Abstract
Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased postthawmotility in stallion semen [1]. The aimof the studywas to determine effects of experimental addition of activeMPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Semen was collected four times from three stallions. Extended (INRA96 ) semen was processed for density gradient centrifugation (Equipure Bottom Layer ) [2]. Purified pellet was re-extended to 100 x1 06spermatozoa/ ml in INRA96 and divided in 3 samples. One sample was used for control, and active human MPO (Calbiochem, Merck) was added in the other two samples to a final concentration of 5 or 50ng/ml. After incubation (2 hours, 20 C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (SigmaAldrich). Statistical differences (P < 0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (P < 0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects on in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation. References
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