C. elegans spermatogenesis is a model for cellular differentiation. In order to identify more genes that control spermatogenesis, we have used microarray screening to find previously unidentified sperm-specific genes. We are using double-stranded RNA interference to disrupt the function of these genes to learn more about their normal activities. We prepared poly-A(+) RNA from well-fed
fem-1(
hc17) (only produce oocytes) and
fem-3(
q23) (only produce sperm) adults to use for screening. Stuart Kim and colleagues generated differentially fluorochrome-labeled cDNA probes. They hybridized these to a microarray chip representing 1200 ESTs from Yuji Kohara at National Institute of Genetics, Japan. The relative proportion of hybridization to each EST was measured by a scanning confocal-laser microscope at two wavelengths. An algorithm identified those ESTs that showed at least a twofold difference in hybridization between probe pools and that were reproducible among several trials with independent RNA preparations. This experiment identified more than 30 genes upregulated during spermatogenesis and an even larger set upregulated during oogenesis. All of the genes on the chip already known to be sperm-specific, including eight major sperm protein ESTs, were among this spermatogenesis-upregulated set. We hope that some of these sperm-specific genes include genes active in the sperm activation signaling pathway which is of particular interest in our lab. To explore this possibility, we will examine their disrupted phenotype in vivo using double-stranded RNA interference (RNAi), recently described by Craig Mello and Andrew Fire. To see if this screening can work for testis-specific genes we have done dsRNA injections of known spermatogenesis genes previously characterized in our lab.
spe-26 shows reproducible RNAi effects although not as robustly as do Mello's and Fire's
unc-22 tests.
spe-12 and
spe-27, expressed later in sperm development, are strongly resistant to RNAi; only a few worms to be scored after injection with dsRNA (and a few of their F1 progeny) have shown disrupted function of
spe-12 and
spe-27. We are testing differentially-expressed spermatogenesis genes with RNAi, injecting worms at different larval stages, and using dsRNA that is more resistant to degradation. If we are successful at producing reproducible RNAi effects, we will identify the disrupted phenotypes of the newly-identified sperm-specific genes.