title: Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm.
abstract: The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/μg protein in fresh samples to 6.7 ± 0.8 nmol/μg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.
authors.affiliation: Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Flores
authors.forename: L
authors.initials: L
authors.lastname: Ramió-Lluch
authors.forename: D
authors.initials: D
authors.lastname: Bucci
authors.forename: J M
authors.initials: JM
authors.lastname: Fernández-Novell
authors.forename: A
authors.initials: A
authors.lastname: Peña
authors.forename: J E
authors.initials: JE
authors.lastname: Rodríguez-Gil
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015925:Cryopreservation / Q000662:veterinary*; D003545:Cysteine; D004247:DNA / Q000378:metabolism*; D004220:Disulfides; D005615:Freezing; D006657:Histones / Q000378:metabolism*; D008297:Male; D011479:Protamines / Q000378:metabolism; D011485:Protein Binding; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D013094:Spermatozoa / Q000502:physiology*; D013552:Swine / Q000502:physiology*
chemical_list: D004220:Disulfides; D006657:Histones; D011479:Protamines; D004247:DNA; D003545:Cysteine