Kawarasaki T, Kohsaka T, Sone M, Yoshida M, Bamba K
Shizuoka Swine and Poultry Experiment Station, Japan.
Mol Reprod Dev. 1995 Apr;40(4):455-9. doi: 10.1002/mrd.1080400409.
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.
本研究旨在确定是否可以通过原位杂交，使用经聚合酶链反应（PCR）产生的、与Y染色体特异的地高辛（Dig）标记DNA探针，来检测携带Y染色体的猪精子。使用一组寡核苷酸引物（5'-AAGTGGTCAGCGTGTCCATA-3'和5'-TTTCTCCTGTATCCTCCTGC-3'），对从一头公猪获得的1.25×10⁴个模板白细胞进行常规PCR（使用Dig-dUTP），扩增猪雄性特异性DNA序列的236 bp片段。当将用Dig标记的DNA探针进行荧光原位杂交应用于公猪和后备母猪制备的中期染色体铺片时，仅在Y染色体的长臂上检测到荧光信号。此外，将用Dig标记的DNA探针和碱性磷酸酶标记的抗Dig进行免疫细胞化学检测，应用于经二硫苏糖醇预处理的精子细胞核和白细胞；从公猪获得的51%的精子细胞核和96%的白细胞被标记，而从后备母猪获得的白细胞均未被Dig标记的DNA探针标记。结果表明，通过PCR产生的猪雄性特异性DNA探针进行原位杂交，使得通过原位杂交直接观察携带Y染色体的猪精子成为可能。
