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

Flores, E.; Cifuentes, D.; Fernández-Novell, J.M.; Medrano, A.; Bonet, S.; Briz, M.D.; Pinart, E.; Peña, A.; Rigau, T.; Rodríguez-Gil, J.E.

doi:10.1016/j.theriogenology.2008.01.022

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Volume 69, Issue 9 , Pages 1083-1094, June 2008

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

E. Flores
- Unit of Animal Reproduction, Department Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain
D. Cifuentes
- Department Biochemistry and Molecular Biology and IRRB, Barcelona Science Park, University of Barcelona, Barcelona, Spain
J.M. Fernández-Novell
- Department Biochemistry and Molecular Biology and IRRB, Barcelona Science Park, University of Barcelona, Barcelona, Spain
A. Medrano
- Unit of Animal Reproduction, Department Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain
S. Bonet
- Unit of Biotechnology of Porcine Reproductión, University of Girona, Girona, Spain
M.D. Briz
- Unit of Biotechnology of Porcine Reproductión, University of Girona, Girona, Spain
E. Pinart
- Unit of Biotechnology of Porcine Reproductión, University of Girona, Girona, Spain
A. Peña
- Unit of Animal Reproduction, Department Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain
T. Rigau
- Unit of Animal Reproduction, Department Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain
J.E. Rodríguez-Gil
- Unit of Animal Reproduction, Department Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain
- Corresponding author. Tel.: +34 935811045; fax: +34 935812006.

Received 1 October 2007; received in revised form 28 November 2007; accepted 23 January 2008. published online 25 March 2008.

Abstract

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.

Keywords: Boar sperm, Freezing–thawing, Protamine-1, DNA fragmentation