ACROSOMAL ENZYMES AND THEIR ROLE IN FERTILIZATION

顶体酶及其在受精中的作用

• 批准号: 3312888
• 负责人: PRAKASH N SRIVASTAVA
• 金额: $ 13.76万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312888
• 关键词: acrosin; acrosome; alpha galactosidase; arylsulfatases; biomarker; cell membrane; egg /ovum; enzyme inhibitors; enzyme mechanism; exo alpha sialidase; exocytosis; fertilization; galactosyltransferases; gel electrophoresis; glycoproteins; granule; hyaluronidase; hydrolase; immunologic techniques; laboratory rabbit; membrane activity; membrane fusion; membrane proteins; oligosaccharides; phospholipase C; protein purification; sialate; sperm; sperm capacitation; tissue /cell culture

The ovum is surrounded by the cumulus, the corona, the zona and vitelline membrane. Capacitated rabbit sperm undergo the acrosome reaction during the passage through the cumulus. Enzymes released following the acrosome reaction or enzymes still bound to the vesicles composed of the plasma (PM) and the outer acrosomal membranes (OAM) hydrolyse the cumulus matrix and allow sperm to reach the corona cells and sperm which are left with a limiting inner acrosomal membrane (IAM) encounter the zona pellucida. The sperm binding to the zona and its subsequent penetration may involve a sequential action of binding to the zona and its subsequent penetration may involve a sequential action of acrosomal hydrolases bound to IAM. The fusion of spermatozoa with vitelline membrane probably involves phospholipases of the persistent PM and OAM on the equatorial segment (ES) and the post acrosomal region (PAR). The subsequent exocytosis of corticle granules may also be triggered by sperm enzymes. This sequence of events provides the rationale for this proposal. The overall objective of this proposal is to elucidate further the role of acrosomal enzymes in fertilization. Specifically, we will isolate and compare enzymes associated with acrosomes; determine the localization of key enzymes involved in sperm-egg binding; isolate acrosomal enzymes from rabbit spermatozoa and determine their effects on rabbit ova. After the extraction of acrosomal enzymes, sperm still contain nearly one-half of neuraminidase, one third of arylsulfatase, and one third of hyaluronidase. these enzymes can be extracted by further extraction of the denuded sperm with 1% Triton X-100 and 0.1 M CaCl2. This treatment also solubilizes the equatorial segment. We will follow this extraction procedure by studying simultaneous changes in sperm morphology at the ultrastructural level. In view of increasing importance of glycosyltransferase and sialic acid in gamete interactions, we will attempt localization of these and other acrosomal enzymes by immunofluorescence, radiolabeling, and cytochemical techniques. The enzyme purification will employ affinity chromatography on hydrophobic columns, high pressure liquid chromatography coupled with conventional methods. The role of acrosomal enzymes in sperm-egg binding, and the ovum penetration will be studied. Enzymes will also be tested on isolated zona.

卵子被卵丘、冠、带和卵黄包围 膜。 获能的兔精子在过程中经历顶体反应 通过积云的通道。 顶体后释放的酶 仍与血浆 (PM) 组成的囊泡结合的反应或酶 顶体外膜（OAM）水解卵丘基质并 让精子到达冠状细胞并留下精子 限制内顶体膜（IAM）遇到透明带。 这 精子与透明带的结合及其随后的渗透可能涉及 与透明带结合的连续作用及其随后的渗透可能 涉及与 IAM 结合的顶体水解酶的连续作用。 这 精子与卵黄膜的融合可能涉及 赤道段 (ES) 上持久性 PM 和 OAM 的磷脂酶 和顶体后区（PAR）。 随后皮质的胞吐作用 颗粒也可能由精子酶触发。 这一系列的事件 提供了该提案的理由。 本次活动的总体目标 建议进一步阐明顶体酶在 施肥。 具体来说，我们将分离并比较酶 与顶体相关；确定关键酶的定位 参与精子与卵子的结合；从兔体内分离顶体酶 精子并确定它们对兔子卵子的影响。 之后 提取顶体酶后，精子中仍含有近二分之一 神经氨酸酶、芳基硫酸酯酶的三分之一和透明质酸酶的三分之一。 这些酶可以通过进一步提取裸露的精子来提取 含有 1% Triton X-100 和 0.1 M CaCl2。 这种治疗还可以溶解 赤道段。 我们将通过研究来遵循这个提取过程 精子形态在超微结构水平上同时发生变化。 在 鉴于糖基转移酶和唾液酸的重要性日益增加 配子相互作用，我们将尝试这些和其他的本地化 通过免疫荧光、放射性标记和细胞化学检测顶体酶 技术。 酶纯化将采用亲和层析 疏水柱、高压液相色谱联用 常规方法。 顶体酶在精卵结合中的作用， 并将研究卵子的穿透性。 酶也将在分离的透明带上进行测试。