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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.


Related in: MedlinePlus

Reversible transition of the ZP meshwork status by substitution treatment with dcp11 and gpp2.(a) RCAI staining of the VE substitutionally treated with dcp11 and gpp2. gpp2 → dcp11; eggs were pretreated first with gpp2 (4 μM), followed by rinse and treatment with dcp11 (8 μM). dcp11 → gpp2; eggs were pretreated first with dcp11 (4 μM), followed by rinse and treatment with gpp2 (8 μM). (Left) Representative confocal image of unfertilized egg. (Right) Intensities across the VE (black) (n = 15). RCAI reactivity of the VE pretreated with dcp11 (blue) and gpp2 (red) were also shown. (b) A schematic model of the transition of ZP meshwork between fertilization competent and incompetent statuses. Our TEM studies revealed that ZP filaments pretreated with dcp11 were arranged parallel to the egg plasma membrane, exhibiting a “pin-stripe” pattern, while ZP filaments pretreated with gpp2 were arranged oblique to the egg plasma membrane, occasionally forming a “herring-bone” pattern. These results implies that the ZP filaments pretreated with dcp11 was a well-organized sheet-like structure, while the ZP filaments pretreated with gpp2 was randomly disoriented organization. On the basis of these two-dimensional images, we hypothesized three-dimensional meshwork model of ZP filaments. Treatment with gpp2 induces a randomized disoriented ZP meshwork that allows sperm to fit into the three-dimensional structure (i.e. capture of sperm), and enables acrosome reaction, while treatment with dcp11 alters a better-organized meshwork, forming parallel sheet of ZP filaments where sperm may not fit to the structure and therefore sperm move away from the VE, resulting in fertilization failure.
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f5: Reversible transition of the ZP meshwork status by substitution treatment with dcp11 and gpp2.(a) RCAI staining of the VE substitutionally treated with dcp11 and gpp2. gpp2 → dcp11; eggs were pretreated first with gpp2 (4 μM), followed by rinse and treatment with dcp11 (8 μM). dcp11 → gpp2; eggs were pretreated first with dcp11 (4 μM), followed by rinse and treatment with gpp2 (8 μM). (Left) Representative confocal image of unfertilized egg. (Right) Intensities across the VE (black) (n = 15). RCAI reactivity of the VE pretreated with dcp11 (blue) and gpp2 (red) were also shown. (b) A schematic model of the transition of ZP meshwork between fertilization competent and incompetent statuses. Our TEM studies revealed that ZP filaments pretreated with dcp11 were arranged parallel to the egg plasma membrane, exhibiting a “pin-stripe” pattern, while ZP filaments pretreated with gpp2 were arranged oblique to the egg plasma membrane, occasionally forming a “herring-bone” pattern. These results implies that the ZP filaments pretreated with dcp11 was a well-organized sheet-like structure, while the ZP filaments pretreated with gpp2 was randomly disoriented organization. On the basis of these two-dimensional images, we hypothesized three-dimensional meshwork model of ZP filaments. Treatment with gpp2 induces a randomized disoriented ZP meshwork that allows sperm to fit into the three-dimensional structure (i.e. capture of sperm), and enables acrosome reaction, while treatment with dcp11 alters a better-organized meshwork, forming parallel sheet of ZP filaments where sperm may not fit to the structure and therefore sperm move away from the VE, resulting in fertilization failure.

Mentions: We further examined whether the above two states could switch by repeated treatment of the VE: dcp11 treatment following gpp2 pretreatment (gpp2 → dcp11) or gpp2 treatment following dcp11 pretreatment (dcp11 → gpp2). Treatment with gpp2 → dcp11 augmented RCAI reactivity to a similar extent as with dcp11 treatment alone, whereas treatment with dc11 → gpp2 did not alter RCAI reactivity, similar to gpp2 treatment alone (Fig. 5a). As for the WGA ligand, the staining patterns of gpp2 → dc11 and dc11 → gpp2 were similar to those of dcp11 or gpp2 treatment alone, respectively (Supplementary Fig. S5). These results demonstrated that the peptide-induced fertilization competent status could be converted, and therefore fertilization competent state has a structural plasticity determined by the dicalcin-gp41 interaction. A schematic representation describing the possible three-dimensional meshwork model of ZP filaments regarding fertilization competence is shown in Fig. 5b.


