The major sperm protein (MSP) triggers oocyte meiotic maturation in C.elegans. Previously, we showed that somatic G-protein(G<font face=symbol>a</font><sub>s</sub> and G<font face=symbol>a</font><sub>o/i</sub>) signaling pathways function in parallel to the oocyte VAB-1 MSP/Eph receptor to regulate oocyte meiotic maturation (Govindan et al.,2006). Using RNAi, we found that
gsa-1 function is necessary and sufficient in the soma to promote meiotic maturation in the presence of sperm. We further showed that
gsa-1 might function to promote meiotic maturation by antagonizing inhibitory sheath/oocyte gap junctions. To determine the focus of action of
gsa-1, we conducted genetic mosaic analysis using a
gsa-1(+) plasmid and
sur-5::gfp extrachromosomal array. Germline-loss mosaics are fertile demonstrating that
gsa-1 is not required in the germ line for meiotic maturation. Critically, array loss in the sheath-spermathecal cell lineages results in sterility due to a failure of oocytes to undergo meiotic maturation despite the presence of sperm. Thus, mosaic analysis supports the hypothesis that
gsa-1 is required in the gonadal sheath cells for oocyte meiotic maturation. To analyze the mechanism by which somatic G-protein signaling promotes meiotic maturation, we conducted a forward genetic screen for suppressors of the
gsa-1 meiotic maturation defect.
gsa-1(RNAi) in wild-type hermaphrodites blocks meiotic maturation resulting in sterility. By contrast,
gsa-1(RNAi) in
ceh-18(
mg57),
inx-22(
tm1661), or
kin-2(
ce179) hermaphrodite genetic backgrounds has no effect. To identify additional regulators of meiotic maturation, we mutagenized wild-type hermaphrodites and subjected the F2 progeny to
gsa-1(RNAi) by feeding. From a screen of ~80,000 EMS-mutagenized haploid genomes, we obtained 127 strains that are fertile following
gsa-1(RNAi). Of these, sixty-five strains do not respond to
unc-22(RNAi) and thus have a generalized RNAi defect, which we will not pursue. Of the remaining sixty-two strains, fourty-two are single-gene mutations that we are assigning to complementation groups, and the rest appear to be multiple hits. Preliminary data suggests that
tn1281 is a
ceh-18 allele, thereby validating our approach. Unexpectedly, several mutations exhibit resistance to germline, but not somatic RNAi. Possibly, these mutations confer a subtle somatic RNAi defect, despite responding fully to
unc-22(RNAi). Alternatively, an endogenous germline RNAi pathway might regulate meiotic maturation,consistent with recent findings in mice(Murchison et al. 2007). This poster will describe progress in characterizing our mutant collection. Govindan et al. Curr Biol 16: 1257,2006. Murchison et al. Genes and Dev 21: 682, 2007.