Fluorescence in situ hybridization with y chromosome-specific probe in decondensed bovine spermatozoa

This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreito...

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Published inTheriogenology Vol. 52; no. 6; pp. 1043 - 1054
Main Authors Kobayashi, J., Kohsaka, T., Sasada, H., Umezu, M., Sato, E.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 15.10.1999
Subjects
Animals
bovine
bulls
Cattle
decondensation
Digoxigenin
Dithiothreitol - pharmacology
DNA Probes
fluorescence
fluorescence in situ hybridization
glutathione
Glutathione - pharmacology
heparin
Heparin - pharmacology
In Situ Hybridization, Fluorescence
Male
rapid methods
Sex Determination Analysis - methods
Sex Determination Analysis - veterinary
Sperm Head - drug effects
Sperm Head - ultrastructure
spermatozoa
Spermatozoa - cytology
Y Chromosome
Y chromosome-bearing sperm
fluorescence in situ hybridization
bovine
decondensation
Y chromosome-bearing sperm
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Summary:This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa.
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ISSN:0093-691X
1879-3231
DOI:10.1016/S0093-691X(99)00193-4