FUNCTIONAL ASPECTS OF SPERM CHROMATIN STRUCTURE

这项提议的目的是检验这样一个假设，即完全成熟的哺乳动物精子对其环境做出反应的染色质的操纵能力有限，从而提供一种机制来防止在细胞生命周期中受到破坏的DNA的传播。具体地说，我们认为精子可以经历一种类型的凋亡性DNA降解，这种能力的结果是精子细胞也与外源DNA相互作用。这对许多领域的研究具有重要意义，如体外受精(IVF)精子的操纵、男性避孕药的开发、疾病的性传播、转基因动物的生产和基因治疗。我们提出了一个精子染色质结构的模型，该模型为精子特异性的凋亡和与外源DNA的相互作用提供了可能的机制，其中鱼精蛋白结合的DNA环通过组蛋白结合的间隔区DNA片段连接起来。我们认为，这些组蛋白结合的间隔区，我们称之为活性染色质焦点，是发生凋亡DNA降解的部位，也是精子染色质与外源DNA相互作用的部位。我们将测试特定的假设，即哺乳动物精子有能力经历特定类型的凋亡DNA降解并可以与DNA相互作用，并且这些活动发生在组蛋白结合的活性染色质焦点。我们将首先通过脉冲场凝胶电泳和Southern印迹分析来确定组蛋白结合的DNA的分布，然后将这种分布与细胞凋亡的DNA降解模式进行比较。然后我们将确定小鼠精子如何与外源DNA相互作用。我们的初步数据表明，活精子与核基质的特定区域结合并结合外源DNA。我们将确定这种DNA是否整合到精子基因组中。最后，我们将分离和鉴定我们在仓鼠精子核基质中发现的一种类似拓扑异构酶II的蛋白。这是一种可能同时参与细胞凋亡、DNA结合和整合的酶。这一应用代表了我们实验室的一个新方向，将引导我们专注于治疗和避孕研究。

The goal of this proposal is to test the hypothesis that the fully mature mammalian spermatozoon has a limited capacity to manipulate its chromatin in response to its environment, providing a mechanism to prevent the propagation of DNA that has been damaged during the life of the cell. Specifically, we propose that the spermatozoon can undergo a type of apoptotic DNA degradation, and a consequence of this ability is that the sperm cell also interacts with exogenous DNA. This has significant implications for research in many areas such as the manipulation of spermatozoa for in vitro fertilization (IVF), the development of male contraceptives, sexual transmission of diseases, the production of transgenic animals, and gene therapy. We have proposed a model for sperm chromatin structure that provides a possible mechanism for the spermatozoa-specific apoptosis and interaction with exogenous DNA, in which the protamine bound DNA toroids are linked by histone bound "spacer" DNA segments. We propose that these histone bound spacers, which we term active chromatin foci, are the sites where apoptotic DNA degradation takes place, and the sites of sperm chromatin interaction with exogenous DNA. We will test the specific hypothesis that mammalian spermatozoa have the ability to undergo a specific type of apoptotic DNA degradation and can interact with DNA, and that these activities occur at the histone bound active chromatin foci. We will first determine the distribution of histone bound DNA by pulse-field gel electrophoresis and Southern blot analysis, then compare this distribution to the apoptotic DNA degradation pattern. We will then determine how mouse spermatozoa interact with exogenous DNA. Our preliminary data suggests that live spermatozoa bind to and incorporate exogenous DNA onto a specific region of the nuclear matrix. We will determine whether this DNA is integrated into the sperm genome. Finally, we will isolate and characterize a topoisomerase II- like protein that we have identified in hamster sperm nuclear matrices. This is an enzyme that might be expected to be involved in both apoptosis, DNA binding, and integration. This application represents a new direction for our laboratory that will lead us to focus on therapeutic and contraceptive studies.