Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa1

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.

Reactive oxygen species as mediators of sperm capacitation and pathological damage

“…Egg yolk contains sufficient quantities of free aromatic amino acids to activate L‐Amino acid oxidase, so significant ROS generation could ensue in the presence of sufficient numbers of dead spermatozoa in the immediate vicinity of this enzyme. Given the substantial numbers of non‐viable spermatozoa are frequently observed in cryostored samples, sufficient oxidative stress may be created via L‐Amino acid oxidase activity to compromise the functionality of live spermatozoa in the same ejaculate (Aitken, Naumovski, et al, ). Amino acid oxidases are not, however, major physiological drivers of sperm capacitation.…”

Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines

“…While cells are under stress due the presence of snake venoms, the phenomenon of DNA damage is amplified multifold. In the same line of research, Aitken et al38 reported the generation of free oxygen species which leads to the state of oxidative stress by l -amino acid oxidase in equine spermatozoa. In addition, Park et al39 demonstrated that in neuroblastoma cells an increase in ROS due to treatment with snake venoms increases pro-apoptotic proteins including Bax, and there was a downregulation of anti-apoptotic Bcl-2 protein upon treatment with venom.…”

Non-viable sperm in the ejaculate: Lethal escorts for contemporary viable sperm

“…One of the earliest modifications of a destabilized/deteriorating plasma membrane is the switching of the phospholipid phosphatidylserine (PS) from the inner to the outer face of the membrane (Peña et al, 2003;Aitken et al, 2015), a process related to capacitation and/or apoptosis . Sperm capacitation, particularly in pigs, but also in other species, including human, is triggered by high levels of bicarbonate, which renders the plasma membrane more fluid and leads to the exteriorization of PS, particularly in the sperm head domain (Harrison and Gadella, 2005;Rodriguez-Martinez, 2007).…”

“…(Bull: Erickson et al, 2015;ram: Martí et al, 2008;stallion: Gallardo-Bolaños et al 2012;boar: Juarez et al, 2011). The markers can simply be remnants of the apoptosis during spermatogenesis (Shaha et al, 2010) showing which cells had escaped the process, but are still condemned to die post-ejaculation (Aitken and Baker, 2013;Aitken et al, 2015). In contrast to apoptosis, necrosis is an accidental cell death following severe cell stress, normally of external source, that either disrupt the functionality of the male genitalia or that directly harm sperm membranes integrity during or after ejaculation (Guthrie and Welch, 2005).…”

“…A further activation of present caspases might occur; whose increase anticipates further ROS attacks on the plasma membrane and the externalization of the PS. The PS is considered the main apoptosis-status marker for external phagocytes, leading to the concept of "silent death" because it does not cause cell lysis and therefore does not elicit an inflammatory/immune reaction/response during in vivo phagocytosis (Aitken and Baker, 2013;Aitken et al, 2015). Sperm necrosis, on the other hand, could trigger an immune response (Lu et al, 2013) as when sperm granulomas are built up.…”