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

基本信息

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
项目类别：
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

项目摘要

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.

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（HYASE）对精子功能和繁殖的贡献。该R03应用的目的是使用可用的hyal2和Hyal3 Null小鼠研究两种新型精子透明酶，Hyal2和Hyal3在小鼠精子功能中的作用，然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）崩溃的6种哺乳动物透明酶中，已知在睾丸中大量表达。在这5、2酸活性和3个中性活性中，只有通过精子粘附分子1（SPAM1或PH-20）示例的多功能蛋白的后者被认为是精子霍斯。尚无关于精子中2种酸性舌酶存在的报道，我们的初步研究表明，在人和小鼠中，它们（透明和透明3）存在于精子中，并且可能有助于对受精至关重要的整体性能活性。此外，它们可能负责精子 - Zona结合，该结合归因于SPAM1，但最近证明在小鼠中并非如此。重要的是，Hyal2可能会发挥重要的独特作用，因为它的缺乏症（与其他霍斯斯不同）所无法补偿的缺乏症会导致不育。基于我们的初步数据，我们的中心假设是透视和透明3存在于小鼠和人类精子中，它们在酶促和非酶促作用中扮演着酶促作用，并且透明度在受精中具有独特的功能（其他透均透均霍斯酶）。由于我们使用HYASE的经验，我们的支持性初步数据以及可用的突变体，因此，理想的定位可以追求和完成以下两个特定目的，以检验我们的中心假设：I。确定hyal2和hyal3是否存在于鼠标精子和hyal3中，是否存在于精子中，可以用精子和透明度的基础来获取hyal2 nulls2 nulls的基础。 a）在野生型（WT）精子中定位并确定蛋白质的亚细胞分布，并比较pH 3-7的呼吸活性与零值。 b）通过自然交配对wt评估雄动参数和透明效应的生育指数。 c）比较零和WT精子结合HA，穿透积云并执行Adrosos反应的能力。 d）将Hyal2和Hyal3的Zona结合亲和力与其他Hyase进行比较。 e）研究附睾/附睾液中的透明2和蛋白质以及蛋白质在体外暴露后是否可以在精子血浆和内部脊髓膜内获取蛋白质。 ii。确定透明2和透明3是否存在，并且在人类精子中具有催化活性。 a）评估人精子中蛋白质的亚细胞分布。 b）分析在pH 7、3和4处的人类精子瘤活动，这是酸性舌酶的最佳选择。显然，对精子中这些霍斯酶的研究是高度保证的，因为酶特异性功能的表征对于诊断和治疗诊所中的精子病理至关重要。公共卫生相关性：由于透明质酸酶在精子 - 蛋之间的重要作用中，这项工作对人类生育有重要意义。它将识别并帮助在人类中的两个新型精子瘤中发挥作用，迄今为止，Spam1是唯一报道的人。在使用重组SPAM1的体外受精（IVF）的成功中，均均需要临时水平。透明2和3（酸性透舌酶）也有望在IVF中发挥关键作用，现在可以使用重组蛋白通过添加到精子表面来促进辅助生殖技术，以治疗精子病理学。