Combination of Dithiothreitol and Detergent Treatment of Spermatozoa Causes Paternal Chromosomal Damage1
Combination of Dithiothreitol and Detergent Treatment of Spermatozoa Causes Paternal Chromosomal Damage1
复制标题
二硫苏糖醇和洗涤剂联合处理精子会导致父本染色体损伤1
作者：
M. Szczygiel;W. Ward
摘要 使用非离子去污剂 Triton X-100 (TX) 或二硫苏糖醇 (DTT) 处理精子可提高小鼠和人类胞浆内单精子注射 (ICSI) 的成功率。在这里，据我们所知，我们首次尝试同时使用这两种试剂，并发现这会导致父本原核严重的染色体断裂。我们通过使用 DTT 的几种组合（含或不含清洁剂）处理小鼠精子，进一步记录了这种效应。用强力移液处理精子以诱导膜破裂，或用 TX 或混合烷基三甲基溴化铵 (ATAB) 的离子去污剂处理。使用游泳的精子作为对照。在每次处理中，测试两个样品，在处理过程中添加或不添加 DTT。在所有使用 DTT 的样品中，通过去缩合测定证实了鱼精蛋白的减少。将经过不同处理后获得的精子核注射到卵母细胞中进行细胞遗传学分析，并对受精卵的父本和母本染色体进行可视化和检查。我们发现，单独用 DTT (87%, 153/176)、TX (79%, 112/142) 或 ATAB (85%, 99/116) 处理的精子 ICSI 产生的正常父本核板数量与游泳对照 (92%, 103/112) 相似。然而，在 TX + DTT 或 ATAB + DTT 治疗中，分别只有 22% (23/103) 和 40% (59/149) 的检查中期评分为正常。在存在 DTT 的情况下，通过剧烈移液破坏膜的精子的正常染色体频率略有降低 (61%, 64/104)，而没有 DTT 的精子则正常 (79%, 125/159)。然而，这种差异在统计学上并不显着。当在 EGTA 或 EGTA 和 EDTA 混合物存在的情况下用 TX + DTT 处理精子时，正常染色体的频率分别为 39% (45/114) 和 47% (38/81)，表明内源性精子核酸酶可能在染色体损伤中发挥作用。我们的结果表明，用去污剂和 DTT 同时处理精子会导致广泛的染色体断裂，因此不应在 ICSI 中尝试。
Abstract Treatment of spermatozoa with either the nonionic detergent Triton X-100 (TX) or dithiothreitol (DTT) has been suggested to confer enhanced success on intracytoplasmic sperm injection (ICSI) in mice and humans. Here, we attempted to use both reagents together, to our knowledge for the first time, and found that this caused severe chromosomal breaks in paternal pronuclei. We documented this effect further by treating mouse spermatozoa with several combinations of DTT with and without detergent. Spermatozoa were treated with vigorous pipetting to induce membrane disruption or with TX or the ionic detergent mixed alkyltrimethylammonium bromide (ATAB). Swim-up spermatozoa were used as controls. In each treatment, two samples were tested, with or without the addition of DTT during the treatment procedure. In all samples with DTT, protamine reduction was confirmed by the decondensation assay. Sperm nuclei obtained after different treatments were injected into oocytes for cytogenetic analysis, and paternal and maternal chromosomes of the zygote were visualized and examined. We found that the numbers of normal paternal karyoplates resulting from ICSI with spermatozoa treated with either DTT (87%, 153/176), TX (79%, 112/142), or ATAB (85%, 99/116) alone were similar to swim-up controls (92%, 103/112). However, only 22% (23/103) and 40% (59/149) of examined metaphases were scored as normal in TX + DTT or ATAB + DTT treatments, respectively. Spermatozoa in which the membranes were disrupted by vigorous pipetting in the presence of DTT had a slightly reduced frequency of normal chromosomes (61%, 64/104), whereas those without DTT were normal (79%, 125/159). However, this difference was not statistically significant. When spermatozoa were treated with TX + DTT in the presence of EGTA or a mixture of EGTA and EDTA, the frequency of normal chromosomes was 39% (45/114) and 47% (38/81), respectively, suggesting that endogenous sperm nucleases may play a role in chromosomal damage. Our results indicate that simultaneous treatment of spermatozoa with detergent and DTT induces extensive chromosomal breakage and, therefore, should not be attempted in ICSI.
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影响因子：
3.6
作者：
KIMURA, Y;YANAGIMACHI, R
通讯作者：
YANAGIMACHI, R
影响因子：
3.6
作者：
Kuretake, S;Kimura, Y;Yanagimachi, R
通讯作者：
Yanagimachi, R
影响因子：
6.1
作者：
Summers, MC;McGinnis, LK;Biggers, JD
通讯作者：
Biggers, JD
影响因子：
3.6
作者：
Kimura, Y;Yanagimachi, R;Yanagimachi, H
通讯作者：
Yanagimachi, H