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活性透明质酸酶（hyases）对精子功能和生殖的贡献。本R03申请的目的是利用现有的Hyal2和Hyal3缺失小鼠来研究两种新型精子酶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种哺乳动物酶中，已知有5种在睾丸中大量表达。在这5,2个酸活性蛋白和3个中性活性蛋白中，只有后者是多功能蛋白，如精子粘附分子1 （SPAM1或PH-20）被认为是精子酶。目前还没有关于在精子中存在这两种酸活性酶的报道，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，可能有助于受精过程中必不可少的总体HYAL2活性。此外，它们可能负责精子带结合，这被归因于SPAM1，但最近在小鼠中发现并非如此。重要的是，HYAL2可能发挥重要的独特作用，因为它的缺乏不能由家庭成员补偿（与其他HYAL2不同）导致不孕。根据我们的初步数据，我们的中心假设是HYAL2和HYAL3存在于小鼠和人类精子中，它们同时发挥酶促和非酶促作用，并且HYAL2在受精中具有独特的功能（未被其他酶补偿）。根据我们研究hyases的经验，我们的支持性初步数据，以及我们现有的突变体，我们理想地定位于追求并实现以下两个特定目标，以验证我们的中心假设：1 .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 null的不育基础。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 3-7时与空白精子的酶活性。B)通过与WT自然交配来评估Hyal2无精子的雄性参数和生育指数。C)比较无精子和WT精子结合HA、穿透积云和进行顶体反应的能力。D)比较HYAL2和HYAL3与其他酶的区结合亲和力。E)研究附睾/附睾液中是否存在Hyal2和Hyal3 mrna和蛋白，以及体外暴露后这些蛋白是否能在精子血浆和顶体内膜上获得。2。确定HYAL2和HYAL3是否存在并在人类精子中具有催化活性。A)评估人类精子中蛋白质的亚细胞分布。B)分析人类精子在pH为7、3和4时的酶活性，pH值为酸性酶的最佳值。显然，对精子中这些酶的研究是非常必要的，因为酶特异性功能的表征对临床诊断和治疗精子病理至关重要。

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.