Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa
Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa
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DOI：
10.1095/biolreprod.114.126052
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发表时间：
2015-05-01
影响因子：
3.6
通讯作者：
Aitken, R. John
中科院分区：
文献类型：
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作者：
Aitken, Joanna B.;Naumovski, Nenad;Aitken, R. John
这项研究首次证明马精子中存在 L-氨基酸氧化酶 (LAAO)，它能够产生大量活性氧 (ROS) 并产生氧化应激状态。 RT-PCR 分析表明，该酶的 mRNA 存在于马睾丸和精子中，而免疫细胞化学研究表明，成熟的 LAAO 蛋白位于精子头部，特别是顶体和顶体后区域。实验研究表明，芳香族氨基酸（L-苯丙氨酸 > L-色氨酸 > L-酪氨酸）是该酶的底物，通过细胞死亡增强的机制引发剂量和时间依赖性的 ROS 生成。这一意想不到的结果通过对亚细胞精子组分中 ROS 生成的分析得到了证实，该分析再次将大部分 LAAO 活性定位于精子头部。马冷冻保存培养基含有足够量的芳香族氨基酸，可以激活 LAAO 系统并产生 ROS。这种活性的生物学意义是在一项实验中确立的，其中发现生理浓度的芳香族氨基酸会抑制精子活力，但前提是同一悬浮液中存在死精子。还发现芳香氨基酸和非活细胞的组合可以提高活精子中的脂质过氧化水平。这些结果表明 LAAO 活性在产生与马精子冷冻保存相关的氧化应激方面具有潜在意义。该酶系统的抑制剂可能有助于开发改进的冷冻保存方案，用于体内临床验证。
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 post-acrosomal 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.