Fertilization, the fusion of sperm and oocyte to form a zygote, is the first and arguably the most important cell–cell interaction event in an organism’s life. Forward and reverse genetic approaches in the nematode Caenorhabditis elegans have identified many genes that are required for gametogenesis and fertilization and thus are beginning to elucidate the molecular pathways that underlie these processes. We identified an allele of the spe-49 gene in a second filial generation (F 2 ) mutagenesis screen for spermatogenesis-defective (spe) mutants. Mutant worms for spe-49 produce sperm that have normal morphology, activate to form ameboid spermatozoa, and can migrate to and maintain their position in the hermaphrodite reproductive tract but fail to fertilize oocytes. This phenotype puts spe-49 in the spe-9 class of late-acting genes that function in sperm at the time of fertilization. We cloned the spe-49 gene through a combination of deficiency mapping, transgenic rescue, and genomic sequencing. spe-49 messenger RNA (mRNA) is enriched in male germ cells, and the complementary DNA (cDNA) encodes a predicted 772-amino-acid six-pass transmembrane protein that is homologous to SPE-42. Indeed, SPE-49 and SPE-42 have identical predicted membrane topology and domain structure, including a large extracellular domain with six conserved cysteine residues, a DC-STAMP domain, and a C-terminal cytoplasmic domain containing a C4–C4 RING finger motif. The presence of two SPE-42 homologs in animal genomes from worms to humans suggests that these proteins are highly conserved components of the molecular apparatus required for the sperm–oocyte recognition, binding, and fusion.
Bibliographical noteFunding Information:
We thank Dr. Shannon Stevenson and Dr. Karen Stine for critical reading of the manuscript and helpful comments. Some nematode strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Strains containing the nDf18, sDf36, and sDf44 deficiencies were generated by the Genetic Toolkit project, which is funded by the NIH National Center for Research Resources (USA) to Ann M. Rose, David L. Baillie, and Donald L. Riddle. We thank the Mitani Lab and the National Bioresource Project for the Experimental Animal Nematode C. elegans (Tokyo, Japan) for providing the tm4655 and tm4967 mutant strains. This work was supported by a grant from the NSF (IOS-0918464), Auburn University at Montgomery Startup Funds, an Auburn University at Montgomery Faculty Grant-in-Aid, and an Auburn University at Montgomery Faculty Equipment Grant-in-aid to T.L.K. The authors declare that no conflicts of interest exist.
- RING finger
- gamete interactions