Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors

Olga Blanco-Prieto; Beatrice Mislei; Felipe Martínez-Pastor; Marcella Spinaci; Gaetano Mari; Diego Bucci

doi:10.1016/j.theriogenology.2023.05.021

Study of mitochondrial function in thawed bull spermatozoa using selective electron transfer chain inhibitors

Theriogenology. 2023 Sep 15:208:8-14. doi: 10.1016/j.theriogenology.2023.05.021. Epub 2023 Jun 1.

Authors

Olga Blanco-Prieto¹, Beatrice Mislei², Felipe Martínez-Pastor³, Marcella Spinaci¹, Gaetano Mari⁴, Diego Bucci⁵

Affiliations

¹ Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell'Emilia, BO, Italy.
² INFA-AUB, University of Bologna, Via Gandolfi 16, Cadriano, BO, Italy.
³ INDEGSAL and Molecular Biology (Cell Biology), Universidad de León, Campus de Vegazana, 24071, León, Spain.
⁴ Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell'Emilia, BO, Italy; INFA-AUB, University of Bologna, Via Gandolfi 16, Cadriano, BO, Italy.
⁵ Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell'Emilia, BO, Italy. Electronic address: diego.bucci3@unibo.it.

PMID: 37290146
DOI: 10.1016/j.theriogenology.2023.05.021

Abstract

Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 10⁶ mL^-1 in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 μM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 μM (CCCP), uncoupling agent; antimycin A 1 μg/mL (ANTI), complex III inhibitor; oligomycin 5 μM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O₂^•- production and H₂O₂ intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.

Keywords: Sperm metabolism.

MeSH terms

Animals
Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
Cattle
Cryopreservation / veterinary
Electrons
Hydrogen Peroxide* / pharmacology
Male
Mitochondria
Semen
Semen Preservation* / methods
Semen Preservation* / veterinary
Sperm Motility
Spermatozoa

Substances

Carbonyl Cyanide m-Chlorophenyl Hydrazone
Hydrogen Peroxide
SYBR-14