Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies.
OBJECTIVE
To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies.


METHODS
The two variables were examined by double probing of the same spermatozoa.


METHODS
Academic Andrology Laboratory.


METHODS
Semen samples subjected for analyses were examined.


METHODS
We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes.


METHODS
Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated.


RESULTS
The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm.


CONCLUSIONS
The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.
Ovári
Laszlo
L
Yale Sperm Physiology Laboratory, Yale University, School of Medicine, New Haven, Connecticut 06510, USA.
Sati
Leyla
L
Stronk
Jill
J
Borsos
Antal
A
Ward
David C
DC
Huszar
Gabor
G
D000782:Aneuploidy; D000814:Aniline Compounds / Q000494:pharmacology*; D004249:DNA Damage / Q000502:physiology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006657:Histones / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007248:Infertility, Male / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007621:Karyotyping / Q000379:methods; D008297:Male; D055101:Semen Analysis / Q000379:methods; D013094:Spermatozoa / Q000166:cytology* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D006657:Histones; C017006:aniline blue