An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova.
An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova.
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一种使用无透明带仓鼠卵子分析人类精子染色体的改进、高效方法。
DOI：
10.1097/00006254-198701000-00015
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发表时间：
1986
影响因子：
9.8
通讯作者：
K. Mikamo
中科院分区：
文献类型：
--
作者：
Y. Kamiguchi;K. Mikamo
我们建立了一种改进的用去透明带仓鼠卵分析人精子染色体的方法。（1）用5-15 μ M离子载体A23187溶液和含3.5%HSA的获能培养基（BWW培养基）连续处理精子，可显著提高人精子对仓鼠卵母细胞的受精率。从几种市售的HSA产品中选择诱导获能最有效的HSA。(2)通过将高获能精子以低浓度短时间授精卵母细胞来确保单精子受精。(3)TC 199培养基用于卵的授精后培养。(4)使用含有鬼臼毒素和长春碱（各0.04 μ g/ml）的培养基来阻断核融合和第一卵裂纺锤体形成。(5)用我们的逐步固定-空气干燥法代替Tarkowski法制备染色体玻片。在每次实验中，成功地对92至177个精子进行了核型分析，其数量相当于受精卵母细胞的43%-79%（平均值：62%）。尽管有上述的定量改进，Q显带的质量不一定令人满意，在我们的幻灯片。改进成带技术是本方法需要解决的一个重要问题。研究了从4名供体的9份精液样本中获得的总计1，091个精子的染色体畸变自发发生率。非整倍体和结构异常的发生率分别为0.9%（超单倍体，0.45%;低单倍体，0.45%）和13.0%。结构畸变包括断裂（45.1%）、片段（32.4%）、交换（21.8%）和缺失（0.7%）。X精子与Y精子的比例为53%比47%。这些结果进行了讨论与以前报道的比较。
We have developed an improved method for analyzing human sperm chromosome, using zona-free hamster ova. Our main improvements of methodology are as follows: (1) Fertilization rate of hamster oocytes by human spermatozoa was markedly raised by successive treatments of the spermatozoa with 5-15 microM ionophore A23187 solutions and a capacitation medium (BWW medium) containing 3.5% HSA. The HSA most effective in inducing capacitation was selected from several kinds of HSA products commercially available. (2) Monospermic fertilization was ensured by inseminating oocytes with highly capacitated spermatozoa at a low concentration for a short time. (3) TC medium 199 was used for postinsemination culture of the eggs. (4) A medium containing podophyllotoxin and vinblastine (0.04 micrograms/ml each) was used to block karyogamy and first-cleavage spindle formation. (5) Chromosome slides were prepared with our gradual fixation-air-dry method instead of Tarkowski's method. Ninety-two to 177 spermatozoa corresponding in number to 43%-79% (mean: 62%) of the inseminated oocytes were successfully karyotyped in each experiment. In spite of above-mentioned quantitative improvements, quality of Q-banding was not necessarily satisfactory in our slides. Improvement of banding technique is an important problem to be solved in our method. Spontaneous incidence of chromosome aberrations was studied in a total of 1,091 spermatozoa obtained from nine semen samples from four donors. Incidences of aneuploidy and structural anomaly were 0.9% (hyperhaploidy, 0.45%; hypohaploidy, 0.45%) and 13.0%, respectively. Structural aberrations included breaks (45.1%), fragments (32.4%), exchanges (21.8%), and deletions (0.7%). Ratio of X-sperm to Y-sperm was 53% to 47%. These results were discussed in comparison with those reported previously.