Figure 4. cil-1 positively regulates sperm activation and motility.

(A) C. elegans sperm activation summarized in this cartoon depicting pathways and genes functioning during sperm activation and fertilization. In a spermatid, MOs containing a TRPC receptor TRP-3 are located just below the PM. During WT activation (solid arrow), MOs fuse to the PM and a pseudopod develops, producing a motile spermatozoon. In fer-1 mutant sperm (upper dotted arrow), MOs do not fuse with the PM and a short pseudopod forms, resulting in immotile sperm. A cil-1 mutant sperm (lower dotted arrow) is normal in MO fusion, but develops into immotile spermatozoon with a short pseudopod. TRPC TRP-3 translocation from MO to the PM appear normal in cil-1 mutant sperm. spe-9, spe-38, spe-41/trp-3, and spe-42 encode various membrane proteins required for sperm-egg interactions. Loss of any of these genes results in motile but infertile spermatozoa. (B-C) Nomarski images of isolated male-derived sperm before and after in vitro activation and endogenously activated hermaphrodite-derived sperm. (B) A round WT spermatid. (B′) WT spermatozoa after 15 minute of pronase activation. Spermatozoa extend full-length pseudopods (yellow arrowheads). Yellow bars depict the length of WT spermatozoa measured. (B″) WT hermaphrodite-derived sperm that are endogenously activated. (B‴) WT male-derived sperm are activated to spermatozoa with pseudopods (arrow arrowheads) within 10 minutes upon 100nM wortmannin application. (C) cil-1(my15) mutant spermatids before activation are slightly smaller than WT spermatids. (C′) Upon activation, cil-1 mutant sperm develop stubby pseudopods. The length of sperm is indicated with orange bars shorter than WT (compare to yellow bars). (C″) Hermaphrodite-derived sperm from cil-1(my15) mutant occasionally develop short pseudopods. (C‴) my15 male-derived sperm are not activated by 100nM wortmannin but exhibit subtle morphological changes. (D) Sperm Tracking Assay. The majority of WT male-derived sperm are deposited and retained within the spermatheca in the hermaphroditic reproductive tract at 10 and 16 hours. In contrast, cil-1(my15) male-derived sperm are not found in the spermatheca at 16 hours. (E-E′) Ultrastructure of him-5(e1490) (E) and cil-1(my15) him-5(e1490) (E′) spontaneously activated spermatozoa.

Abbreviations: lm, laminar membranes; mo, membranous organelles; n, nucleus; p, pseudopod. While the cytoplasm in this cil-1 pseudopod (compare E to E′) appears denser than that of the WT control, the significance, if any, of this observation is unclear. (F-G) Monitoring MO fusion during sperm activation with a lipophilic FM1-43 dye. (F) The PM of WT spermatids is stained with FM1-43. (F′) In WT spermatozoa, the dye concentrates at the MO fusion sites. MO fusion events are restricted to the PM of cell body (arrowheads) but not pseudopod (bracket). (G) FM1-43 dye marks the PM of cil-1 spermatids. (G′) In short my15 spermatozoa with visible pseudopods, MO fusion sites are concentrated on the cell body (arrowheads) and excluded from the pseudopod PM (bracket). (H-I) Immunohistochemistry of sperm using the MO antibody 1CB4 (red), anti-TRP-3 (green), and DAPI (blue). The triple labeled image were generated by overlaying three confocal images from the same Z-section. (H) In WT spermatids, MOs (red) are located around the cell periphery below the PM. Location of TRP-3 (green) partially overlaps with 1CB4 labeled MOs. (H′) In WT spermatozoa, 1CB4 positive MOs are primarily located around the PM, but absent from pseudopods (compare with bracket area in H″). (H″) In WT spermatozoa, anti-TRP-3 staining is detectable in the cell body and pseudopod PM (bracket). (H‴) Overlay of WT spermatozoa. (I) In my15 spermtids, MOs (red) are localized to the cell periphery just below the PM as in WT. Anti-TRP-3 staining (green) overlaps with MOs. (I′) In my15 spermatozoa, similarly to WT, MOs are found in the cell body but not pseudopod. (I″) In my15 spermatozoa, TRP-3 protein is detected both in the cell body and pseudopod, as in WT. (I‴) Overlay of H′ and H″ with the DAPI image. Scale bar, 5um