The role of SPE-11 in C. elegans egg activation

为了确定sp11突变胚胎的主要缺陷，我们进行了详细的表型分析。鉴于spe-11末端表型是一个圆形的，脆弱的1细胞胚胎，我们检查了两种蛋壳完整性的诊断。首先，我们已经证明了spe-11（hc90）突变胚胎具有渗透敏感性，这表明蛋壳内层（赋予渗透屏障）发生了破坏。其次，在这些突变体中，蛋壳的几丁质层有缺陷。在零胚11型（hc90）胚胎中，几丁质仅在胚胎表面呈有限的新月形，而野生型胚胎的几丁质则在胚胎周围存在。作为早期胚胎发生的另一个标志，我们研究了称为皮质颗粒的细胞内囊泡的运输，这些囊泡在卵子激活期间经历了特征性的易位。sp11突变体在依赖细胞周期的皮质颗粒运动过程中没有受到损害，因为CAV-1::GFP（皮质颗粒的标记物）在sp11突变体中表现出正常的运输。我们还询问在没有SPE-11的情况下，其他卵子激活基因的定位是否正常。在spe-11（hc90）突变体中，EGG-3::GFP通常在受精后定位。因此，我们在spe-11突变体中发现的最早缺陷是在蛋壳形成中。

To identify the primary defect in the spe-11 mutant embryos, we have undertaken a detailed phenotypic analysis. Given that the spe-11 terminal phenotype is a round, fragile 1-cell embryo, we have examined two diagnostics of eggshell integrity. First, we have shown that spe-11(hc90) mutant embryos are osmotically sensitive, indicating a disruption in the inner layer of the eggshell, which confers the osmotic barrier. Second, the chitin layer of the eggshell is defective in these mutants. Chitin is observed only in a restricted crescent at the surface of the embryo in the null spe-11(hc90) embryos, in contrast to wild type embryos where chitin is present around the periphery of the embryo. As an additional marker of early embryogenesis, we have investigated the trafficking of intracellular vesicles called cortical granules, which undergo a characteristic translocation during egg activation. The spe-11 mutants are not compromised in the cell cycle dependent process of cortical granule movement because CAV-1::GFP, a marker of cortical granules, exhibits normal trafficking in spe-11 mutants. We also asked if the localization of other egg activation genes was normal in the absence of SPE-11. EGG-3::GFP is normally localized following fertilization in spe-11(hc90) mutants. Thus, the earliest defects we have detected in spe-11 mutants are in eggshell formation. It has been shown that injection of a spe-11 transgene into the hermaphrodite germline is sufficient to rescue the loss of spe-11 in sperm (Browning and Strome, 1996). We have investigated if a stable spe-11::GFP transgene is able to perform the same function. spe-11(hc90) hermaphrodites expressing this transgene are fertile, indicating rescue of the egg activation defect. In addition, hermaphrodites that have exhausted their sperm supply produce live progeny when mated to spe-11(hc90) males. Thus, the spe-11::GFP transgene expressed in oocytes or sperm is competent to replace endogenous SPE-11, pointing to the existence of a regulatory mechanism to ensure proper activation of SPE-11 activity following fertilization. We are currently performing a non-complementation screen in order to recover a strong temperature sensitive allele of spe-11 that can be used for a genetic suppressor screen.