Freeze-thawing induces alterations in the protamine-1/DNA overall structure in boar sperm

The main aim of this work was to test the effects that freeze-thawing could have on the overall nuclear structure of boar sperm. This was done by analyzing both the DNA fragmentation and the protamine-1–DNA interaction of the boar-sperm nucleus. Our results indicate that freezing–thawing did not induce a significant degree of DNA fragmentation, as manifested through both the Sperm–Sus–Halomax® stain and a random primed analysis prior to partial DNA digestion with enzymes BamHI–HinDIII. On the other hand, freeze-thawing induced significant changes in the protamine-1–DNA interaction, as revealed through both Western blot analysis and immunocytochemistry for protamine-1. These alterations caused, in turn, significant changes in the overall nuclear structure of boar sperm after thawing. Protamine-1–DNA alterations started to be apparent during the cooling phase of the freeze-thawing protocol. These results imply that one of the alterations that may be responsible for the loss of fertilizing ability of boar sperm after freeze-thawing may be an alteration in the correct formation of the overall nuclear structure, which, in turn, would induce alterations in the correct formation of the first nuclear structure after oocyte penetration.