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)

Reversible transition of the ZP meshwork status by substitution treatment with dcp11 and gpp2.(a) RCAI staining of the VE substitutionally treated with dcp11 and gpp2. gpp2 → dcp11; eggs were pretreated first with gpp2 (4 μM), followed by rinse and treatment with dcp11 (8 μM). dcp11 → gpp2; eggs were pretreated first with dcp11 (4 μM), followed by rinse and treatment with gpp2 (8 μM). (Left) Representative confocal image of unfertilized egg. (Right) Intensities across the VE (black) (n = 15). RCAI reactivity of the VE pretreated with dcp11 (blue) and gpp2 (red) were also shown. (b) A schematic model of the transition of ZP meshwork between fertilization competent and incompetent statuses. Our TEM studies revealed that ZP filaments pretreated with dcp11 were arranged parallel to the egg plasma membrane, exhibiting a “pin-stripe” pattern, while ZP filaments pretreated with gpp2 were arranged oblique to the egg plasma membrane, occasionally forming a “herring-bone” pattern. These results implies that the ZP filaments pretreated with dcp11 was a well-organized sheet-like structure, while the ZP filaments pretreated with gpp2 was randomly disoriented organization. On the basis of these two-dimensional images, we hypothesized three-dimensional meshwork model of ZP filaments. Treatment with gpp2 induces a randomized disoriented ZP meshwork that allows sperm to fit into the three-dimensional structure (i.e. capture of sperm), and enables acrosome reaction, while treatment with dcp11 alters a better-organized meshwork, forming parallel sheet of ZP filaments where sperm may not fit to the structure and therefore sperm move away from the VE, resulting in fertilization failure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525147&req=5

f5: Reversible transition of the ZP meshwork status by substitution treatment with dcp11 and gpp2.(a) RCAI staining of the VE substitutionally treated with dcp11 and gpp2. gpp2 → dcp11; eggs were pretreated first with gpp2 (4 μM), followed by rinse and treatment with dcp11 (8 μM). dcp11 → gpp2; eggs were pretreated first with dcp11 (4 μM), followed by rinse and treatment with gpp2 (8 μM). (Left) Representative confocal image of unfertilized egg. (Right) Intensities across the VE (black) (n = 15). RCAI reactivity of the VE pretreated with dcp11 (blue) and gpp2 (red) were also shown. (b) A schematic model of the transition of ZP meshwork between fertilization competent and incompetent statuses. Our TEM studies revealed that ZP filaments pretreated with dcp11 were arranged parallel to the egg plasma membrane, exhibiting a “pin-stripe” pattern, while ZP filaments pretreated with gpp2 were arranged oblique to the egg plasma membrane, occasionally forming a “herring-bone” pattern. These results implies that the ZP filaments pretreated with dcp11 was a well-organized sheet-like structure, while the ZP filaments pretreated with gpp2 was randomly disoriented organization. On the basis of these two-dimensional images, we hypothesized three-dimensional meshwork model of ZP filaments. Treatment with gpp2 induces a randomized disoriented ZP meshwork that allows sperm to fit into the three-dimensional structure (i.e. capture of sperm), and enables acrosome reaction, while treatment with dcp11 alters a better-organized meshwork, forming parallel sheet of ZP filaments where sperm may not fit to the structure and therefore sperm move away from the VE, resulting in fertilization failure.
Mentions: We further examined whether the above two states could switch by repeated treatment of the VE: dcp11 treatment following gpp2 pretreatment (gpp2 → dcp11) or gpp2 treatment following dcp11 pretreatment (dcp11 → gpp2). Treatment with gpp2 → dcp11 augmented RCAI reactivity to a similar extent as with dcp11 treatment alone, whereas treatment with dc11 → gpp2 did not alter RCAI reactivity, similar to gpp2 treatment alone (Fig. 5a). As for the WGA ligand, the staining patterns of gpp2 → dc11 and dc11 → gpp2 were similar to those of dcp11 or gpp2 treatment alone, respectively (Supplementary Fig. S5). These results demonstrated that the peptide-induced fertilization competent status could be converted, and therefore fertilization competent state has a structural plasticity determined by the dicalcin-gp41 interaction. A schematic representation describing the possible three-dimensional meshwork model of ZP filaments regarding fertilization competence is shown in Fig. 5b.

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.


Related in: MedlinePlus