Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei.
Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Wyrobek
A J
AJ
Molecular Biology Section, Lawrence Livermore National Laboratory, University of California, Livermore 94550.
Alhborn
T
T
Balhorn
R
R
Stanker
L
L
Pinkel
D
D
D002467:Cell Nucleus / Q000648:ultrastructure; D002843:Chromatin / Q000648:ultrastructure*; D015342:DNA Probes; D004229:Dithiothreitol; D005453:Fluorescence; D006801:Humans; D007463:Iodobenzoates; D008094:Lithium / Q000494:pharmacology; D008297:Male; D000644:Quaternary Ammonium Compounds; D012459:Salicylates / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013094:Spermatozoa / Q000187:drug effects / Q000648:ultrastructure*; D014998:Y Chromosome / Q000187:drug effects / Q000648:ultrastructure*
D002843:Chromatin; D015342:DNA Probes; D007463:Iodobenzoates; D000644:Quaternary Ammonium Compounds; D012459:Salicylates; C020891:3,5-diiodosalicylic acid; D012965:Sodium Chloride; D008094:Lithium; D004229:Dithiothreitol