The Role of Acid-active Hyaluronidases in Sperm Function

描述(由申请人提供)：我们的长期目标是确定酸性活性透明质酸酶(Hyase)对精子功能和生殖的总体贡献。此R03应用程序的目的是使用现有的Hyal2和Hyal3缺失小鼠来研究两种新的精子水合酶Hyal2和HYAL3在小鼠精子功能中的作用，并随后确定它们在人类精子中的存在。在催化细胞外基质中透明质酸(HA)分解的6种哺乳动物水合酶中，有5种已知在睾丸中大量表达。在这5个蛋白中，有2个是酸性的，3个是中性的，只有后者是以精子黏附分子1(SPAM1或PH-20)为代表的多功能蛋白，被认为是精子水合酶。目前还没有关于精子中存在这两种酸性活性水合酶的报道，我们的初步研究表明，在人类和小鼠中，它们(Hyal2和HYAL3)存在于精子中，可能与受精所必需的整体水合酶活性有关。此外，它们可能负责精子与小带的结合，这被认为是SPAM1所致，但最近在小鼠身上显示不是这样。重要的是，Hyal2可能发挥着重要的独特作用，因为它的缺陷不能得到家庭成员的补偿(不像其他Hyase)，会导致不孕。根据我们的初步数据，我们的中心假设是，Hyal2和HYAL3存在于小鼠和人类的精子中，它们既扮演着酶的角色，也扮演着非酶的角色，并且Hyal2在受精过程中具有独特的功能(不被其他Hyase所补偿)。由于我们使用Hyase的经验，我们的支持性初步数据，以及我们可用的突变体，我们处于理想的位置来追求和实现以下两个特定目标来测试我们的中心假设：i.确定Hyal2和HYAL3是否存在于小鼠精子和附睾中，在那里它们可以被精子获得，以及Hyal2空不育的基础。A)定位和确定野生型(WT)精子中蛋白质的亚细胞分布，并比较pH值为3-7的水合酶活性与空精子中的水合酶活性。B)通过与WT的自然交配，评估Hyal2 nulus的雄参数和生育指数。C)比较空精子和WT精子与透明质酸结合、穿透卵丘和进行顶体反应的能力。D)比较Hyal2和HYAL3与其他水合酶与透明带结合的亲和力。E)研究附睾液/附睾液中是否存在Hyal2和Hyal3的mRNAs和蛋白质，以及体外暴露后这些蛋白质是否可以在精子浆和顶体内膜上获得。确定人类精子中是否存在Hyal2和HYAL3并具有催化活性。A)评估这些蛋白质在人类精子中的亚细胞分布。B)分析人精子水合酶活性的最适pH值为7、3和4。显然，对精子中这些水合酶的研究是非常有必要的，因为酶的特定功能的特征对于临床上诊断和治疗精子病理至关重要。 公共卫生相关性：由于透明质酸酶在精子-卵子相互作用中扮演的重要角色，这项工作对人类生育能力有重要的影响。这将有助于确定两种新的精子水合酶在人类中的作用，目前为止，SPAM1是唯一报道的一种。在已经使用重组SPAM1的体外受精(IVF)中，关键水平的水合酶是必需的。酸性水合酶Hyal2和Hyal3也有望在试管受精中发挥关键作用，这些重组蛋白现在可以通过添加到精子表面来推进辅助生殖技术，以治疗精子病理。

DESCRIPTION (provided by applicant): Our long-term objective is to determine the contribution of acid-active hyaluronidases (hyases) to sperm function and reproduction in general. The purpose of this R03 application is to use available Hyal2 and Hyal3 null mice to investigate the role of two novel sperm hyases, HYAL2 and HYAL3 in mouse sperm function, and subsequently determine their presence in human sperm. Of the 6 mammalian hyases that catalyze the breakdown of hyaluronic acid (HA) in the extracellular matrix, 5 are known to be abundantly expressed in the testis. Of these 5, 2 acid-active and 3 neutral-active, only the latter which are multifunctional proteins exemplified by Sperm Adhesion Molecule 1 (SPAM1 or PH-20) are considered to be sperm hyases. There have been no reports of the presence of the 2 acid-active hyases in sperm, and our preliminary studies suggest that in humans and mice they (HYAL2 and HYAL3) are present in sperm and may contribute to the overall hyase activity that is essential in fertilization. Moreover, they may be responsible for sperm-zona binding, which has been attributed to SPAM1, but recently shown not to be so in mice. Importantly, HYAL2 may perform an essential unique role in that its deficiency which is not compensated for by family members (unlike other hyases) leads to infertility. Based on our preliminary data, our central hypothesis is that HYAL2 and HYAL3 are present in mouse and human sperm where they play both enzymatic and non- enzymatic roles and that HYAL2 has a unique function (not compensated for by other hyases) in fertilization. Due to our experience working with hyases, our supportive preliminary data, and our available mutants we are ideally positioned to pursue and accomplish the following two Specific aims to test our central hypothesis: I. Determine if HYAL2 and HYAL3 are present in mouse sperm and epididymis where they can be acquired by sperm, and the basis of the infertility of Hyal2 nulls. A) Localize and determine the subcellular distribution of the proteins in wild-type (WT) sperm and compare hyase activity at pH 3-7 with those of the nulls. B) Assess the andrological parameters and the fertility index of Hyal2 nulls by natural mating to WT. C) Compare the ability of the nulls and WT sperm to bind HA, penetrate the cumulus, and perform the acrosome reaction. D) Compare the zona-binding affinities of HYAL2 and HYAL3 with other hyases. E) Investigate the presence of Hyal2 and Hyal3 mRNAs and proteins in the epididymis/epididymal fluid and if the proteins can be acquired on sperm plasma and inner acrosomal membranes after in vitro exposure. II. Determine if HYAL2 and HYAL3 are present and are catalytically active in human sperm. A) Assess the subcellular distribution of the proteins in human sperm. B) Analyze human sperm hyase activity at pH 7, 3 and 4 which is the optimum for acidic hyases. Clearly a study of these hyases in sperm is highly warranted since the characterization of enzyme-specific functions is crucial for the diagnosis and treatment of sperm pathology in the clinic. PUBLIC HEALTH RELEVANCE: Due to the essential roles played by hyaluronidases in sperm-egg interactions, there are important implications of this work for human fertility. It will identify and help establish a role for two novel sperm hyases in humans where SPAM1 has so far been the only one reported. Critical levels of hyases are required for the success of in vitro fertilization (IVF) where recombinant SPAM1 has been used. HYAL2 and 3, acidic hyases, are also expected to play a critical role in IVF and the recombinant proteins can now be used to advance assisted reproductive technology via their addition to the sperm surface, to treat sperm pathology.