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Characterization of an L-amino acid oxidase in equine spermatozoa.
Abstract: This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptophan > L-tyrosine) were substrates for this enzyme, eliciting the dose- and time-dependent generation of ROS via mechanisms that were enhanced by cell death. This unexpected result was confirmed by analyses of ROS generation in subcellular sperm fractions, which again located a majority of LAAO activity to the sperm head. Equine cryopreservation medium was shown to contain sufficient quantities of aromatic amino acids to activate the LAAO system and generate ROS. The biological significance of this activity was established in an experiment in which physiological concentrations of aromatic amino acids were found to suppress sperm motility but only if dead spermatozoa were present in the same suspension. The combination of aromatic amino acids and nonviable cells was also found to enhance the levels of lipid peroxidation in live spermatozoa. These results suggest the potential significance of LAAO activity in generating the oxidative stress associated with the cryopreservation of equine spermatozoa. It is possible that inhibitors of this enzyme system may facilitate the development of modified cryostorage regimes for clinical validation in vivo.
© 2015 by the Society for the Study of Reproduction, Inc.
Publication Date: 2015-03-04 PubMed ID: 25740544DOI: 10.1095/biolreprod.114.126052Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research paper demonstrates for the first time that an enzyme called L-amino acid oxidase (LAAO) is present in horse sperm cells, where it influences the production of reactive oxygen species, causing oxidative stress. This enzyme could be significant in the cryopreservation process of horse sperm cells, meaning those involved in this process need to be aware of its existence and potentially modify their storage protocols.
Existence of L-amino acid oxidase in equine spermatozoa
- The study was designed to investigate the existence of L-amino acid oxidase (LAAO) in equine or horse sperm and its impact on those cells. This is the first time such a study was conducted.
- The researchers detected the LAAO enzyme’s mRNA, the molecule that carries codes from the DNA, in the horse’s testis and in sperm cells, revealing its existence for the first time.
- Additionally, the mature LAAO protein was found to be located in the head of the sperm cells, primarily in the acrosomal and postacrosomal domains. This is important because it gives a physical location for the enzyme in the sperms.
LAAO’s role and impact
- LAAO was found to generate reactive oxygen species (ROS), which can cause a state of oxidative stress – a situation in which cells are exposed to harmful oxygen molecules which can damage them. Oxidative stress is often associated with the cellular damage process that occurs during cryopreservation.
- LAAO’s activity is stimulated in the presence of aromatic amino acids (specifically, L-phenylalanine, L-tryptophan, and L-tyrosine), resulting in ROS generation that increases in a dose- and time-dependent way.
- LAAO activity is enhanced during cell death, which is an unexpected result.
LAAO in the context of sperm cryopreservation
- The study also showed that the traditional equine cryopreservation medium contains enough amino acids to trigger the LAAO system and generate ROS.
- Physiological concentrations of aromatic amino acids were found to suppress sperm cell movement but only in the presence of dead sperm cells in the same suspension.
- The combination of aromatic amino acids and nonviable cells increased lipid peroxidation levels in live sperm cells. Lipid peroxidation is a process where free radicals ‘steal’ electrons from the lipids in cell membranes, causing cell damage.
- This suggests that LAAO activity might contribute to oxidative stress associated with the cryopreservation of horse sperm cells.
Future implications and applications
- Based on the findings, inhibitors of LAAO enzymes might be used in future to improve cryopreservation techniques.
- These inhibitors may aid in developing modified cryostorage protocols for clinical validation, with the aim of improving the viability and quality of cryopreserved sperm.
Cite This Article
APA
Aitken JB, Naumovski N, Curry B, Grupen CG, Gibb Z, Aitken RJ.
(2015).
Characterization of an L-amino acid oxidase in equine spermatozoa.
Biol Reprod, 92(5), 125.
https://doi.org/10.1095/biolreprod.114.126052 Publication
Researcher Affiliations
- Faculty of Veterinary Science, University of Sydney, Camperdown, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
- Faculty of Veterinary Science, University of Sydney, Camperdown, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia john.aitken@newcastle.edu.au.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- Gene Expression Regulation, Enzymologic / physiology
- Horses / physiology
- Hydrogen Peroxide
- L-Amino Acid Oxidase / genetics
- L-Amino Acid Oxidase / metabolism
- Male
- Spermatozoa / enzymology
Citations
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