Sperm DNA damage in fertilization

描述（由申请人提供）：本申请的主要目的是评估由于各种精子处理而观察到的精子DNA降解现象。假设在哺乳动物受精过程中存在一种机制，通过破坏父亲染色体来阻止潜在受损DNA向胚胎的传递，这种机制是基于内源性核酸酶的活性，并且它可以在精子细胞中活跃。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被破坏。我们还表明，当离子螯合剂EGTA或/和EDTA存在于精子处理培养基中时，可以部分或完全阻止这种DNA降解，这表明内源性核酸酶/s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。在本应用中，我们将通过扩展我们的初步数据，并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系染色体断裂，检查经过ICSI处理的精子在受精后的早期事件，并测试在没有ICSI的情况下是否可以观察到父系染色体断裂，以排除观察到的染色体断裂是ICSI结合操作的人工产物的可能性。特异性目标2将回答这个应用中最关键的问题：DNA降解是发生在精子还是卵母细胞中？我们将测试受精后是否会在卵母细胞中诱导父系DNA断裂。我们将分析精子DNA合成前后的完整性。我们还将评估母体（卵母细胞）DNA是否在精子、卵母细胞或两者暴露的损伤中被降解。最后，我们将确定精子是否能够诱导其他DNA的降解，这是一个直接的测试，以确认DNA降解机制存在于精子头部的可能性。在特异性目标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。我们将确定精子所暴露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什么，以及在什么条件下提供最大的保护。我们还将确定内源性Ca2+和Mg2+依赖性核酸酶是否存在于卵母细胞中，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，它将检验一种新颖的观点，即精子是能够对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潜在受损DNA传递给胚胎的机制的一部分。该研究将促进我们对哺乳动物生殖生物系统的认识，从而对基础科学产生影响。这对于临床研究和不育夫妇的健康和福利也有重要意义，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。

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.