Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring
Long-term effects of mouse intracytoplasmic sperm injection with DNA-fragmented sperm on health and behavior of adult offspring
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DOI：
10.1095/biolreprod.107.065623
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发表时间：
2008-04-01
影响因子：
3.6
通讯作者：
Gutierrez-Adan, Alfonso
中科院分区：
文献类型：
--
作者：
Fernandez-Gonzalez, Raul;Moreira, Pedro Nuno;Gutierrez-Adan, Alfonso
遗传和环境因素对精子的DNA损伤程度不同。通常，DNA片段化精子（DFS）用于人类生殖的卵胞浆内单精子注射（ICSI）治疗，DFS的使用仍然是一个值得关注的问题。本研究的目的是探讨长期的后果，对小鼠的发展和行为的ICSI与DFS。使用CD 1和B6D2F1小鼠品系，卵母细胞注射新鲜精子或不含冷冻保护剂的冷冻解冻精子。该处理增加了TUNEL阳性精子的百分比、通过彗星试验测量的尾长和通过定量PCR测量的端粒损失。将ICSI产生的胚胎在KSOM中培养24小时，并将2细胞胚胎转移到CD 1雌性中。DFS降低了植入前胚胎发育率和后代数量。用5-甲基胞嘧啶抗体免疫荧光染色显示，ICSI产生的胚胎中雄性原核的主动去甲基化延迟2小时。此外，ICSI还影响了一些表观遗传调控基因的转录和甲基化，如印记基因、X连锁基因和逆转录转座子基因。在3月龄和12月龄时，对采用DFS产生的动物和体内受精对照的ICSI进行行为测试：自发活动（旷场）、探索/焦虑行为（高架十字迷宫，旷场）和空间记忆（Y迷宫中的自由选择探索范式）。ICSI组女性在旷场试验中表现为焦虑增加、缺乏习惯性模式、短时空间记忆缺陷和年龄依赖性运动功能减退（P <0.05）。用新鲜或冷冻精子行ICSI的小鼠从16周龄起体重增长显著高于对照组（P <0.01）。16月龄动物的解剖病理学分析显示一些大器官和病理学增加（33%的DFS产生的CD 1雌性动物在肺和背部或颈部真皮中出现一些实体瘤）。此外，20%的B6D2F1小鼠在出生后的前5个月内死亡，25%的存活动物表现出早衰症状，70%的B6D2F1小鼠在不同类型的肿瘤中比对照组更早死亡。我们认为，根据DFS的水平，卵母细胞可以部分修复片段化的DNA，产生能够植入并产生活后代的囊胚。然而，不完全修复可能导致长期病变。我们的数据表明，在ICSI中使用DFS可以产生仅在以后的生活中出现的影响，例如异常生长，过早衰老，异常行为和间质肿瘤。
Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intracytoplasmic sperm injection (ICSI) treatments in human reproduction, and use of DFS is still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or with frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL-positive spermatozoa, tail length as measured by comet assay, and loss of telomeres as measured by quantitative PCR. The ICSI-generated embryos were cultured for 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. The DFS reduced both the rate of preimplantation embryo development and number of offspring. Immunofluorescence staining with an antibody against 5-methylcytosine showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 mo of age, ICSI with DFS-produced animals and in vivo-fertilized controls were submitted to behavioral tests: locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, and age-dependent hypolocomotion in the open-field test (P < 0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than that of their control counterparts from 16 wk on (P < 0.01). Anatomopathological analysis of animals at 16 mo of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors in lungs and dermis of back or neck). Moreover, 20% of the B6D2F1 mice generated with DFS died during the first 5 mo of life, with 25% of the surviving animals showing premature aging symptoms, and 70% of the B6D2F1 mice generated with DFS died earlier than controls with different kind of tumors. We propose that depending on the level of DFS, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. The incomplete repair, however, may lead to long-term pathologies. Our data indicate that use of DFS in ICSI can generate effects that only emerge during later life, such as aberrant growth, premature aging, abnormal behavior, and mesenchymal tumors.