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.
