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Hydroxyflutamide alters the characteristics of live boar spermatozoa
Marta Zarzycka1, Malgorzata Kotwicka2, Magdalena Jendraszak2
1Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
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.
Insights
Hydroxyflutamide (OH-Flu) exposure in vitro negatively impacts boar sperm function, reducing mitochondrial potential and motility while increasing superoxide production and calcium levels. These adverse effects on sperm viability and function worsen over time.
Area of Science:
- Reproductive Biology
- Spermatozoa Physiology
- Environmental Toxicology
Background:
- Previous studies indicated hydroxyflutamide (OH-Flu) alters boar sperm plasma membrane integrity and mitochondrial function.
- Further investigation is needed to understand the direct cellular and molecular effects of OH-Flu on spermatozoa.
Purpose of the Study:
- To investigate the direct effects of OH-Flu on boar sperm mitochondrial membrane potential, superoxide anion production, and phosphatidylserine translocation (PST).
- To analyze OH-Flu's impact on intracellular calcium ion dynamics and sperm motility.
- To elucidate the dose- and time-dependent cellular responses of spermatozoa to OH-Flu exposure.
Main Methods:
- Boar spermatozoa were incubated with varying concentrations of OH-Flu (5, 50, 100 μg/mL) for 2 and 24 hours.
- Mitochondrial membrane potential and superoxide production were assessed using JC-1 dye and MitoSOX Red.
- Phosphatidylserine membrane translocation (PST) was measured via annexin-V binding.
- Intracellular calcium dynamics were evaluated using Fluo-3.
- Sperm motility and velocity were analyzed using computer-assisted systems and flow cytometry/confocal microscopy.
Main Results:
- A significant decrease in mitochondrial membrane potential and a rise in mitochondrial superoxide anion production were observed after 2 hours of exposure to 50 μg/mL OH-Flu.
- Adverse effects intensified over time, with increased PST observed after 24 hours.
- OH-Flu (50 and 100 μg/mL) reduced sperm motility, fast subpopulation percentage, and straight-line velocity.
- All concentrations of OH-Flu rapidly increased intracellular calcium ion concentration.
Conclusions:
- Hydroxyflutamide directly impairs boar sperm mitochondrial function, induces oxidative stress, and disrupts membrane integrity.
- OH-Flu negatively affects sperm motility and intracellular calcium homeostasis.
- These findings highlight the detrimental in vitro effects of OH-Flu on spermatozoa, impacting their functional characteristics.