A crucial event in the life of sexually reproducing organisms is the specification of sexual identity. C. elegans is an excellent system to explore sex specification, as the species has two sexes, XX hermaphrodites and XO males. Hermaphrodites have a female soma and a hermaphroditic germline that transiently produces sperm in L4 larvae then undergo a switch to oogenesis in the late L4 stage; sperm are stored within the spermatheca and used for self-fertilization. Germline sex is established by a complex network of repressive transcriptional and post-transcriptional regulation; however, how this network transforms the X chromosome to autosome ratio into sperm and eggs is still not clear. Through our studies on the role of the nuclear hormone receptor, NHR-23, in molting we generated a GFP-fusion at the 3' end of the endogenous
nhr-23 gene using CRISPR-mediated genome engineering. This knock-in also included a degron sequence that allows inducible, tissue-specific degradation when the small molecule auxin was administered (Zhang et al., 2015). Surprisingly, we observed a novel expression of NHR-23:GFP-degron-3xFLAG in the germline nuclei of hermaphrodites throughout the L4 stage. Using a transgene that permitted auxin-mediated depletion of transgenes specifically in the germline, we demonstrated that NHR-23 depletion in this tissue results in unfertilized oocytes. This phenotype appeared to be due to a defect in sperm production, as DIC microscopy revealed an absence of sperm following NHR-23 depletion in the germline. Moreover, the introduction of wildtype sperm was sufficient to rescue infertility following NHR-23 depletion in hermaphrodites, confirming that infertility was due to defective spermatogenesis. Taken together, our data indicate that NHR-23 is necessary for fertility and sperm development and that NHR-23 is critical for specifying sperm in hermaphrodites or for the very early stages of spermatogenesis. Ongoing efforts involve using a combination of ChIP-seq and RNA-seq to determine the direct targets of NHR-23 in the germline which promote sperm formation.