Shilling FM et al. (1998), Voltage-dependent activation of frog eggs by a ...

XB-ART-14175

Dev Biol 1998 Oct 01;2021:113-24. doi: 10.1006/dbio.1998.8982.

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Voltage-dependent activation of frog eggs by a sperm surface disintegrin peptide.

Shilling FM , Magie CR , Nuccitelli R .

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Fertilin, a sperm protein of the metalloprotease/disintegrin/cysteine-rich (MDC) family, plays a critical role in sperm-egg binding in mammals. Peptides corresponding to the disintegrin domain of fertilin and antibodies against fertilin have been shown to inhibit mammalian sperm-egg binding and fusion. A protein from the same family, xMDC16, was recently cloned from frog (Xenopus laevis) testis and was found to be involved in frog sperm-egg binding. Here we report that xMDC16 is localized predominantly on the posterior surface of egg jelly-activated sperm, and peptides from the disintegrin domain of this protein activate eggs when applied near the egg surface. Egg activation was dependent on (1) specific amino acid residues (KTX); (2) the presence of divalent cations, but not external Ca2+ alone; and (3) voltage across the egg plasma membrane. This is the first demonstration of egg activation in vertebrates by the surface application of a peptide derived from a sperm surface protein, supporting a model for egg activation that involves a signal transducing receptor for sperm in the egg's plasma membrane.

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Species referenced: Xenopus laevis
Genes referenced: adam21 adam28.2 gopc mbp slc5a5

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	FIG. 1.Labeling of liveXenopus laevissperm with a rabbitantibody generated against a fusion protein containing the disinte-grin and cysteine-rich portions of xMDC16 (secondary antibodycoupled to FITC). (a) A differential interference contrast image of asingle sperm showing the head region (10mm long); the posteriorend of the head is indicated, as is the tail. (b) Live sperm werelabeled with immune serum following activation by egg jelly–water. The label was found predominantly on the posterior regionof the helical sperm head. (c) Sperm without egg jelly treatment,exposed to the xMDC16 antiserum, showed no labeling. (d) Spermlabeling was eliminated by previous incubation of the antiserumwith a fusion protein containing the disintegrin and cysteine-richdomains of xMDC16. (e) Preincubation of the antiserum with MBPalone had no effect on antibody labeling of the sperm. Scale bar(10mm) applies to all confocal fluorescent images.115Egg Activation by a Sperm Surface Disintegrin PeptideCopyright © 1998 by Academic Press. All rights of reproduction in any form reserved.
	FIG. 2.Quantitation of the immunofluorescence labeling accom-plished by measuring the summed labeling intensity within thelabeled sperm as the amount of competing fusion protein wasvaried. The error bars are equal to the 95% confidence intervalsaround the mean;nis indicated in parentheses above each bar.Sperm from five males were used. The asterisk refers to a statisti-cally significant difference from the immune serum value (P,0.05, analysis of variance). These data suggest that the antibodies toxMDC16 recognize a protein on the surface of live, egg jelly-activated sperm and support the specificity of labeling by the rabbitimmune serum since the label intensity on the sperm is reducedwhen the serum is preexposed to the indicated amount of xMDC16fusion protein, but not with exposure to MBP alone.
	FIG. 3.Western blot ofXenopus laevissperm proteins separatedby PAGE and blotted onto nitrocellulose. Sperm protein was boiledin reducing sample buffer for 10 min (0.1 M DTT). The numbersnext to blot indicate the molecular weights in kDa of standards runon the blot. (A) Blot was treated with immune and preimmune serafrom rabbits injected with a fusion protein containing the cysteine-rich and disintegrin domains of xMDC16 (the rabbit serum was akind gift from Dr. Carl Blobel). Arrow indicates a band around 56kDa that labels in the immune but not preimmune blot. (B) Whenthe immune serum is pretreated with the xMDC16 fusion protein(1 mg/ml) and applied to the blot, the 56-kDa band no longer labels,compared to pretreatment with the same amount of maltose-binding protein (MBP) alone. This result was observed in fourseparate Western blots.117Egg Activation by a Sperm Surface Disintegrin PeptideCopyright © 1998 by Academic Press. All rights of reproduction in any form reserved.
	FIG. 4.Peptide-induced activation ofX. laeviseggs. (a) Activationof dejellied eggs by increasing concentrations of xMDC16 peptide,with KTE or AAA sequences. The eggs were placed into wellscontaining the indicated concentration of each peptide. The verti-cal bars indicate the 95% confidence intervals. (b) Activation ofvitelline envelope-intact (1VE) and vitelline envelope-free eggs(2VE) after local application of peptide containing the KTE se-quence or the AAA and ATA substitution. Replacement of the KTEregion by either AAA or ATA resulted in loss of the ability toactivate eggs. The number of eggs treated is indicated beside eachpoint.119Egg Activation by a Sperm Surface Disintegrin PeptideCopyright © 1998 by Academic Press. All rights of reproduction in any form reserved.
	FIG. 5.Peptide-induced changes in [Ca21]iin theXenopusegg. (a) Peptide-induced changes in [Ca21]iin a dejellied, albino egg injected withfura-2– dextran (50 –100mM final concentration) in Ca21-free F1 medium detected by ratio imaging. Response to soluble xMDC16 peptide(20 nmol) added from direction indicated (arrowhead). The fluorescence emitted was calibrated as described previously (Nuccitelliet al.,1993) and converted into a pseudocolor representation of the calcium concentration. Egg diameter, 1.2 mm. (b) Comparison of intracellularcalcium levels in dejellied albino eggs treated with xMDC16 (upper trace, no external Ca21) or sperm (lower trace, normal Ca21). Theinterval between data points is 10 s, and the time of sperm or peptide addition is indicated. Intracellular free calcium in eggs is elevatedin response to xMDC16 peptide in a manner identical to that at fertilization.
	FIG. 6.Two-electrode voltage clamp study of peptide-induced egg activation. Activation current across the plasma membrane in eggs clampedat220 mV. The traces were copied from a chart recording and correspond to the current passed to keep the voltage constant. The times ofaddition of peptide or sperm are indicated with an arrow. (Upper trace) Egg clamped at220 mV and treated with 20 nmol of xMDC16 (1mlof20 mM stock). (Lower trace) An egg clamped at220 mV and treated with sperm. Asterisk (*) indicates initial rapid inward current.
	FIG. 7.Percentage of egg activation by xMDC16 disintegrinpeptide in eggs voltage clamped at different membrane potentials.The number of eggs per data point is indicated. Activation wasscored by the elicitation of an activation current, vitelline envelopeelevation, and cortical contraction. Peptide-induced egg activationincludes an inward activation current similar to that observed atfertilization and the ability of the peptide to activate is dependenton the voltage across the egg’s plasma membrane