The Caenorhabditis elegans spe‐49 gene is required for fertilization and encodes a sperm‐specific transmembrane protein homologous to SPE‐42

Abstract

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 ) 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.

Sperm membrane proteins DCST1 and DCST2 are required for the sperm-egg fusion process in mice and fish

“…DC-STAMP-like domain-containing proteins, namely the testis-enriched SNKY, SPE-42, and SPE-49, are necessary for male fertility in Drosophila 21,22 and C. elegans 23–25 , respectively. Specifically, sneaky -disrupted fly spermatozoa can enter the egg, but fail to break down the sperm plasma membrane: the male pronucleus thus does not form, and embryonic mitotic divisions do not occur 22 .…”

“…Specifically, sneaky -disrupted fly spermatozoa can enter the egg, but fail to break down the sperm plasma membrane: the male pronucleus thus does not form, and embryonic mitotic divisions do not occur 22 . Spe-42 and spe-49 mutant C. elegans spermatozoa can migrate into the spermatheca, the site of fertilization in worms, but these mutants are nearly or completely sterile, respectively, suggesting that SPE-42 and SPE-49 are involved in the ability of spermatozoa to fertilize eggs 23–25 . SNKY, SPE-42 and SPE-49 have homologs in vertebrates called DCST1 and DCST2, but the roles of these proteins have remained undetermined.…”

The EGF-motif containing protein SPE-36 is a secreted protein required for sperm function at fertilization inC. elegans

“…Similarly, both IZUMO1 and SPACA6 are proteins with Ig-like domains that are required for mammalian fertilization (Barbaux et al, 2020; Inoue et al, 2005; Lorenzetti et al, 2014; Noda et al, 2020). Fertilization in both worms and mammals also requires a pair of DC-STAMP domain- containing proteins: SPE-42 and SPE-49 in C. elegans , DCST1 and DCST2 in mammals (Inoue et al, 2021; Kroft et al, 2005; Noda et al, 2021; Wilson et al, 2018). Mammalian ADAM3, Integrin Beta, SED1, and C. elegans SPE-9 and SPE-36, all contain one or more EGF domains (Ensslin and Shur, 2003; Singson et al, 1998; Takagi et al, 2001; Yuan et al, 1997).…”

The molecular underpinnings of fertility: Genetic approaches in Caenorhabditis elegans

“…Other times sterile mutants were misclassified as embryonic lethal and sometimes discarded. For example, the spe‐49 gene was initially named let‐479 because the phenotype was thought to be embryonic lethality 21 . Therefore, community mutation collections could be a rich source of uncharacterized fertility mutants.…”

“…To characterize spermatogenesis, L’Hernault and coworkers designed a screen for spe mutants. 17 , 21 They described two types of strategies. One strategy is similar to the one described above.…”