Kawarasaki T, Sone M, Yoshida M, Bamba K
Shizuoka Swine and Poultry Experiment Station, Japan.
Mol Reprod Dev. 1996 Apr;43(4):548-53. doi: 10.1002/(SICI)1098-2795(199604)43:4<548::AID-MRD18>3.0.CO;2-V.
This study was carried out to develop a rapid and simultaneous detection system of chromosome Y- and 1-bearing porcine spermatozoa by fluorescence in situ hybridization (FISH). Chromosome Y- and 1-specific DNA probes were produced by polymerase chain reaction with digoxigenin (Dig)- or biotin-dUTP. The hybridization probe mixture of labeled Y-chromosome and chromosome 1-specific DNA was applied to the preparation, immediately denatured at 75 degrees C for 8 min, hybridized for 5 min at 37 degrees C and overall FISH steps were done within a few hours. When double FISH with Dig-labeled chromosome Y-specific and biotin-labeled chromosome 1-specific probes was applied to sperm nuclei pretreated with dithiothreitol, the average of 50.9% of sperm nuclei had the Dig-signal, 99.2% of the sperm nuclei had the biotin-signal and the average of 0.3% of sperm nuclei showed no signal. The putative rate of Y-bearing spermatozoa ranged from 49.8% to 52.8% among 5 boars and the average putative rate of Y-bearing spermatozoa was 51.0%. The results indicated that a rapid and simultaneous FISH with chromosome Y- and 1-specific porcine DNA probes produced by PCR made possible more accurate assessment of Y-bearing porcine spermatozoa.
本研究旨在通过荧光原位杂交（FISH）开发一种快速同时检测携带Y染色体和1号染色体的猪精子的系统。通过用洋地黄毒苷（Dig）或生物素-dUTP进行聚合酶链反应制备Y染色体和1号染色体特异性DNA探针。将标记的Y染色体和1号染色体特异性DNA的杂交探针混合物应用于样本制备，立即在75℃变性8分钟，在37℃杂交5分钟，整个FISH步骤在数小时内完成。当将用Dig标记的Y染色体特异性探针和生物素标记的1号染色体特异性探针进行双重FISH应用于用二硫苏糖醇预处理的精子核时，平均50.9%的精子核有Dig信号，99.2%的精子核有生物素信号，平均0.3%的精子核无信号。5头公猪中携带Y染色体精子的推定率在49.8%至52.8%之间，携带Y染色体精子的平均推定率为51.0%。结果表明，用PCR制备的猪Y染色体和1号染色体特异性DNA探针进行快速同时FISH能够更准确地评估携带Y染色体的猪精子。
