FUNCTION OF MACAQUE SPERM PH 20 IN FERTILIZATION

意义PH-20蛋白是生物分子中的一种 与卵母细胞包被物相互作用的哺乳动物精子表面 在受精之前。PH-20似乎具有多种功能和 是目前正在开发的避孕疫苗的目标 并在灵长类动物身上进行了有效性测试。本项目的目标 我们正在研究PH-20在体内的作用机制。 灵长类受精，因为它适用于哺乳动物 受精生物学，并将在设计 多决定簇免疫原避孕疫苗。结果 PH-20是一种透明质酸酶，是PH-20在体内发挥作用所必需的。 精子对卵丘细胞外基质(ECM)的穿透率很高 很有可能。猕猴精子PH-20的两种主要亚型可能是 它们的分子量分别为kDa和53 kDa。两者都有 这些形式的PH-20在三个不同的地点或 精子的隔间。透明质酸处理精子的实验研究 (HA)导致精子蛋白的磷酸化，并增加 细胞内钙离子。这些HA诱导的改变可以通过以下方式抑制 用抗PH-20免疫球蛋白Fab片段治疗。我们可以显示涨幅 在穿透过程中在单个细胞中测得的精子钙 猕猴卵丘细胞外基质的体外实验。房委会在调停这方面的角色 细胞内钙的变化是由类似的观察提示的 含有透明质酸的人造凝胶中的单个精子。生物学的 细胞内钙离子变化的重要性可能是“启动”精子 用于立即诱导顶体反应，只要精子 与透明带发生结合。PH-20可能具有必需的 在受精过程中的作用，使其成为 避孕药。未来的方向决定着 猕猴PH-20亚型、透明质酸酶与透明质酸结合特性 活动并研究与之交互过程中的信号功能 卵丘ECM和透明带。关键词精子、受精、 美国国立卫生研究院避孕经费拨款RR00169，HD29125；安德鲁·梅隆 基础

Significance The PH-20 protein is one of several molecules on the surface of mammalian sperm which interact with the oocyte investments prior to fertilization. PH-20 appears to have multiple functions and is a target for contraceptive vaccines that are now being developed and tested for effectiveness in primates. Objectives In this project we are studying the cellular mechanisms of action of PH-20 during primate fertilization because it is applicable to mammalian fertilization biology and will be particularly useful in the design of contraceptive vaccines with multi determinant immunogens. Results PH-20 is a hyaluronidase and a requirement for PH-20 function during sperm penetration of the cumulus extra cellular matrix (ECM) is highly likely. Two primary isoforms of macaque sperm PH -20 can be distinguished by their molecular weights of 64 kDa and 53 kDa. Both of these forms of PH-20 are found in three separate locations or compartments of the sperm. Treatment of sperm with hyaluronic acid (HA) results in phosphorylation of sperm proteins, and an increase in intracellular calcium. These HA-induced changes can be inhibited by treatment with Fab fragments of anti-PH-20 IgG. We can show increases in sperm calcium as measured in individual cells during penetration of the macaque cumulus ECM in vitro. The role of HA in mediating such intracellular calcium changes is suggested by similar observations on individual sperm in artificial gels containing HA. The biological importance of intracellular calcium changes may be to "prime" sperm for immediate induction of the acros ome reaction as soon as sperm binding to the zona pellucida takes place. PH-20 may have a required function in fertilization, making it a vulnerable target for contraceptive agents. Future Directions Determine structures of macaque PH-20 isoforms, characterize hyaluronidase and HA binding activities and investigate signaling functions during interaction with the cumulus ECM and zona pellucida. KEY WORDS sperm, fertilization, contraception FUNDING NIH Grant RR00169, HD29125; Andrew Mellon Foundation