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

Zannoni S, L'Hernault SW, Singson AW - BMC Dev. Biol. (2003)

Bottom Line: This leads to an accumulation of SPE-9 on the pseudopod of mature sperm.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Waksman Institute and Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA. szannoni@hotmail.com

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.

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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.
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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.

Mentions: SPE-9 is segregated to spermatids where, depending on the plane of focus, we see a ring of staining (Fig. 2A,2B,2C,2D). In addition to the identical staining patterns observed with the EX and C antisera, we stained sperm isolated from worms carrying a allele of spe-9 to further validate specificity. The eb19 allele of spe-9 encodes a premature stop codon before the amino acid sequences used to generate our antibodies [15]. spe-9(eb19) animals are completely sterile and produce spermatids that show no immunoreactivity to both the EX and C sera (Fig. 2E,2F,2G,2H). To make sure that SPE-9 localization patterns are not temperature dependent, we immunostained sperm isolated from worms raised at 16°C, 20°C and 25°C. The staining pattern of SPE-9 was not altered by culture temperature (data not shown). The SPE-9 staining pattern is very different from staining patterns seen with sera that stain internal sperm structures [17,18]. We also labeled spermatids with the monoclonal antibody 1CB4 [19] (Fig. 2I,2J). The epitope recognized by 1CB4 has been shown to reside on membranous organelles (MOs). MOs are located in the cytoplasm close to the plasma membrane. This MO vesicle-staining pattern is distinct from SPE-9 staining (compare 2B and 2D with 2J).


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

Zannoni S, L'Hernault SW, Singson AW - BMC Dev. Biol. (2003)

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.
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Related In: Results  -  Collection

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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.
Mentions: SPE-9 is segregated to spermatids where, depending on the plane of focus, we see a ring of staining (Fig. 2A,2B,2C,2D). In addition to the identical staining patterns observed with the EX and C antisera, we stained sperm isolated from worms carrying a allele of spe-9 to further validate specificity. The eb19 allele of spe-9 encodes a premature stop codon before the amino acid sequences used to generate our antibodies [15]. spe-9(eb19) animals are completely sterile and produce spermatids that show no immunoreactivity to both the EX and C sera (Fig. 2E,2F,2G,2H). To make sure that SPE-9 localization patterns are not temperature dependent, we immunostained sperm isolated from worms raised at 16°C, 20°C and 25°C. The staining pattern of SPE-9 was not altered by culture temperature (data not shown). The SPE-9 staining pattern is very different from staining patterns seen with sera that stain internal sperm structures [17,18]. We also labeled spermatids with the monoclonal antibody 1CB4 [19] (Fig. 2I,2J). The epitope recognized by 1CB4 has been shown to reside on membranous organelles (MOs). MOs are located in the cytoplasm close to the plasma membrane. This MO vesicle-staining pattern is distinct from SPE-9 staining (compare 2B and 2D with 2J).

Bottom Line: This leads to an accumulation of SPE-9 on the pseudopod of mature sperm.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Waksman Institute and Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA. szannoni@hotmail.com

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.

Show MeSH