Functionally non-redundant paralogs spe-47 and spe-50 encode FB-MO associated proteins and interact with him-8.

The activation of C. elegans spermatids to crawling spermatozoa is affected by a number of genes including spe-47. Here, we investigate a paralog to spe-47: spe-50, which has a highly conserved sequence and expression, but which is not functionally redundant to spe-47. Phylogenetic analysis indicates that the duplication event that produced the paralogs occurred prior to the radiation of the Caenorhabditis species included in the analysis, allowing a long period for the paralogs to diverge in function. Furthermore, we observed that knockout mutations in both genes, either alone or together, have little effect on sperm function. However, hermaphrodites harboring both knockout mutations combined with a third mutation in the him-8 gene are nearly self-sterile due to a sperm defect, even though they have numerous apparently normal sperm within their spermathecae. We suggest that the sperm in these triple mutants are defective in fusing with oocytes, and that the effect of the him-8 mutation is unclear but likely due to its direct or indirect effect on local chromatin structure and function.

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans.

Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed "the brake model." Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a ß-strand that mediates MSP-MSP dimerization. Both N- and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.

SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans.

BACKGROUND: The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid. RESULTS: Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background. CONCLUSIONS: These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.