title: Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy.
abstract: This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60  kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.
authors.affiliation: Department of Health Science, Metropolitan Autonomous University, Campus Iztapalapa, 09340 Mexico DF, Mexico.
authors.forename: María Dolores
authors.initials: MD
authors.lastname: Saavedra
authors.forename: Irene
authors.initials: I
authors.lastname: Mondéjar
authors.forename: Pilar
authors.initials: P
authors.lastname: Coy
authors.forename: Miguel
authors.initials: M
authors.lastname: Betancourt
authors.forename: Humberto
authors.initials: H
authors.lastname: González-Márquez
authors.forename: María
authors.initials: M
authors.lastname: Jiménez-Movilla
authors.forename: Manuel
authors.initials: M
authors.lastname: Avilés
authors.forename: Raquel
authors.initials: R
authors.lastname: Romar
mesh_terms: D000818:Animals; D037282:Calreticulin / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism*; D046149:Embryo Culture Techniques; D005089:Exocytosis; D005306:Fertilization; D005307:Fertilization in Vitro / Q000662:veterinary; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013552:Swine / Q000378:metabolism; D014018:Tissue Distribution; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D037282:Calreticulin