ACROSOMAL ENZYMES AND THEIR ROLE IN FERTILIZATION

顶体酶及其在受精中的作用

基本信息

批准号：
3312887
负责人：
PRAKASH N SRIVASTAVA
金额：
$ 12.45万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-04-01 至 1988-03-31
项目状态：
已结题

项目摘要

The principal aims of proposal fall into four major categories: 1) The isolation and characterization of sperm acrosomal membranes. 2. The purification, characterization and localization of acrosomal enzymes. 3. The determination of the role of these enzymes in the gamete interaction, ie. the acrosome reaction, the proenzyme activation and the penetration of the ovum. 4. The investigation on the immunogenecity of the isolated membranes and acrosomal enzymes as a potential contraceptive tool. The procedures for the isolation of acrosomal membranes and related enzymes have been developed in our laboratory. In short, the acrosomal components are sequentially extracted using methods such as osmotic shock, nitrogen pressure, salt and detergent extractions, and the density gradient centrifugation. The purification of enzymes is done by the conventional chromatography procedures as well as the specific substrate and inhibitor affinity chromatography procedures. For the penetration of the ovum, sperm must undergo the acrosome re-action in order to activate the enzymes and ensure their release during the penetration. We will determine the role of acrosomal phospholipase C in the induction of the acrosome reaction and the role of Cathepsin D and other enzymes in activating proenzyme(s). The effect of purified enzymes will be tested on the rabbit and hamster ova. The enzymes will be used alone and in combination to determine their cooperativity during the sperm penetration of the ovum. The synthetic and natural inhibitors of these enzymes will be screened and their contraceptive potential assessed. The antibodies to the isolated acrosomal components will be raised both in male and female rabbits. The antisera will be used to localize the antigens. The immunized rabbits will also be evaluated for the duration of the circulating antibodies and its effect on their fertility. These studies are expected our knowledge of the events leading to fertilization at the molecular level.

提案的主要目标分为四大类： 1) 精子顶体膜的分离和表征。 2. 的顶体酶的纯化、表征和定位。 3. 确定这些酶在配子相互作用中的作用， IE。顶体反应、酶原激活和渗透卵子。 4. 分离株的免疫原性考察膜和顶体酶作为潜在的避孕工具。这顶体膜和相关酶的分离程序已在我们的实验室开发。简而言之，顶体成分依次采用渗透休克、氮气等方法提取压力、盐和洗涤剂萃取以及密度梯度离心。酶的纯化采用常规方法进行色谱程序以及特定的底物和抑制剂亲和层析程序。用于卵子、精子的穿透必须经历顶体反应才能激活酶确保它们在渗透过程中释放。我们将确定角色顶体磷脂酶 C 在顶体反应的诱导中的作用组织蛋白酶 D 和其他酶在激活酶原中的作用。这纯化酶的效果将在兔子和仓鼠卵子上进行测试。这些酶将单独或组合使用以确定其精子穿透卵子过程中的协同作用。合成的和将筛选这些酶的天然抑制剂并研究它们的作用评估避孕潜力。分离顶体的抗体这些成分将在雄性和雌性兔子中饲养。抗血清将用于定位抗原。免疫后的兔子也将被评估循环抗体的持续时间及其对他们的生育能力。这些研究是我们对事件的了解导致分子水平上的受精。