Fertilization is the fusion of sperm and oocyte to initiate embryonic development. It can occur internally or externally, depending on the animal species. In internally fertilizing animals, sperm must locate freshly ovulated oocytes within the convoluted architecture of the female reproductive tract. These sperm guidance mechanisms are not well understood. Our lab has shown that C. elegans oocytes secrete F-series prostaglandins (PGFs) to guide sperm to the spermatheca. These PGFs are synthesized independent of the canonical cyclooxygenase cascade, which catalyzes PGF synthesis in mammals. We have recently shown that cyclooxygenase null mice and worms produce similar PGF isomers, suggesting that a second PGF enzymatic pathway exists. Moreover, we have identified these PGF isomers in human ovarian follicular fluid. The goal of my project is to identify genes critical for C. elegans sperm guidance and in particular, PGF metabolism.
In order to identify genes essential for sperm guidance in C. elegans, we have devised an in vivo screening method using RNAi and a one hour sperm guidance assay. C. elegans oocytes synthesize sperm-guiding PGFs from PUFA precursors provided in yolk, so the screen will focus on genes expressed in the adult germ line (Reinke et al., 2004). N2 hermaphrodites are administered RNAi by the feeding method. L4 stage worms are added to the plates and kept at 25ºC for 36-48 hours. RNAi hermaphrodites are anesthetized and incubated with wild-type males stained with MitoTracker Red CMXRos. Mated hermaphrodites are removed from males and allowed to rest for an hour. Fluorescent sperm distribution within the uterus is viewed on plates using a stereomicroscope. In controls, sperm are observed in two foci roughly equidistant to the vulva, corresponding to the two spermathecae. Deficiency in PGF synthesis causes more uniform sperm distribution throughout the uterus. This strategy permits screening of about 50-100 RNAi clones per month. My project focuses on screening 266 germline genes on Chromosome II. Out of 75 RNAi clones screened to date, I have identified 10 clones that caused strong sperm guidance defects, and 11 clones that caused moderate defects. These RNAi clones correspond to genes implicated in diverse functions, including receptor-mediated endocytosis, protein secretion, calcium ion binding, and lipid storage. For selected positive clones, null mutations will be generated using CRISPR/Cas9. Transgenic and genetic mosaic experiments will be used to determine where these genes are expressed and function. Liquid chromatography tandem mass spectrometry methods will be used to determine if the genes modulate PGF metabolism.