A flow cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure

Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased post-thaw motility in stallion semen [1]. The aim of the study was to determine effects of experimental addition of active MPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen.
Three stallions were used and semen was collected four times. Extended (INRA96TM) semen was processed for density gradient centrifugation (Equipure Bottom Layer®) [2]. Purified pellet was re-extended to 100x106spermatozoa/ml in INRA96TM 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 final concentration of 5 or 50 ng/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) (Sigma-Aldrich). 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 of 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
[1] Ponthier J, Desvals M, Franck T, de la Rebiere de Pouyade G, Spalart M, Palmer E, Serteyn D, Deleuze S. Myeloperoxidase in equine semen: Concentration and Localization during freezing processing. Journal of Equine Veterinary Science 2012;32: 32-37.
[2] Edmond AJ, Teague SR, Brinsko SP, Comerford KL, Waite JA, Mancill SS, Love CC, Varner DD. Effect of density-gradient centrifugation on quality and recovery rate of equine spermatozoa. Animal Reproduction Science 2008;107: 318-318.
[3] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000;21: 895-902.