title: Hydroxyflutamide alters the characteristics of live boar spermatozoa.
abstract: Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 μg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the "C-Ruch" computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P<0.05) in sperm mitochondrial membrane potential and a concomitant increase (P<0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 μg OH-Flu compared with the respective controls and other doses used (P<0.05). The adverse effects of OH-Flu become strengthened over time (P<0.05). Notably, 50 and 100 μg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P<0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P<0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P<0.05) only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility.
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Zarzycka
authors.affiliation: Department of Cell Biology, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Kotwicka
authors.affiliation: Department of Cell Biology, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Jendraszak
authors.affiliation: Department of Cell Biology, Poznan University of Medical Sciences, Poznan, Poland.
authors.forename: Izabela
authors.initials: I
authors.lastname: Skibinska
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Kotula-Balak
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland. Electronic address: barbara.bilinska@uj.edu.pl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bilinska
mesh_terms: D000818:Animals; D005485:Flutamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology; D013481:Superoxides; D013552:Swine / Q000502:physiology*
chemical_list: D013481:Superoxides; C014290:hydroxyflutamide; D005485:Flutamide
keywords: Boar spermatozoa; Hydroxyflutamide; Intracellular free calcium ion concentration; Mitochondrial membrane potential; Mitochondrial superoxide anion; Phosphatidylserine membrane translocation