In C. elegans, the MSP hormone triggers meiotic maturation and MAP kinase (MAPK) activation in oocytes. In the absence of sperm, VAB-1 Eph receptor and the CEH-18 sheath cell pathway inhibit meiotic maturation. MSP antagonizes these regulatory circuits to promote meiotic maturation and MAPK activation. Previous results indicated that binding of MSP to oocytes was reduced in
vab-1(
dx31) null mutants and that expression of VAB-1 on the surface of mammalian cells was sufficient to confer specific binding. To test whether MSP can directly bind VAB-1, we expressed the VAB-1 ectodomain in mammalian cells and partially purified it from the culture supernatant. In pull-down assays, the VAB-1 ectodomain bound MSP. We are currently determining the binding constant for this interaction. We are taking two approaches to identify additional regulators of meiotic maturation and oocyte growth control. In the first approach, we conducted a genome wide RNAi screen in a
fog-2(
q71) female background for MSP-independent maturation. This screen identified several known regulators of meiotic maturation, including
ceh-18,
cgh-1, and
emo-1, thereby validating the approach. We classified the positive clones based on their effect on gonadal morphology. We are most interested in the class 1 positives (20 genes) that have little or no effect on gonadal structure. RNAi of class 2 and class 3 genes had moderate or severe effects on gonadal structure, respectively. In addition to
ceh-18, class 1 contains 19 new regulators of meiotic maturation, including
goa-1,
gpb-1,
gsa-1,
kin-2, and AC7.1 (a GPCR). These results suggest that G-protein signaling negatively regulates meiotic maturation. To determine whether negative regulators function in the germ line or the soma, we performed RNAi in the
rrf-1 mutant background, which is insensitive to somatic RNAi. This analysis indicates that G-protein signaling functions in the soma to negatively regulate meiotic maturation. Sheath/oocyte gap junctional communication is likely involved because class 1 contains two innexins (
inx-14 and
inx-22). In the second approach, we conducted a genetic screen for mutations that affect meiotic maturation and oocyte growth. We are screening a collection of ~1200 temperature-sensitive mutants for strains that produce abnormally large oocytes. To date, we have identified eight mutants, which have significantly larger oocytes and reduced rates of meiotic maturation. Currently, we are continuing the screen and conducting phenotypic and genetic analyses of MSP signaling.