ACROSOMAL ENZYME BIOSYNTHESIS BY MAMMALIAN SPERMATIDS

哺乳动物精细胞顶体酶的生物合成

• 批准号: 3322826
• 负责人: George L. Gerton
• 金额: $ 13.23万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3322826
• 关键词: acrosin; acrosome; beta galactosidase; enzyme biosynthesis; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; guinea pigs; immunofluorescence technique; immunoprecipitation; laboratory mouse; lysosomes; mannose; meiosis; monoclonal antibody; phosphates; pinocytosis; receptor; sperm

The biosynthesis of acrosomal enzymes by isolated spermatogenic cells will be investigated to determine whether these enzymes are processed in a manner similar to that used to deliver acid hydrolases to lysosomes. Guinea pig sperm will be prepared and induced to undergo the acrosome reaction with the ionophore A23187. The soluble acrosomal components released from the sperm will be collected and used as the source for two acrosomal enzymes: proacrosin/acrosin and beta-galactosidase. Monoclonal antibodies will be prepared against these enzymes and used to study their synthesis by purified populations of guinea pig pachytene spermatocytes, round spermatids, and condensing spermatids. Using immunoblotting of cell extracts, immunofluorescence, and immunoprecipitation of extracts from metabolically labeled spermatogenic cells, the monoclonal antibodies will be used to determine what cell types are capable of synthesizing proacrosin and beta-galactosidase. Using pulse- chase labeling followed by immunoprecipitation, the processing of precursor forms of proacrosin and beta-galactosidase will be examined in spermatogenic cell cultures. Experiments will also be performed to determine whether mannose-6-phosphate groups are attached to the newly-synthesized proacrosin and beta- galactosidase. Each spermatogenic cell population will be assayed for mannose-6-phosphate receptor by immunoblotting, immunofluorescence, and as receptor activity assay. Spermatogenic cells will also be tested for their ability to internalize bovine testicular beta-galactosidase (a model lysosomal enzyme), guinea pig proacrosin, and guinea pig sperm beta-galactosidase via mannose-6-phosphate receptor-mediated endocytosis. The experiments performed during this project will provide important new information concerning the synthesis, processing and transport of acrosomal enzymes. Increased information concerning the development of the acrosome will advance our understanding of the regulation of male fertility.

分离生精过程中顶体酶的生物合成 将对细胞进行研究，以确定这些酶是否 以类似于输送酸的方式进行处理 水解酶转化为溶酶体。将准备好豚鼠精子并 被诱导与离子载体发生顶体反应 A23187。从顶体中释放的可溶性顶体成分 精子将被收集并用作两个顶体的来源 酶：顶体酶原/顶体酶和β-半乳糖苷酶。单克隆 将制备针对这些酶的抗体，并用于 用纯化的豚鼠群体研究它们的合成 粗线型精母细胞、圆形精细胞和凝聚 精子细胞。通过对细胞提取液进行免疫印迹， 免疫荧光法和免疫沉淀法测定丹参提取物 代谢标记的生精细胞，单克隆性 抗体将被用来确定哪些细胞类型能够 合成顶体酶原和β-半乳糖苷酶。使用脉冲- Chase标记后免疫沉淀，处理 原顶体酶和β-半乳糖苷酶的前体形式将是 在生精细胞培养中进行检测。实验还将 以确定甘露糖-6-磷酸基团 附着在新合成的顶体酶原和β- 半乳糖苷酶。将对每个生精细胞群进行检测 通过免疫印迹对甘露糖-6-磷酸受体， 免疫荧光和AS受体活性测定。 生精细胞也将接受测试，以确定它们是否有能力 内化牛睾丸β-半乳糖苷酶(模型 溶酶体酶)、豚鼠顶体原和豚鼠精子 甘露糖-6-磷酸受体介导的β-半乳糖苷酶 内吞作用。在这个项目中进行的实验将 提供有关合成的重要新信息， 顶体酶的加工和运输。增加了 关于顶体意志发育的信息 提高对男性生育调控的认识。