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Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

Miwa N, Ogawa M, Hanaue M, Takamatsu K - Sci Rep (2015)

Bottom Line: Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively.Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork.These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Toho University, Ohmori-nishi 5-21-16, Ohta-ku, Tokyo 143-8540, Japan.

ABSTRACT
Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

No MeSH data available.


Dicalcin- and gp41-derived peptides induce alterations in in vivo lectin reactivities of the VE.(a) Blot of VE proteins treated with RCAI. CBB, CBB-stained VE; RCAI, Rhodamine-labeled RCAI blot. (b) Representative confocal images of a Xenopus egg treated with RCAI and averaged intensities across the VE. (Left) RCAI stained the outermost region of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of RCAI staining across the VE (dashed line in Control in the left, n = 15). The position where RCAI-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (c) RCAI staining of the VE pretreated with dcp11. (Left) RCAI reactivity of the VE pretreated with dcp11. (Right) Intensities across the VE (black) (n = 15). RCAI reactivities pretreated with dicalcin (blue) and BSA (red) were also shown. (d) RCAI staining of the VE pretreated with gpp2. (Left) RCAI reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15). (e) Blot of VE proteins treated with WGA. WGA recognized gp41 as well as gp69/64 and gp120. CBB, CBB-stained VE; WGA, FITC-labeled WGA blot. (f) Representative confocal images of a Xenopus egg treated with WGA and averaged intensities across the VE. (Left) WGA stained the outermost and midsection regions of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of WGA staining across the VE (n = 15). The position where WGA-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (g) WGA staining of the VE pretreated with dcp11. (Left) WGA reactivity of the VE pretreated with dcp11. (Right) Intensities of the VE across the VE (black) (n = 15). WGA reactivities of the VE pretreated with dicalcin (blue) and BSA (red) were also shown. (h) WGA staining of the VE pretreated with gpp2. (Left) WGA reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15).
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f3: Dicalcin- and gp41-derived peptides induce alterations in in vivo lectin reactivities of the VE.(a) Blot of VE proteins treated with RCAI. CBB, CBB-stained VE; RCAI, Rhodamine-labeled RCAI blot. (b) Representative confocal images of a Xenopus egg treated with RCAI and averaged intensities across the VE. (Left) RCAI stained the outermost region of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of RCAI staining across the VE (dashed line in Control in the left, n = 15). The position where RCAI-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (c) RCAI staining of the VE pretreated with dcp11. (Left) RCAI reactivity of the VE pretreated with dcp11. (Right) Intensities across the VE (black) (n = 15). RCAI reactivities pretreated with dicalcin (blue) and BSA (red) were also shown. (d) RCAI staining of the VE pretreated with gpp2. (Left) RCAI reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15). (e) Blot of VE proteins treated with WGA. WGA recognized gp41 as well as gp69/64 and gp120. CBB, CBB-stained VE; WGA, FITC-labeled WGA blot. (f) Representative confocal images of a Xenopus egg treated with WGA and averaged intensities across the VE. (Left) WGA stained the outermost and midsection regions of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of WGA staining across the VE (n = 15). The position where WGA-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (g) WGA staining of the VE pretreated with dcp11. (Left) WGA reactivity of the VE pretreated with dcp11. (Right) Intensities of the VE across the VE (black) (n = 15). WGA reactivities of the VE pretreated with dicalcin (blue) and BSA (red) were also shown. (h) WGA staining of the VE pretreated with gpp2. (Left) WGA reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15).

Mentions: Carbohydrate-dependent recognition has been repeatedly implicated to play an important role for establishment of an appropriate sperm-egg interaction25. Indeed, alterations in the staining pattern of a lectin have been observed in human ZP of fertilization-failed oocytes15. Our previous study has also revealed that pretreatment with dicalcin increases in vivo reactivity of the VE to the Gal/GalNAc-sensitive lectin, Ricinus communis agglutinin I (RCAI), suggesting that dicalcin regulates the distribution pattern of oligosaccharides within the VE through its binding to gp4116. To verify whether dicalcin- and gp41-derived peptides also alter the distribution pattern of oligosaccharides, we examined the lectin reactivity of the VE following peptide pretreatment. Consistent with our previous study, RCAI reacted only with gp41 in our lectin blot analysis (Fig. 3a), and its reactivity increased following pretreatment with dicalcin when RCAI-staining signals were quantified across the VE (Fig. 3b). Pretreatment with dcp11 augmented RCAI reactivity to the same extent as with dicalcin pretreatment (Fig. 3c), indicating that dicalcin and dcp11 caused similar changes in the distribution pattern of the RCAI ligand.


Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

Miwa N, Ogawa M, Hanaue M, Takamatsu K - Sci Rep (2015)

Dicalcin- and gp41-derived peptides induce alterations in in vivo lectin reactivities of the VE.(a) Blot of VE proteins treated with RCAI. CBB, CBB-stained VE; RCAI, Rhodamine-labeled RCAI blot. (b) Representative confocal images of a Xenopus egg treated with RCAI and averaged intensities across the VE. (Left) RCAI stained the outermost region of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of RCAI staining across the VE (dashed line in Control in the left, n = 15). The position where RCAI-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (c) RCAI staining of the VE pretreated with dcp11. (Left) RCAI reactivity of the VE pretreated with dcp11. (Right) Intensities across the VE (black) (n = 15). RCAI reactivities pretreated with dicalcin (blue) and BSA (red) were also shown. (d) RCAI staining of the VE pretreated with gpp2. (Left) RCAI reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15). (e) Blot of VE proteins treated with WGA. WGA recognized gp41 as well as gp69/64 and gp120. CBB, CBB-stained VE; WGA, FITC-labeled WGA blot. (f) Representative confocal images of a Xenopus egg treated with WGA and averaged intensities across the VE. (Left) WGA stained the outermost and midsection regions of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of WGA staining across the VE (n = 15). The position where WGA-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (g) WGA staining of the VE pretreated with dcp11. (Left) WGA reactivity of the VE pretreated with dcp11. (Right) Intensities of the VE across the VE (black) (n = 15). WGA reactivities of the VE pretreated with dicalcin (blue) and BSA (red) were also shown. (h) WGA staining of the VE pretreated with gpp2. (Left) WGA reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15).
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Related In: Results  -  Collection

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f3: Dicalcin- and gp41-derived peptides induce alterations in in vivo lectin reactivities of the VE.(a) Blot of VE proteins treated with RCAI. CBB, CBB-stained VE; RCAI, Rhodamine-labeled RCAI blot. (b) Representative confocal images of a Xenopus egg treated with RCAI and averaged intensities across the VE. (Left) RCAI stained the outermost region of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of RCAI staining across the VE (dashed line in Control in the left, n = 15). The position where RCAI-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (c) RCAI staining of the VE pretreated with dcp11. (Left) RCAI reactivity of the VE pretreated with dcp11. (Right) Intensities across the VE (black) (n = 15). RCAI reactivities pretreated with dicalcin (blue) and BSA (red) were also shown. (d) RCAI staining of the VE pretreated with gpp2. (Left) RCAI reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15). (e) Blot of VE proteins treated with WGA. WGA recognized gp41 as well as gp69/64 and gp120. CBB, CBB-stained VE; WGA, FITC-labeled WGA blot. (f) Representative confocal images of a Xenopus egg treated with WGA and averaged intensities across the VE. (Left) WGA stained the outermost and midsection regions of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of WGA staining across the VE (n = 15). The position where WGA-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (g) WGA staining of the VE pretreated with dcp11. (Left) WGA reactivity of the VE pretreated with dcp11. (Right) Intensities of the VE across the VE (black) (n = 15). WGA reactivities of the VE pretreated with dicalcin (blue) and BSA (red) were also shown. (h) WGA staining of the VE pretreated with gpp2. (Left) WGA reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15).
Mentions: Carbohydrate-dependent recognition has been repeatedly implicated to play an important role for establishment of an appropriate sperm-egg interaction25. Indeed, alterations in the staining pattern of a lectin have been observed in human ZP of fertilization-failed oocytes15. Our previous study has also revealed that pretreatment with dicalcin increases in vivo reactivity of the VE to the Gal/GalNAc-sensitive lectin, Ricinus communis agglutinin I (RCAI), suggesting that dicalcin regulates the distribution pattern of oligosaccharides within the VE through its binding to gp4116. To verify whether dicalcin- and gp41-derived peptides also alter the distribution pattern of oligosaccharides, we examined the lectin reactivity of the VE following peptide pretreatment. Consistent with our previous study, RCAI reacted only with gp41 in our lectin blot analysis (Fig. 3a), and its reactivity increased following pretreatment with dicalcin when RCAI-staining signals were quantified across the VE (Fig. 3b). Pretreatment with dcp11 augmented RCAI reactivity to the same extent as with dicalcin pretreatment (Fig. 3c), indicating that dicalcin and dcp11 caused similar changes in the distribution pattern of the RCAI ligand.

Bottom Line: Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively.Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork.These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Toho University, Ohmori-nishi 5-21-16, Ohta-ku, Tokyo 143-8540, Japan.

ABSTRACT
Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

No MeSH data available.