title: Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction.
abstract: Previously, we reported the involvement of tyrosine phosphorylation in events that lead to ram sperm capacitation. In this study, we carried out a comparative analysis of the localization of tyrosine, serine and threonine phosphoproteins in different functional stages of ram spermatozoa (after the swim-up procedure, in vitro capacitation, and ionophore-induced acrosome reaction) by immunofluorescence, immunocytochemistry and confocal microscopy. Capacitation increased protein tyrosine, serine and threonine phosphorylation whereas the induction of the acrosome reaction resulted in significantly decreased phosphorylation, mainly in those proteins that increased following capacitation. Control samples showed tyrosine-phosphorylated proteins restricted to the head, mainly distributed at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization. In vitro capacitation promoted both tail and acrosome phosphorylation, and the acrosome reaction induced the loss of labeling on the acrosome and the subsequent increase in the post-acrosomal region and flagellum. The preferential localization of serine- and threonine-phosphorylated proteins in the equatorial and acrosomal regions found in control samples changed during capacitation, which induced tail phosphorylation in a sequential manner. After the acrosome reaction, the labeling of both phosphoamino acids decreased in the acrosome and increased in the post-acrosome. The obtained results were proved by two immunodetection techniques and strengthened by confocal microscopy, and indicate that changes in phosphorylated proteins during capacitation and acrosome reaction of ram spermatozoa may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration.
authors.affiliation: Department of Biochemistry and Molecular and Cell Biology Animal Pathology and Anatomy Embryology and Genetics, School of Veterinary Medicine, University of Zaragoza, Zaragoza, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Grasa
authors.forename: Carmen
authors.initials: C
authors.lastname: Colas
authors.forename: Margarita
authors.initials: M
authors.lastname: Gallego
authors.forename: Luís
authors.initials: L
authors.lastname: Monteagudo
authors.forename: Teresa
authors.initials: T
authors.lastname: Muiño-Blanco
authors.forename: José Alvaro
authors.initials: JA
authors.lastname: Cebrián-Pérez
mesh_terms: D020101:Acrosome Reaction; D000818:Animals; D000001:Calcimycin; D061207:Calcium Ionophores; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012756:Sheep / Q000378:metabolism*; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D061207:Calcium Ionophores; D010750:Phosphoproteins; D000001:Calcimycin; D017346:Protein Serine-Threonine Kinases