C. elegans spermatozoa lacking spe-45 are incapable of fusing with the oocyte plasma membrane

Abstract

C. elegans spe-9 class genes encode sperm proteins with indispensable roles during fertilization. We have previously reported that spe-45 belongs to the spe-9 class, based on the finding that self-sperm of spe-45(tm3715) hermaphrodites were not consumed by fertilization. In this study, we directly observed live fertilization in the spermatheca of fem-1(hc17) females after mating with spe-45(tm3715) males. As expected, it was clearly shown that spe-45 mutant spermatozoa failed to fuse with the oocyte plasma membrane. Thus, our live imaging system for C. elegans fertilization seems to be useful for evaluation of the functions of male and female gametes.

Figure 1 spe-45(tm3715) spermatozoa fail to fuse with oocytes in the spermatheca of fem-1(hc17) females

(A) Gross structures of mouse IZUMO1 and SPACA6 and C. elegans SPE-45. All the proteins shown here are sperm transmembrane (TM) proteins with a single immunoglobulin (Ig)-like domain that are essentially required for gamete fusion. The domain architectures of each protein were predicted by the SMART program (http://smart.embl-heidelberg.de). AA, amino acid; N, the amino-terminus; C, the carboxy-terminus; +, positively charged region; and –, negatively charged region. Numbers of “+” and “–” symbols represent relative numbers of basic and acidic amino acids, respectively. This panel was prepared based on the previous reports (Singaravelu et al., 2015; Nishimura et al., 2015; Nishimura and L’Hernault, 2016). (B) Time-lapse analysis on the number of self-sperm in the spermatheca of spe-45 and other spe-9 class mutant hermaphrodites. Data shown in this table are based on the assumption that the number of self-sperm in each spermatheca of N2 (wild type, WT), spe-9(eb19), spe-42(tm2421), and spe-45(tm3715) worms at 24 h post the fourth larval stage (L4) is 100%. This table was prepared according to the previously reported data (Nishimura et al., 2015). (C) Live imaging of fertilization between WT or spe-45 mutant spermatozoa and WT oocytes. We generated oxIs318; him-5(e1490) and oxIs318; spe-45(tm3715); him-5(e1490) males as WT and spe-45 mutant, respectively, and these two strains produce spermatozoa of which nuclei are fluorescently labeled with mCherry (magenta). We also raised fem-1(hc17); ltIs38 hermaphrodites at 25°C as WT females, in which the oocyte plasma membrane (PM) carries a green fluorescent protein (GFP, green). After mating of the females with WT (left) or spe-45 mutant (right) males, live fertilization occurring in the spermatheca was observed by two-color 3D time-lapse spinning-disc confocal microscopy, as previously reported (Takayama and Onami, 2016). Still images were then acquired from movies recording the live fertilization. The white arrowhead indicates a fluorescence gap, which presumably shows that sperm-oocyte fusion occurs at the site. While the fluorescence gap region constantly appeared on the oocyte PM during fertilization by WT spermatozoa (n = 8), spe-45 mutant spermatozoa could not yield the arc-shaped GFP signals on the oocyte surface (n = 8). Scale bar, 20 µm.