Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa

Joanna Aitken, Nenad NAUMOVSKI, Ben Curry, Christopher Grupen, Zamira Gibb, R John Aitken

Research output: Contribution to journalArticle

25 Citations (Scopus)

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.
Original languageEnglish
Article number125
Pages (from-to)1-13
Number of pages13
JournalBiology of Reproduction
Volume92
Issue number5
DOIs
Publication statusPublished - 2015
Externally publishedYes

Fingerprint

L-Amino Acid Oxidase
Horses
Spermatozoa
Aromatic Amino Acids
Reactive Oxygen Species
Sperm Head
Cryopreservation
Oxidative Stress
Enzymes
Subcellular Fractions
Sperm Motility
Enzyme Inhibitors
Phenylalanine
Tryptophan
Lipid Peroxidation
Tyrosine
Testis
Suspensions
Cell Death
Polymerase Chain Reaction

Cite this

Aitken, Joanna ; NAUMOVSKI, Nenad ; Curry, Ben ; Grupen, Christopher ; Gibb, Zamira ; Aitken, R John. / Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa. In: Biology of Reproduction. 2015 ; Vol. 92, No. 5. pp. 1-13.
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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.",
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Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa. / Aitken, Joanna; NAUMOVSKI, Nenad; Curry, Ben; Grupen, Christopher; Gibb, Zamira; Aitken, R John.

In: Biology of Reproduction, Vol. 92, No. 5, 125, 2015, p. 1-13.

Research output: Contribution to journalArticle

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T1 - Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa

AU - Aitken, Joanna

AU - NAUMOVSKI, Nenad

AU - Curry, Ben

AU - Grupen, Christopher

AU - Gibb, Zamira

AU - Aitken, R John

N1 - © 2015 by the Society for the Study of Reproduction, Inc.

PY - 2015

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AB - 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.

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KW - Spermatozoa/enzymology

KW - Gene Expression Regulation, Enzymologic/physiology

KW - Cryopreservation/veterinary

KW - Animals

KW - Horses/physiology

KW - sperm motility and transport

KW - gamete biology

KW - sperm

KW - cryopreservation

KW - oxidative stress

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