Biotinylation of proteins on the surface of zona-free mouse oocytes.
In this study, we have labelled proteins on the surface of unfertilized, zona-free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow-bore glass pipette. Surface proteins were labelled using sulfo-NHS-biotin (sulfosuccinimidobiotin), a water-soluble, membrane-impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin-FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting using streptavidin-HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of M(r) 82 and 69 kD, but other major proteins of M(r) 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of M(r) 18-100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos.
Flaherty
S P
SP
Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, South Australia.
Swann
N J
NJ
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D013084:Sperm-Ovum Interactions; D015044:Zona Pellucida / Q000648:ultrastructure
D008565:Membrane Proteins; D001710:Biotin