Sperm DNA damage in fertilization

描述（由申请方提供）：本申请的主要目的是评价各种精子处理导致的精子DNA降解现象。该假说认为哺乳动物受精过程中存在一种机制，通过破坏父本染色体来防止潜在受损DNA传递到胚胎，该机制基于内源性核酸酶的活性，并且它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，当精子用各种化学品（洗涤剂，DTT，外源DNA）处理或受到机械损伤（冷冻干燥和冷冻无冷冻保护），然后注入卵母细胞时，受精卵中的父本染色体被破坏。我们还表明，当精子处理培养基中存在离子螯合剂EGTA或/和EDTA时，可以部分或完全防止这种DNA降解，这表明内源性核酸酶参与了这一过程。精子可能具有依赖核酸酶的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。在本申请中，我们将通过扩展我们的初步数据和回答这些数据带来的第一个主要问题，开始对精子DNA损伤的起源和功能的探索。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。我们将探讨在什么条件下，父亲的染色体断裂，检查早期受精后事件ICSI处理的精子，并测试，如果父亲的染色体断裂可以观察到在没有ICSI，排除可能性，观察到的染色体断裂是一个人工制品的ICSI结合操作。具体目标2将回答这个应用程序中最关键的问题：DNA降解发生在精子还是卵母细胞？我们将测试受精后卵母细胞中是否能诱导父本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、卵母细胞或两者暴露的损伤而降解。最后，我们将确定精子是否能够诱导其自身以外的DNA降解，这是一个直接的测试，以确认精子头部内存在DNA降解机制的可能性。在特定目标3中，我们将测试父本DNA降解是否取决于内源性Ca 2+和Mg 2+依赖性核酸酶或拓扑异构酶II的活性。我们将确定精子所暴露的应激是否可以被螯合剂和其他核酸酶抑制剂的存在所中和，核酸酶活性的范围是什么，以及在什么条件下提供最大的保护。我们还将确定卵母细胞中是否存在内源性Ca 2+和Mg 2+依赖性核酸酶，它们是否可以在那里被抑制，以及这种抑制对父本DNA完整性的影响。这一提议的意义在于它将检验一个新的想法，即精子是能够对其环境做出反应的活跃细胞。这种反应被怀疑是机制的一部分，该机制可以防止在受精过程中将潜在受损的DNA传递给胚胎。这项研究将通过促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。这也将对不孕夫妇的临床研究和健康和福利具有重要意义，为人类辅助生殖诊所提供线索，以了解哪些治疗引起精子中的DNA降解机制，因此应该避免。

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.