title: The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa.
abstract: Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h-30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (P<0.05) increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h) of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance.
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Yeste
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
authors.forename: Efrén
authors.initials: E
authors.lastname: Estrada
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
authors.forename: Maria-Montserat
authors.initials: MM
authors.lastname: Rivera Del Álamo
authors.affiliation: Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
authors.forename: Sergi
authors.initials: S
authors.lastname: Bonet
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Rigau
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Barcelona, Spain.
authors.forename: Joan-Enric
authors.initials: JE
authors.lastname: Rodríguez-Gil
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D010766:Phosphorylation; D012662:Semen Preservation; D012694:Serine / Q000378:metabolism; D013048:Specimen Handling / Q000379:methods; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D013552:Swine; D013696:Temperature
chemical_list: D018797:Cell Cycle Proteins; D018840:HSP70 Heat-Shock Proteins; D012694:Serine; D038362:Glycogen Synthase Kinase 3