Molecular Biology of Mammalian Fertilization

哺乳动物受精的分子生物学

• 批准号: 7593420
• 负责人: JURRIEN DEAN
• 金额: $ 53.5万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593420
• 关键词: Acrosome; Acrosome Reaction; Binding; Biological Assay; Cell membrane; Cells; Cytoplasmic Granules; Embryo; Ensure; Event; Exocytosis; Fertilization; Germ Cells; Herpes zoster disease; Hour; Human; In Vitro; Link; Live Birth; Modeling; Modification; Molecular; Molecular Biology; Mus; N-terminal; Penetration; Peptides; Perivitelline Space; Reaction; Refractory; Research Design; Role; Signal Transduction; Sperm Penetration; Staging; Vitelline Membrane; ZP2; Zona Pellucida; base; disulfide bond; egg; embryo cell; extracellular; molecular site; pup; scaffold; sperm cell; three dimensional structure; zona pellucida glycoprotein

We assessed Acr3-EGFP sperm binding to normal and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2, but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a 'zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for ≥ 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction. The only documented modification of the zona pellucida that temporally correlates with the zona block in mice and humans is cleavage of ZP2 which results in an N-terminal peptide (30 kD) that remains covalently linked to the parental fragment via a disulfide bond(s). Fortuitously, ZP2 remains uncleaved in huZP2 rescue mice in which human ZP2 replaces endogenous mouse ZP2 and the presence of only two pronuclei in one-cell embryos, their progression to the two-cell stage in vitro, and the birth of live pups attests to an effective block to polyspermy. Even though sperm continue to bind after fertilization, the absence of excessive sperm in the perivitelline space suggests that part of this block is attributable to the zona pellucida. If correct, the presence of intact huZP2 following fertilization suggests that the zona block observed with the humanized zona pellucida may be independent of huZP2 cleavage. Thus, it appears that cleavage of ZP2 is not required to block post-fertilization sperm penetration of the zona pellucida.

我们评估了Acr 3-EGFP精子与正常和huZP 2拯救卵子的结合，其中人ZP 2取代小鼠ZP 2，但在受精后保持未切割。所观察到的Acr 3-EGFP精子与来自huZP 2救援小鼠的胚胎的从头结合支持精子-卵子识别的“支架”模型，其中完整的ZP 2决定了支持精子结合的三维结构，独立于受精和皮质颗粒胞吐作用。令人惊讶的是，结合精子的顶体在未裂解的人ZP 2存在下保持完整24小时，无论精子是在受精之前还是之后加入。完整顶体的持久性表明精子与透明膜的结合不足以诱导顶体胞吐。过滤器渗透试验表明了另一种机制，其中渗透到血管基质中启动了触发顶体反应所必需的机械感觉信号转导。 在小鼠和人类中，唯一记录到的与β-阻断在时间上相关的透明质酸修饰是ZP 2的切割，其产生通过二硫键与亲本片段保持共价连接的N-末端肽（30 kD）。幸运的是，在huZP 2拯救小鼠中，ZP 2保持未切割，其中人ZP 2取代内源性小鼠ZP 2，并且在单细胞胚胎中仅存在两个原核，它们在体外发展到两细胞阶段，以及活幼崽的出生证明了对多精受精的有效阻断。尽管精子在受精后继续结合，但在卵周间隙中没有过多的精子，这表明这种阻碍部分归因于透明膜。如果正确的话，受精后完整huZP 2的存在表明用人源化透明质酸观察到的β-阻断可能不依赖于huZP 2切割。因此，似乎不需要ZP 2的分裂来阻止受精后精子穿透透明卵。