Sperm DNA damage in fertilization

受精过程中精子 DNA 损伤

• 批准号: 6988265
• 负责人: Monika A Ward
• 金额: $ 13.52万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6988265
• 关键词: DNA damage; DNA replication; chromosome aberrations; developmental genetics; egg /ovum; embryogenesis; fertilization; laboratory mouse; male; nuclease; nucleic acid metabolism; nucleic acid quantitation /detection; sperm; sperm analysis; spermatogenesis

DESCRIPTION (provided by applicant): The major goal of this application is to evaluate the phenomenon of sperm DNA degradation observed as the result of various sperm treatments. The hypothesis is that there exists a mechanism in mammalian fertilization that prevents the transmission of potentially damaged DNA to the embryo by disruption of the paternal chromosomes that this mechanism is based on the activity of endogenous nucleases, and that it can be active within sperm cells. In our preliminary studies we have shown that when spermatozoa are treated with various chemicals (detergents, DTT, exogenous DNA) or are subjected to mechanical insults (freeze drying and freezing without cryoprotection), and then injected into the oocytes, the paternal chromosomes in the zygotes are broken. We have also shown that this DNA degradation could be partially or completely prevented when ion chelators, EGTA or/and EDTA, were present in the sperm handling medium, which suggests the involvement of endogenous nuclease/s in this process. The idea that spermatozoa may have nuclease-dependent mechanism for DNA degradation differs from current thinking on the function of mammalian male gametes. In this application we will initiate the exploration on the origin and function of sperm DNA damage by expanding our preliminary data and answering the first major questions that this data brought. In Specific Aim 1, we will determine the causes of sperm DNA damage, and whether they are inherent to the cell or, are the result of experimental manipulation. We will explore what are the conditions under which the paternal chromosome breaks, examine early post-fertilization events after ICSI with treated spermatozoa, and test if paternal chromosome breakage can be observed in the absence of ICSI, to exclude the possibility that observed chromosome breakage is an artifact of ICSI combined with manipulation. Specific Aim 2 will answer the most crucial question of this application: Does DNA degradation occur in the spermatozoa or in the oocyte? We will test whether paternal DNA breakage can be induced in the oocytes after fertilization. We will analyze sperm DNA integrity before and after DNA synthesis. We will also evaluate if maternal (oocyte) DNA is degraded in the result of insults to which sperm, oocyte, or both are exposed. Finally, we will establish whether spermatozoa are able to induce degradation of DNA other than their own, a direct test to confirm the possibility that DNA degradation mechanism exist within sperm heads. In Specific Aim 3, we will test if paternal DNA degradation depends on the activity of endogenous Ca2+ and Mg2+dependent nucleases or topoisomerase II. We will establish if stresses to which spermatozoa are exposed can be neutralized by the presence of on chelators and other nuclease inhibitors, what is the range of nuclease activity, and what are the conditions under which the maximum protection is provided. We will also establish if endogenous Ca2+ and Mg2+dependent nucleases are present in the oocytes, if they can be inhibited there, and what will be the effects of this inhibition on paternal DNA integrity. The significance of this proposal is that it will test a novel idea that spermatozoa are active cells able to respond to their environment. This response is suspected to be a part of the mechanism that allows preventing the transmission of potentially damaged DNA to the embryo during fertilization. The study will have impact on basic science by advancing our understanding of biological system of mammalian reproduction. It will also have significance for clinical research and health and welfare of infertile couples providing clues for human assisted reproduction clinics as to what treatments evoke DNA-degrading mechanisms in spermatozoa and therefore should be avoided.

描述（由申请人提供）：本申请的主要目标是评估由于各种精子处理而观察到的精子 DNA 降解现象。该假设认为，哺乳动物受精中存在一种机制，可以通过破坏父本染色体来防止可能受损的 DNA 传输到胚胎，该机制基于内源核酸酶的活性，并且可以在精子细胞内活跃。在我们的初步研究中，我们已经表明，当精子用各种化学物质（洗涤剂、DTT、外源DNA）处理或受到机械损伤（冷冻干燥和无冷冻保护的冷冻），然后注射到卵母细胞中时，受精卵中的父本染色体就会断裂。我们还表明，当精子处理介质中存在离子螯合剂 EGTA 或/和 EDTA 时，可以部分或完全阻止这种 DNA 降解，这表明内源性核酸酶参与了这一过程。精子可能具有核酸酶依赖性 DNA 降解机制的观点与目前对哺乳动物雄性配子功能的看法不同。在此应用中，我们将通过扩展我们的初步数据并回答该数据带来的首要问题，开始探索精子 DNA 损伤的起源和功能。在具体目标 1 中，我们将确定精子 DNA 损伤的原因，以及它们是细胞固有的还是实验操作的结果。我们将探索父本染色体断裂的条件，检查处理精子的 ICSI 后早期受精后事件，并测试在没有 ICSI 的情况下是否可以观察到父本染色体断裂，以排除观察到的染色体断裂是 ICSI 与操作相结合的假象的可能性。具体目标 2 将回答本应用中最关键的问题：DNA 降解发生在精子还是卵母细胞中？我们将测试受精后是否可以在卵母细胞中诱导父本 DNA 断裂。我们将在 DNA 合成前后分析精子 DNA 的完整性。我们还将评估母体（卵母细胞）DNA 是否因精子、卵母细胞或两者均受到损伤而降解。最后，我们将确定精子是否能够诱导自身以外的DNA降解，这是一个直接的测试，以证实精子头部内存在DNA降解机制的可能性。在具体目标 3 中，我们将测试父本 DNA 降解是否取决于内源性 Ca2+ 和 Mg2+ 依赖性核酸酶或拓扑异构酶 II 的活性。我们将确定精子所面临的压力是否可以通过螯合剂和其他核酸酶抑制剂的存在来中和，核酸酶活性的范围是多少，以及在什么条件下提供最大保护。我们还将确定卵母细胞中是否存在内源性 Ca2+ 和 Mg2+ 依赖性核酸酶，它们是否可以被抑制，以及这种抑制对父本 DNA 完整性有何影响。该提案的意义在于，它将检验一个新观点，即精子是能够对其环境做出反应的活跃细胞。这种反应被怀疑是防止受精过程中可能受损的 DNA 传输到胚胎的机制的一部分。这项研究将通过增进我们对哺乳动物生殖生物系统的理解而对基础科学产生影响。它还对临床研究以及不孕夫妇的健康和福利具有重要意义，为人类辅助生殖诊所提供线索，了解哪些治疗会引起精子中的 DNA 降解机制，因此应该避免。