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Detection of Y‐bearing porcine spermatozoa by in situ hybridization using digoxigenin‐labeled, porcine male‐specific DNA probe produced by polymerase chain reaction
Author(s) -
Kawarasaki Tatsuo,
Kohsaka Tetsuya,
Sone Masaru,
Yoshida Mitsutoshi,
Bamba Kimio
Publication year - 1995
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080400409
Subject(s) - dig , microbiology and biotechnology , biology , in situ hybridization , digoxigenin , boar , hybridization probe , dna , semen , fluorescence in situ hybridization , polymerase chain reaction , in situ , oligomer restriction , oligonucleotide , chromosome , biochemistry , chemistry , genetics , messenger rna , gene , organic chemistry
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 × 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. © 1995 Wiley‐Liss, Inc.