Description

Gamete fusion is a pivotal step during fertilization to create an organism of the next generation. In C. elegans, since oocytes have no thick egg coat like the zona pellucida, perhaps spermatozoa directly bind to and fuse with the oocyte plasma membrane (PM). Thus, C. elegans is an excellent model to investigate how a spermatozoon and an oocyte fuse together.

C. elegans spe genes encode proteins needed for male germline functions, such as spermatogenesis (meiosis to produce spermatids), spermiogenesis (transformation of spermatids into spermatozoa), and fertilization (gamete fusion) (L’Hernaut, 1997; Nishimura and L’Hernault, 2010; Singaravelu and Singson, 2011). In particular, spe-9 (Singson et al., 1998), spe-38 (Chatterjee et al., 2005; Singaravelu et al., 2012), spe-41/trp-3 (Xu and Sternberg, 2003; Singaravelu et al., 2012; Takayama and Onami, 2016), spe-42 (Kroft et al., 2005), and spe-49 (Wilson et al., 2018) belong to the spe-9 class, and these genes all play essential roles during fertilization. However, it has been unclear whether members of the spe-9 class have evolutionarily conserved roles during fertilization. To explore this question, we hypothesized that C. elegans has an ortholog of mouse Izumo1. This gene encodes a sperm transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain that is required for sperm-oocyte fusion (Figure 1A) (Inoue et al., 2005; Inoue et al., 2015). Recently, mouse Spaca6 was also identified as a testis-enriched gene encoding an Ig-like TM protein with an indispensable role in gamete fusion, as is the case for mouse Izumo1 (Figure 1A) (Lorenzetti et al., 2014; Barbaux et al., 2020; Noda et al., 2020). Moreover, Ig-like proteins from various species are known to act in gamete interactions (Nishimura and L’Hernault, 2016). Thus, we searched for male germline-enriched genes encoding proteins with the Izumo1-like domain architecture in the C. elegans genome by reverse genetic approaches (Nishimura et al., 2015). spe-45 (formerly named F28D1.8/oig-7) meets these criteria (Figure 1A), and spe-45(tm3715) hermaphrodites exhibit typical Spe phenotypes; the mutant worms are self-sterile but fertile after outcrossing to wild-type (WT) males. Singaravelu et al. also identified the same gene by a forward genetic approach (Singaravelu et al., 2015).

Among the male germline functions, spermatogenesis and spermiogenesis are not impaired in spe-45(tm3715) worms (Singaravelu et al., 2015; Nishimura et al., 2015). As shown in Figure 1B, while self-sperm mostly disappeared in the spermatheca of WT hermaphrodites at 72 h post the fourth larval stage (L4), more than half of self-sperm still reside in the spermatheca of spe-9(eb19), spe-42(tm2421), and spe-45(tm3715) hermaphrodites even at the same time point (Nishimura et al., 2015). Thus, all the tested mutants are presumably incapable of completing fertilization (gamete fusion), indicating that spe-45 is a member of the spe-9 class. Additionally, a domain-swapping approach showed that the Ig-like domains of C. elegans SPE-45 and mouse IZUMO1 share a conserved role during fertilization despite only ~9% identity of the entire amino acid sequences of these two proteins (Nishimura et al., 2015).

The data in Figure 1B provide strong but indirect evidence that spe-45 is involved during gamete fusion. On the other hand, we have succeeded in live imaging of fertilization occurring in the spermatheca by two-color 3D time-lapse spinning-disc confocal microscopy (Takayama and Onami, 2016). In this system, we used spermatozoa from males carrying the oxIs318 allele, which produces mCherry-labeled sperm nuclei, and oocytes from ltIs38 hermaphrodites, of which the PM is tagged with a green fluorescent protein (GFP). Therefore, we produced him-5(e1490) and spe-45(tm3715); him-5(e1490) males in the oxIs318 background as WT-like and spe-45 mutant, respectively, and fem-1(hc17) hermaphrodites in the ltIs38 background as WT-like females.

After mating of fem-1 females with WT or spe-45 males, the spermatheca of the females was observed. From movies recording the live fertilization (see Extended Data), we could obtain still images showing a fluorescence gap on the oocyte PM, which was yielded by WT but not spe-45(tm3715) spermatozoa (Figure 1C). The place indicated by the fluorescence gap region seemed to represent where gamete fusion occurs, based on the previous report (Takayama and Onami, 2016). Therefore, our present findings show direct evidence that spe-45(tm3715) spermatozoa have a defect in sperm-oocyte fusion.

One of the advantages of using C. elegans for reproductive biology is that live fertilization in the spermatheca can be observed through transparent cuticles. Our imaging analysis demonstrates that spermatozoa lacking spe-45 fail to fuse with the oocyte PM, unlike WT spermatozoa. However, it remains to be clarified how each spe-9 class gene acts during gamete fusion. For instance, the data in Figure 1B imply that functional roles of spe-9 and spe-45 might be distinguishable from that of spe-42 during gamete fusion. Our imaging system would provide important clues to solve the point.