PgmNr W4011: Identification of Genes that Regulate the Activation of C. elegans Sperm.

Authors:
A. Greer; D. Chavez; T. Shimko; B. Philpot; A. Duffy; K. Mylroie; G. Stanfield


Institutes
University of Utah, Salt Lake City, UT.


Keyword: Gametogenesis, Fertilization, Egg-embryo transition

Abstract:

Nematode sperm become competent for motility and fertilization during a process termed sperm activation, which is regulated by specific extracellular signals in males and hermaphrodites. While hermaphrodites create their own sperm during their last larval stage and store it for future use, adult males continually create sperm, which activate when the sperm is transferred to a hermaphrodite. Furthermore, hermaphrodites and male sperm activation pathways differ from each other. Hermaphrodite sperm activation requires the activity of a set of sperm genes termed the spe-8 group, which mediate the response to extracellular zinc. The pathway by which male sperm become active involves a signaling pathway including swm-1, try-5, and snf-10. SWM-1 is a serine protease inhibitor. When it is absent, sperm is prematurely activated within the male gonad and is rarely transferred to hermaphrodites. TRY-5 is the next protein in the sperm activation cascade and is a seminal fluid serine protease. When TRY-5 is absent, males do not transfer activator, though they are still fertile due to activation by hermaphrodite signals. The protein SNF-10, an SLC6 transporter, is found in the plasma membrane of sperm and is required for activation of male sperm by TRY-5. To identify additional genes within the sperm activation cascade, the Swm-1 activated-sperm phenotype was used as the basis of a genetic suppressor screen.

Analysis of mutants from the swm-1 suppressor screen suggests that many genes are involved in sperm activation. One goal of this project is to characterize the phenotype of suppressor mutants. First, we are using hermaphrodite brood counts to measure the fertility of each mutant. We find that multiple male pathway mutants have decreased fertility, unlike the previous characterized mutants in this class. We are also performing experiments to distinguish between potential roles in sperm vs. upstream of the TRY-5 protease. We are using in vitro activation assays to test if any of these genes functions in sperm or is needed for response to the protease signaling pathway. We are also examining the effect of these mutations on localization or transfer of TRY-5::GFP. A second current goal of this project is to identify two of these genes, which we have shown to represent two novel regulators in the male (jn19) and hermaphrodite (jn15) pathways. We mapped and sequenced these mutants. Prioritizing candidate genes according to preexisting data for sperm-enriched gene expression, we are using CRISPR to generate candidate gene deletions that may mimic the jn19 and jn15 phenotypes. These experiments will provide insight into protease signaling, which is important for cell motility as well as other processes.



Wormbase Genetic Index
1. swm-1
2. try-5
3. snf-10
4. spe-8