Dynamic localization of SPE-9 in sperm: a protein required for sperm-oocyte interactions in Caenorhabditis elegans

Abstract

Background: Fertilization in Caenorhabditis elegans requires functional SPE-9 protein in sperm. SPE-9 is a transmembrane protein with a predicted extracellular domain that contains ten epidermal growth factor (EGF)-like motifs. The presence of these EGF-like motifs suggests that SPE-9 is likely to function in gamete adhesive and/or ligand-receptor interactions.

Results: We obtained specific antisera directed against different regions of SPE-9 in order to determine its subcellular localization. SPE-9 is segregated to spermatids with a pattern that is consistent with localization to the plasma membrane. During spermiogenesis, SPE-9 becomes localized to spiky projections that coalesce to form a pseudopod. This leads to an accumulation of SPE-9 on the pseudopod of mature sperm.

Conclusions: The wild type localization patterns of SPE-9 provide further evidence that like the sperm of other species, C. elegans sperm have molecularly mosaic and dynamic regions. SPE-9 is redistributed by what is likely to be a novel mechanism that is very fast (approximately 5 minutes) and is coincident with dramatic rearrangements in the major sperm protein cytoskeleton. We conclude that SPE-9 ends up in a location on mature sperm where it can function during fertilization and this localization defines the sperm region required for these interactions.

Figure 1

Schematic representation of SPE-9. Motifs are indicated in the key by grey or black shaded boxes. Regions corresponding to sequences used to generate EX and C sera are indicated by the brackets below the diagram. The extracellular and intracellular domains of SPE-9 are indicated.

Figure 2

Immunofluorescence localization of SPE-9 in spermatids. (A, C, E, G, I) Nomarski DIC images of spermatids. (B, D, F, H) Staining for SPE-9 in red. (J) Staining for 1CB4 in green. Staining with anti-SPE-9-EX (A, B) and anti-SPE-9-C (C, D) at this plane of focus show a ring of staining around the cell. No staining is observed in a spe-9(eb19) mutant background for either anti-SPE-9-EX (E, F) or anti-SPE-9-C (G, H). For contrast, 1CB4 stains membranous organelles (MOs) in the cytoplasm (I, J). Bar in I is 5 μm and applies to all panels.

Figure 3

SPE-9 is a cell surface protein. Non-permeabilized spermatids incubated with anti-SPE-9-EX and 1CB4 (A-C) or anti-SPE-9-C and 1CB4 (D-F). A and D show Nomarski DIC images. B and E show the anti-SPE-9 staining pattern on these cells. C and F show the lack of 1CB4 staining. DAPI staining of nuclei is blue. Dashed lines in C, E and F indicate the positions of the cells. Bar in D is 5 μm and applies to all panels.

Figure 4

SPE-9 is concentrated in spikes during spermiogenesis and pseudopods in mature sperm. (A, C, E, G) Nomarski DIC images. (B, D, F, H) Staining for SPE-9 in red. Arrows point to spikes or pseudopods. (A, B) Wild-type spermatid activated with TFP. (C, D) spe-12(hc76) spermatid treated with pronase. Note the numerous small spots that correspond to arrested or developing spikes. (E, F) Wild-type sperm activated by TEA treatment and stained with anti-SPE-9-EX. (G, H) Wild-type sperm activated by TEA treatment and stained with anti-SPE-9-C. Bar in G is 5 μm and applies to all panels.

Figure 5

Summary of the localization of SPE-9. SPE-9 localization is indicated with red.