Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs.
To analyze sperm surface molecules involved in sperm-egg envelope binding in Xenopus laevis, heat-solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP-ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP-ML. It was found that SP-ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65-130 and 20-30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose-dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization.
Kubo
Hideo
H
Department of Medical Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan. kubo-hd@igakuken.or.jp
Shiga
Keiko
K
Harada
Yuichirou
Y
Iwao
Yasuhiro
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000917:Antibody Formation; D000918:Antibody Specificity; D005260:Female; D006825:Hybridomas / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D010063:Ovum / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013094:Spermatozoa / Q000737:chemistry / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014817:Vitelline Membrane / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism / Q000502:physiology
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins