Description

The gene products of C. elegans ORFs Y59E9AL.6 and C41G7.6 were assigned the names CYLC-1 and CYLC-2, respectively, due to their homology to human cylicins (Lacroix et al.. 2016). In mammals, expression of cylicin I and cylicin II is testis-specific and immunohistochemistry shows the proteins localize to the cytoskeletal calyx of the sperm head (Hess et al. 1993; Hess et al. 1995; Rousseaux-Prèvost et al. 2003). To determine the localization of C. elegans CYLC-1 and CYLC-2 we used CRISPR/Cas9 to endogenously tag each protein with mNeonGreen (mNG). We find that CYLC-2::mNG localizes to sperm in both hermaphrodites and males (Figure 1A-F). Examination of spermatids dissected from males shows CYLC-2::mNG concentrated in puncta (Figure 1F). Based on their size and position in the spermatids, we predict that these puncta correspond to the membranous organelles (MOs). However, further studies will be necessary to confirm whether CYLC-2 is concentrated in the MOs of spermatids and to determine if any changes in subcellular localization take place upon sperm activation.

We present CYLC-2::mNG as another genetically encoded sperm marker that can be used to track sperm presence, transfer, and movement (Figure 1G). For fertility studies it is often important to know if hermaphrodite self sperm are present in the spermathecae or if males have successfully mated and transferred sperm to a hermaphrodite. The creation of bright, sperm-specific fluorescent markers in C. elegans has been notoriously challenging (Wong et al.. 2020). Traditionally, the transfer and movement of sperm has been studied by indiscriminately marking male cells with vital dyes such as Mitotracker Red CMXRos or SYTO 17 (Hu et al. 2019; Singson et al.. 1999; Hill and L’Hernault 2001). Alternatively, fixation and staining with DAPI has been a blunt tool to confirm the presence or absence of sperm (Singaravelu et al. 2015; Krauchunas et al.. 2018). Most recently, the Stanfield lab has shown that combining two unlinked sperm histone::mCherry transgenes into the same strain significantly improved the fluorescent signal without impacting sperm function (Wong et al.. 2020).

CYLC-2::mNG is bright enough that it can be seen with just a fluorescence stereo microscope, allowing for the transfer of sperm to be monitored over time in free living hermaphrodites on a culture plate. It also makes crossing this sperm marker into mutant backgrounds relatively straightforward. Finally, it adds a bright, non-nuclear sperm marker to our community toolkit. By marking one population of sperm with a nuclear marker such as histone::mCherry and another with CYLC-2::mNG we can differentiate between hermaphrodite self sperm and male sperm, or sperm from two different males, within the hermaphrodite reproductive tract. There is no statistically significant difference in the total number of progeny sired by males with tagged CYLC-2 as compared to control males (p-value = 0.4, Figure 1H). In addition, there is no difference in the percentage of progeny produced that are outcross progeny (p-value = 0.58, Figure 1I). From these data, we infer that tagging CYLC-2 with mNG has no discernable effect on the ability of the sperm to migrate, compete with hermaphrodite sperm, or fertilize the egg.

We conclude that CYLC-2::mNG makes an excellent sperm marker to observe sperm transfer and migration in C. elegans. In addition, it is intriguing that the presence of these cylicin-like cytoskeletal proteins in sperm is conserved since C. elegans sperm have neither actin nor tubulin (L’Hernault and Roberts 1995). In the future, mutant studies will help us determine the role of cylicins in spermatogenesis, sperm structure, and sperm function.