Twigg J P, Irvine D S, Aitken R J
MRC Reproductive Biology Unit, Edinburgh.
Hum Reprod. 1998 Jul;13(7):1864-71. doi: 10.1093/humrep/13.7.1864.
We present the first evidence that genetically damaged human spermatozoa are able to form normal pronuclei in oocytes after intracytoplasmic sperm injection (ICSI). The role of reactive oxygen species (ROS) as a cause of chromatin and DNA damage is well recognized. The same class of molecule can be found in the semen of males with severe infertility, who remained infertile until the advent of ICSI. In this study we have investigated the role of ROS in the induction of chromatin damage, DNA strand breakage and the subsequent ability of spermatozoa to decondense and form pronuclei after ICSI. Spermatozoa from normozoospermic men participating in our research programme were exposed to oxidizing environments created by co-incubation with hydrogen peroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH) or activated white cells. The subsequent ability of the spermatozoa to decondense in vitro was examined using sequential incubations in EDTA, dithiothreitol and sodium dodecyl sulphate, and the amounts of DNA strand breakage were assessed using an in-situ nick translation protocol. Finally, cells exposed to hydrogen peroxide, NADPH and activated leukocytes were microinjected into hamster oocytes, and their ability to decondense and form normal pronuclei was determined. The results indicate that human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage, yet the rate of pronucleus formation is no different from that of untreated control cells. The ability of genetically damaged spermatozoa to achieve normal fertilization following ICSI has implications for the practice of this form of assisted conception therapy.
我们提供了首个证据，即基因受损的人类精子在卵胞浆内单精子注射（ICSI）后能够在卵母细胞中形成正常原核。活性氧（ROS）作为染色质和DNA损伤原因的作用已得到充分认识。在严重不育男性的精液中也能发现同一类分子，这些男性在ICSI出现之前一直不育。在本研究中，我们调查了ROS在诱导染色质损伤、DNA链断裂以及ICSI后精子随后的解聚和形成原核能力方面的作用。参与我们研究项目的正常精子男性的精子与过氧化氢、还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）共同孵育或与活化白细胞共同孵育，从而暴露于氧化环境中。随后，通过在乙二胺四乙酸（EDTA）、二硫苏糖醇和十二烷基硫酸钠中进行连续孵育来检测精子在体外解聚的能力，并使用原位缺口平移方案评估DNA链断裂的量。最后，将暴露于过氧化氢、NADPH和活化白细胞的细胞显微注射到仓鼠卵母细胞中，并确定它们解聚和形成正常原核的能力。结果表明，人类精子染色质在氧化应激条件下会发生交联，并表现出DNA链断裂增加，但原核形成率与未处理的对照细胞无异。基因受损的精子在ICSI后实现正常受精的能力对这种辅助受孕治疗形式的实践具有重要意义。
