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First published online 19 December 2007
doi: 10.1242/dev.015503

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Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

Department of Molecular Biology, Cellular Biology, and Biochemistry, Box G-L173, Brown University, Providence, RI 02912, USA.

View larger version (74K): [in this window] [in a new window]   Fig. 1. Peroxidase-mediated crosslinking in the sea urchin fertilization envelope contributes to its permeability barrier. (A-D) Plots show permeability of dextrans into the perivitelline space of zygotes whose embryos were formed as normal (A), after egg dejellying (B), in calcium-free seawater (C), and in 3-AT (D). Plots show the percentage of normalized fluorescence (variable molecular mass Texas Red dextran fluorescence to control 3,000-dalton Cascade Blue dextran) within the perivitelline space versus the media outside the zygote. At least seven individuals were measured for each point plotted. Mean percentages and standard deviation are shown. Data from normal formation conditions (A) are reproduced in greyscale in other plots (B-D). Significant changes in permeability compared with normal zygotes (P<10-10) are indicated (asterisks). (E,F) Merged images of fertilization envelopes formed under normal conditions for S. purpuratus (E) and L. variegatus (F). Neutral Texas Red dextran fluorescence (3,000 to 70,000 daltons; red) overlaid with control Cascade-Blue dextran (3,000 daltons; blue), on top of DIC images (greyscale). Perivitelline space is found between the zygote (zyg) and the fertilization envelope (arrow). Scale bar: 50 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. In vivo incorporation of tyramide-conjugates identifies endogenous ovoperoxidase activity. (A) The chemistry of peroxidase-mediated formation of dityrosine crosslinks between adjacent proteins (R1, R2). This chemistry can be exploited to permanently bind a fluorochrome to proteins, as per the tyramide signal amplification mechanism (Bobrow et al., 1989). (B) The tyramide-fluorochrome conjugates compete with the formation of endogenous dityrosine crosslinks. (C) Eggs pretreated with the ovoperoxidase inhibitor 3-aminotriazole (3-AT; right) (Showman and Foerder, 1979) show significantly less tyramide-Alexa Fluor 488 incorporation in the fertilization envelope than untreated controls (left). Fluorescence images (top) are complemented by DIC images (bottom). (C') Fluorescence images from C overlaid on respective DIC images to show selectivity of incorporation. Scale bar: 100 µm. (D) Quantification of fluorescence intensity between control and 3-AT treated eggs. Mean fluorescence per unit area (AU, arbitrary units) at the equator of the fertilization envelope of each species. Standard deviation per treatment is shown. The number of replicates measured per treatment is indicated.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Kinetics of ovoperoxidase crosslinking is linked to hydrogen peroxide synthesis. The incorporation of fluorescent tyramide analogs can be visualized in real time, and is limited to the fertilization envelope. (A) Representative snapshots of Strongylocentrotus purpuratus fertilization at times shown, where sperm fusion (arrowhead) represents time=0 (seconds). The concentration of tyramide-Alexa Fluor 594 (red) within the fertilization envelope (arrowhead) increases over time. The plasma membrane is counter-stained with FM1-43 (green). Fluorescence images (top) are complemented by DIC images (bottom). Scale bar: 50 µm. (B) Quantification of tyramide-Alexa Fluor 594 incorporation into the fertilization envelope (red) versus hydrogen peroxide production over time (blue) (Wong et al., 2004). Mean accumulation of tyramide fluorescence in the fertilization envelope shown (solid line); squares represent individual data sets per egg (total shown, n=4). Inset: Rate of tyramide-Alexa Fluor 594 incorporation into the fertilization envelope, as a proxy for ovoperoxidase activity (red), versus rate of hydrogen peroxide production by Udx1 (blue) (Wong et al., 2004). Plots show the percentage of final fluorescence intensity (tyramide-Alexa Fluor 594) or the maximal rate of hydrogen peroxide synthesis.