The Role of Acid-active Hyaluronidases in Sperm Function

酸性活性透明质酸酶在精子功能中的作用

• 批准号: 7993488
• 负责人: PATRICIA ANASTASIA DELEON
• 金额: $ 7.65万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993488
• 关键词: Acids; Acrosome Reaction; Affinity; Area; Assisted Reproductive Technology; Binding; Biochemical; Biological; Biology; Cell Adhesion; Cells; Clinic; Data; Diagnosis; Enzymes; Epididymis; Event; Extracellular Matrix; Family; Family member; Fertility; Fertilization; Fertilization in Vitro; Genes; Hand; Herpes zoster disease; Human; Hyaluronic Acid; Hyaluronic Acid Binding; Hyaluronidase; In Vitro; Infertility; Investigation; Knockout Mice; Letters; Liquid substance; Measures; Membrane; Molecular; Mus; Oocytes; Partner in relationship; Pathology; Penetration; Plasma; Play; Positioning Attribute; Proteins; Recombinant Proteins; Recombinants; Reporting; Reproduction; Role; SPAM1 gene; Sperm Adhesion Molecule 1; Sperm Maturation; Sperm-Ovum Interactions; Surface; Testing; Testis; Work; Zona Pellucida; base; egg; experience; indexing; link protein; mutant; novel; protein distribution; public health relevance; sperm cell; sperm function; success

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.

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（透明质酸酶）对精子功能和生殖的贡献。本R 03申请的目的是使用可用的Hyal 2和Hyal 3缺失小鼠来研究两种新型精子透明质酸酶HYAL 2和HYAL 3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6种哺乳动物透明质酸水解酶中，已知5种在睾丸中大量表达。在这5种蛋白质中，2种酸活性蛋白质和3种中性活性蛋白质，只有后者是多功能蛋白质，例如精子粘附分子1（SPAM 1或PH-20）被认为是精子透明质酸酶。目前还没有关于精子中存在2种酸活性透明质酸酶的报道，我们的初步研究表明，在人类和小鼠中，它们（HYAL 2和HYAL 3）存在于精子中，可能有助于受精所必需的整体透明质酸酶活性。此外，它们可能负责精子结合，这已被归因于SPAM 1，但最近在小鼠中显示并非如此。重要的是，HYAL 2可能发挥重要的独特作用，因为它的缺乏无法得到家庭成员的补偿（与其他hyases不同）导致不育。基于我们的初步数据，我们的中心假设是HYAL 2和HYAL 3存在于小鼠和人类精子中，它们在其中发挥酶促和非酶促作用，并且HYAL 2在受精中具有独特的功能（不被其他透明质酸酶补偿）。由于我们在与Hyases合作方面的经验，我们支持的初步数据，以及我们可用的突变体，我们理想地定位于追求和实现以下两个具体目标来测试我们的中心假设：I。确定HYAL 2和HYAL 3是否存在于小鼠精子和附睾中，它们可以被精子获得，以及Hyal 2无效的不育的基础。A）定位并确定野生型（WT）精子中蛋白质的亚细胞分布，并将pH 3-7下的透明质酸酶活性与空值的透明质酸酶活性进行比较。B）通过与WT自然交配来评估Hyal 2无效的雄性学参数和生育力指数。C）比较空精子和WT精子结合HA、穿透卵丘和进行顶体反应的能力。D）比较HYAL 2和HYAL 3与其他透明质酸酶的透明带结合亲和力。E）研究附睾/附睾液中Hyal 2和Hyal 3 mRNA和蛋白质的存在，以及在体外暴露后是否可以在精子血浆和顶体内膜上获得蛋白质。二.确定HYAL 2和HYAL 3是否存在并且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）在pH 7、3和4下分析人精子透明质酸酶活性，pH 7、3和4是酸性透明质酸酶的最佳pH。很明显，对精子中这些透明质酸酶的研究是非常必要的，因为酶特异性功能的表征对于临床上诊断和治疗精子病理学是至关重要的。 公共卫生关系：由于透明质酸酶在精子-卵子相互作用中发挥的重要作用，这项工作对人类生育力有重要意义。它将确定并帮助确定两种新的精子透明质酸在人类中的作用，其中SPAM 1是迄今为止唯一报道的一种。使用重组SPAM 1的体外受精（IVF）成功需要临界水平的透明质酸酶。HYAL 2和3，酸性Hyases，预计也将在IVF中发挥关键作用，重组蛋白现在可以通过添加到精子表面来促进辅助生殖技术，以治疗精子病理。