title: Identification of phospholipase activity in Rhinella arenarum sperm extract capable of inducing oocyte activation.
abstract: Egg activation, which includes cortical granule exocytosis, resumption and completion of meiosis and pronuclear formation culminates in the first mitotic cleavage. However, the mechanism through which the fertilizing sperm induces this phenomenon is still controversial. We investigated the effect of the microinjection of homologous sperm soluble fractions obtained by fast protein liquid chromatography (FPLC) from reacted sperm (without acrosome) and non-reacted sperm on the activation of Rhinella arenarum oocytes matured in vitro. The FPLC-purified sperm fraction obtained from reacted or non-reacted sperm is able to induce oocyte activation when it is microinjected. This fraction has a 24 kDa protein and showed phospholipase C (PLC) activity in vitro, which was inhibited by D-609 but not by n-butanol or neomycin, suggesting that it is a PLC that is specific for phosphatidylcholine (PC-PLC). The assays conducted using inhibitors of inositol triphosphate (IP3) and ryanodine receptors (RyRs) indicate that the fraction with biological activity would act mainly through the cADPr (cyclic ADP ribose) pathway. Moreover, protein kinase C (PKC) inhibition blocks the activation produced by the same fraction. Immunocytochemical studies indicate that this PC-PLC can be found throughout the sperm head.
authors.affiliation: Departamento de Biología del Desarrollo,Instituto Superior de Investigaciones Biológicas (INSIBIO),Chacabuco 461-2 ° piso,T4000ILI San Miguel de Tucumán,República Argentina.
authors.forename: Federico
authors.initials: F
authors.lastname: Bonilla
authors.affiliation: Departamento de Química Biológica - Instituto Superior de Investigaciones Biológicas-INSIBIO.Universidad Nacional de Tucumán,Chacabuco 461 (4000),Tucumán,Argentina.
authors.forename: Carlos
authors.initials: C
authors.lastname: Minahk
authors.affiliation: Departamento de Biología del Desarrollo-INSIBIO. Facultad de Bioquímica,Química y Farmacia.Universidad Nacional de Tucumán,Chacabuco 461 (4000),Tucumán,Argentina.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Ajmat
authors.affiliation: Departamento de Biología del Desarrollo-INSIBIO. Facultad de Bioquímica,Química y Farmacia.Universidad Nacional de Tucumán,Chacabuco 461 (4000),Tucumán,Argentina.
authors.forename: Graciela Sánchez
authors.initials: GS
authors.lastname: Toranzo
authors.affiliation: Departamento de Biología del Desarrollo-INSIBIO. Facultad de Bioquímica,Química y Farmacia.Universidad Nacional de Tucumán,Chacabuco 461 (4000),Tucumán,Argentina.
authors.forename: Marta Inés
authors.initials: MI
authors.lastname: Bühler
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D000818:Animals; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002022:Bufo arenarum; D002850:Chromatography, Gel / Q000379:methods; D005260:Female; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods; D008297:Male; D008845:Microinjections; D009355:Neomycin / Q000494:pharmacology; D009636:Norbornanes; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013077:Sperm Head / Q000378:metabolism; D013084:Sperm-Ovum Interactions / Q000502:physiology*; D013094:Spermatozoa / Q000737:chemistry* / Q000378:metabolism; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D001952:Bridged-Ring Compounds; D009636:Norbornanes; D010713:Phosphatidylcholines; D013859:Thiocarbamates; D013871:Thiones; C046498:tricyclodecane-9-yl-xanthogenate; D020001:1-Butanol; D011493:Protein Kinase C; D010740:Phospholipases; D010738:Type C Phospholipases; D009355:Neomycin