Analysis of human sperm-derived pronuclei by three-colour fluorescent in-situ hybridization.
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 spermatozoa (spermatozoa able to fertilize zona-free hamster eggs and produce a pronucleus) does not imply chromosomal selection.
Martínez-Pasarell
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Departament de Biologia Cellular i Fisiologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Márquez
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C
Coll
M D
MD
Egozcue
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J
Templado
C
C
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure*; D003116:Color; D006224:Cricetinae; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D012730:Sex Chromosomes; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000648:ultrastructure*