Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization

Human Reproduction
O Martínez-PasarellC Templado

Abstract

We describe a modification of the human sperm-zona-free hamster egg fusion method that permits the study of aneuploidy in sperm-derived pronuclei by multicolour fluorescent in-situ hybridization (FISH). Zona-free hamster eggs and human spermatozoa were fused and cultured for 15 h in the presence of colcemid (1 microg/ml of medium) to obtain hamster oocytes arrested at metaphase II and human spermatozoa at the pronuclear stage. By applying a whole human genomic DNA probe we confirmed that 100% of pronuclei tested (372/372) were of human origin. One-colour fluorescent in-situ hybridization using a centromeric 18 probe was applied to 919 pronuclei with different dithiothreitol (DTT) pretreatments: 50 mM (10 min) or 25 mM (20 and 25 min). The highest hybridization efficiency was obtained with treatment with 25 mM DTT for 20 min (90.3%). Sex chromosome aneuploidy was analysed by three-colour FISH in a total of 2596 pronuclei from a normal donor. Hybridization efficiency was 98.6%. The disomy rates for X, Y and XY chromosomes (0.11, 0.04 and 0.08% respectively) were similar to data reported for sperm nuclei by three-colour FISH and to those obtained in sperm chromosomes. These results suggest that selection of potentially fertile spe...Continue Reading

Citations

Feb 8, 2008·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Ewa WilandMaciej Kurpisz
Aug 13, 2008·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Marta OlszewskaMaciej Kurpisz
Sep 7, 2002·Fertility and Sterility·Brian DaleFulvio Zullo
Mar 4, 1999·The Journal of Obstetrics and Gynaecology Research·P Li, H Hoshiai
Sep 30, 2005·Cytogenetic and Genome Research·C TempladoJ Benet

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