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Identification of ovoperoxidase target proteins labeled in vivo. (A) Fertilization envelopes isolated from embryos fertilized in the presence of 5 mM tyramine alone, or with tyramide-Alexa Fluor 594 or tyramide-biotin (1:100 dilution each) were separated by SDS-PAGE. Sixty micrograms of purified fertilization envelopes were used for Coomassie staining, the identification of Alexa Fluor 594 fluorescence, and detection of biotinylated bands. Antisera for rendezvin (RDZ) epitopes or for ovoperoxidase (OVOP) were used to probe 2 µg purified fertilization envelope proteins by immunoblot to identify the major target of ovoperoxidase-dependent dityrosine crosslinking (arrowhead). The major fluorescently labeled protein is boxed. (B) List of unmodified peptide matches to rendezvin120 from mass spectrometry analysis of fluorescently labeled band from the fertilization envelopes (boxed band from A). (C) Fifty micrograms of total zygote lysates fertilized in the presence or absence of 3-AT or the Udx1 inhibitor diphenyleneiodonium (DPI), which abolishes synthesis of the ovoperoxidase substrate hydrogen peroxide (Wong et al., 2004), were separated by SDS-PAGE, and then stained with Coomassie or immunoblotted for different rendezvin (RDZ) epitopes. RDZ anti- is against the amino-terminal fragment whereas RDZ anti- is against the carboxy-terminal portion.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Semi-in vivo crosslinking identifies four major targets of ovoperoxidase. (A) Isolation scheme for in vitro crosslinking assay, based on the ability to inhibit ovoperoxidase-dependent crosslinking using diphenyleneiodonium (DPI). (B) Titration of tyramide-Alexa Fluor 594 incorporation using the in vitro crosslinking assay from A. Fifty micrograms of DPI-isolated SFEs were reacted with various concentrations of tyramide-Alexa Fluor 594 in the presence of 10 mM tyramine (unless noted) and 10 µM hydrogen peroxide. Two-fifths (20 µg) of each reaction was separated by SDS-PAGE, and then visualized for Alexa Fluor 594 fluorescence (red) prior to staining with Coomassie (blue). Images were overlaid in pseudocolor to identify fluorescently labeled proteins (purple). Coomassie staining identifies the major fertilization envelope bands, including proteoliaisin (PLN), SFE1, SFE9, rendezvin isoforms (RDZ60, RDZ90) and ovoperoxidase (OVOP). (C) DPI-SFEs were in vitro crosslinked using 10 µM H2O2, and then separated by SDS-PAGE. Gels were stained for total protein (5 µg, Coomassie) or immunoblotted for individual fertilization envelope components (1 µg). (D) Titration of in vitro fertilization envelope crosslinking of rendezvin. Five micrograms of DPI-SFEs were exposed to serial dilutions of hydrogen peroxide (maximum of 10 µM H2O2) in the presence or absence of the inhibitors 3-AT (1-100 mM) or DPI (10 µM). One-fifth (1 µg) of each reaction was separated by SDS-PAGE and either stained with Coomassie or immunoblotted for rendezvin (RDZ). Individual Coomassie bands of structural fertilization envelope proteins are labeled. Arrow indicates rendezvin120.

View larger version (62K): [in this window] [in a new window]   Fig. 6. Chronology of fertilization envelope formation. Diagram of the time-course of fertilization envelope assembly and modification of the egg cortex, starting from sperm fusion to ovoperoxidase-dependent crosslinking. Milestones (1-6) are detailed in the text. In panel 6, the dashed line denotes non-covalent interaction whereas the solid line denotes dityrosine crosslinking between proteins. CGSP1, cortical granule serine protease 1 (proCGSP1 indicates the zymogenic form); OVOP, ovoperoxidase (ovop indicates the zymogenic form); TG, transglutaminase; Udx1, urchin dual oxidase 1; RDZ, rendezvin; PLN, proteoliaisin; H2O2, hydrogen peroxide; "R-Q=K-R", covalent epsilon (gamma-glutamyl)lysine bonds; "R-Y=Y-R", covalent dityrosine crosslink; 40 kDa, macromolecules of 40,000 daltons.