The PGL proteins are constitutive protein components of P granules and function redundantly in C. elegans germline development. Among them, PGL-1 is the most critical component. PGL-1 has been shown to specifically interact with IFE-1 in vitro and in vivo. IFE-1 is one of the five C. elegans isoforms of eIF4E, the mRNA 5'' cap-binding component of the translation initiation complex eIF4F. We further identified that IFE-1 is specifically required for spermatogenesis using RNAi. These findings suggest that IFE-1 and possibly PGL-1 are involved in translational control of a subset of mRNAs that are required for spermatogenesis. To further analyze the molecular mechanism of action of IFE-1 and PGL-1 on spermatogenesis, we performed a proteomic analysis to search for proteins that are down-regulated in
ife-1(
ok1978) mutant worms, which show the same temperature-sensitive fertilization defective phenotype as
ife-1(RNAi) worms. Worm populations of
ife-1(
ok1978) mutant (
ife-1 single or
ife-1;
fem-3(
q20gf) double mutant) and wild type (N2 or
fem-3(
q20gf)) were synchronized at L1 larval stage, up-shifted and grown to young adult stage at non-permissive 25oC, and harvested. Worm protein lysates prepared from
ife-1(
ok1978) and wild type were labeled with Cy3 and Cy5 fluorescent dyes, and applied to 2D-DIGE analysis. Protein spots significantly down-regulated in
ife-1(
ok1978) mutant worms compared to wild-type control worms were further analyzed by MALDI-TOF mass spectrometry to identify the proteins. Through this analysis, we found that some sperm-specific proteins were indeed significantly down-regulated in
ife-1(
ok1978) mutants at 25oC. Among the genes encoding the down-regulated sperm proteins, RNAi of
gsp-3 caused an
ife-1-like phenotype. That is,
gsp-3(RNAi) hermaphrodites produced unfertilized oocytes or early-arrested embryos, and this defect was rescued by mating with wild-type males, as observed in
ife-1 mutants. Although the
ife-1 phenotype is temperature-sensitive, the
gsp-3(RNAi) phenotype is not. These results suggest that IFE-1 is essential for translation of some sperm-specific proteins, including the predicted phosphatase GSP-3. We are investigating whether the temperature-sensitive fertilization defect of
ife-1 mutant is caused mainly by a reduced level of GSP-3.