiferous tubules were teased apart, treated with collagenase and trypsin, centrifuged in testis isolation medium, dropped on glass slides, and air dried. Micronuclei were evident when DNA was stained with DAPI. Also, to determine their chromosomal origin, DNA probes for the pan-centromeric regions and the X chromosome were labeled with digoxigenin and biotin by nick translation and the signals were detected using a combination of rhodamine and FITC. Two types of micronuclei can be discerned by these multi-probe procedures; one carries a chromosomal fragment missing a centromere, while the other contains a whole chromosome. The developed methods are now being applied to a current study. The effect of aneugen chloral hydrate on the development of the germ cells of male mice is being investigated with the micronuclei and fluorescence in-situ hybridization methods. By sampling mice shortly after exposure, we investigate the sensitivity of methods. By sampling mice shortly after exposure, we investigate the sensitivity of the meiotic has of spermatogenesis, whereas sampling mice at a longer time interval will reveal effects on spermatogonial cells in mitosis. Such information may have important consequences for human males exposed to this anesthetic agent.
- Research Organization:
- Lawrence Livermore National Lab., CA (United States)
- OSTI ID:
- 149610
- Report Number(s):
- UCRL-ID--114972-1; CONF-9312109--Absts.; ON: DE95012452
- Country of Publication:
- United States
- Language:
- English
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