The origin and compartmentalization of core somatic histones and GSTO2 in sperm and their role in fertilization

受精是发育的基石，精子和卵子这两个分化的细胞为胚胎发生奠定了基础，配子的能力是必不可少的。然而，关于精子-卵子相互作用导致合子发育的分子和机制，只有基本的知识可用。我们的长期目标是了解这些机制。我们的方法是从分子上解剖与受精过程有关的精子隔膜，如顶体和核周膜。这些蛋白质的特性为了解它们的意义提供了线索，并导致了特定探针和实验的设计，以测试它们预测的受精功能。这一策略已经取得了成功，因为我们发现了新的精子蛋白，这些蛋白与卵子的穿透和受精过程中的激活有关，导致了用于检测和治疗不育症的生物标志物和合成分子的开发。了解卵子第一次受精时发生的事件有助于应用我们如何影响这些事件并改善牲畜和人类的生殖。利用这种蛋白质组学方法，我的实验室提出，围绕精子核的核周膜(PT)分为两个功能区：顶体下部分和顶体后部分，顶体下部分包含参与顶体组装的蛋白质，顶体后部分包含参与受精过程中精子/卵子相互作用的蛋白质。这一建议的主要目的是确定我们已经确定的几个关键的精子PT蛋白的意义：即一组核心体体组蛋白(H3、H_2B、H_2A和H_4)，它们在精子头的顶体后区域的胞浆中的前所未有的位置表明在受精过程中男性原核的染色质重塑中起作用，以及谷胱甘肽-S-转移酶欧米茄2(GSTO_2)，它们相似的位置表明在调节共价连接的分子的氧化和还原状态中发挥作用，特别是在受精过程中精子核的解体过程中。通过这些蛋白质的功能失活，我们将测试它们在受精过程中的作用机制，并确定它们是否是合子发育所必需的。需要检验的四个具体假设是：1.在PT中存在核心组蛋白和GSTO2是常见的真核精子特征。2.核心组蛋白和GSTO2在精子发生过程中从头合成，并通过微管套筒组装成PT的一部分。3.PT核心组蛋白在受精过程中溶解在卵浆中，并通过稳定解聚精子核的DNA为原核形成做贡献。4.PT锚定的GSTO_2在受精过程中溶解在卵浆中，利用谷胱甘肽还原精蛋白之间的S-S键来解聚精子核。

Fertilization is the cornerstone of development where the two differentiated cells, sperm and egg, set the stage for embryogenesis, for which competency of the gametes is essential. Yet only rudimentary knowledge is available on the molecules and mechanisms involved in sperm-egg interactions leading to zygotic development. Our long term goal is to gain an understanding of these mechanisms. Our approach is to molecularly dissect sperm compartments, such as the acrosome and perinuclear theca, that are implicated in the fertilization process. The identities of these proteins provide clues to their significance and lead to the design of specific probes and experiments to test their predicted fertilization functions. This strategy has been successful as we have discovered novel sperm proteins that are implicated in the penetration of the egg and its activation during fertilization, leading to the development of biomarkers and synthetic molecules for the detection and treatment of infertility. Understanding the events that occur when an egg is first fertilized leads to application of how we can influence those events and improve reproduction in livestock and humans.Utilizing this proteomic approach has led my lab to propose that the perinuclear theca (PT), a proteinaceous cap surrounding the sperm nucleus, is divided into two functional regions: the subacrosomal part, containing proteins involved in acrosomal assembly and the postacrosomal part, containing proteins involved in sperm/egg interactions during fertilization. The major goal of this proposal is to determine the significance of several key sperm PT proteins we have identified: namely, a group of core somatic histones (H3, H2B, H2A and H4), whose unprecedented cytosolic location in the postacrosomal region of the sperm head suggests a role in chromatin remodeling of the male pronucleus during fertilization and Glutathione-S-Transferase Omega 2 (GSTO2), whose similar location suggests a role in regulating the oxidative and reductive state of covalently-linked molecules, especially during the disassembly of the sperm nucleus during fertilization. By functional deactivation of these proteins we will test their proposed mechanisms of action during fertilization and determine if they are required for zygotic development.The four specific hypotheses to be tested are: 1. The presence of Core histones and GSTO2 in the PT is a common eutherian sperm feature. 2. Core histones and GSTO2 are synthesized de novo during spermiogenesis and assembled as part of the PT by the microtubular manchette. 3. PT core histones dissolve in the ooplasm during fertilization and contribute by stabilizing the DNA of the decondensing sperm nucleus in preparation for pronuclear formation. 4. PT anchored GSTO2 dissolves in the ooplasm during fertilization and utilizes glutathione in the reduction of S-S bonds between protamines to decondense the sperm nucleus.