Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled, porcine male-specific DNA probe produced by polymerase chain reaction.
This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5'-AAGTGGTCAGCGTGTCCATA-3' and 5'-TTTCTCCTGTATCCTCCTGC-3') for 236 bp fragment of porcine male-specific DNA sequence and 1.25 x 10(4) template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization.
Kawarasaki
T
T
Shizuoka Swine and Poultry Experiment Station, Japan.
Kohsaka
T
T
Sone
M
M
Yoshida
M
M
Bamba
K
K
D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D015342:DNA Probes; D004076:Digoxigenin; D017403:In Situ Hybridization; D007621:Karyotyping; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013094:Spermatozoa / Q000737:chemistry / Q000648:ultrastructure*; D013552:Swine; D014998:Y Chromosome / Q000737:chemistry / Q000235:genetics*
D015415:Biomarkers; D015342:DNA Probes; D004076:Digoxigenin