143 Stimulated glycolysis is able to maintain ATP levels and motility of bull spermatozoa submitted to mitochondrial depolarisation

Abstract

Studies have reported the importance of mitochondria in sperm metabolism. However, for some species, glycolysis appears to be as essential as oxidative phosphorylation in sperm physiology. On the other hand, these mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and glycolysis in ATP synthesis and sperm kinetics of bovine spermatozoa. For this purpose, sperm from seven bovine epididymides (n = 7) was collected and diluted to a concentration of 100 × 10⁶ spermatozoa mL⁻¹ in Tyrode's albumin lactate pyruvate medium. Then, each sample was divided into 10 aliquots and evaluated in a 2 × 5 factorial design, with the first factor being the presence or absence of glucose (5 mM) to stimulate glycolysis and the second factor being treatment with the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 0, 0.1, 0.3, 1, and 3 µM) to deplete mitochondrial ATP. Sperm samples were subjected to measurements of ATP levels using a luminescence technique (CellTiter-Glokit, Promega), with ATP levels measured in duplicate. In addition, sperm samples were subjected to computerised analysis of total and progressive motilities (ISAS, Proiser). Statistical analysis was performed using SAS (SAS Institute Inc.), where the interaction between the factors was analysed using PROC GLM and the comparison between groups was performed using means analysis of variance (least significant difference test). It was considered significant at 5%. Adenosine triphosphate was lower at FCCP concentrations of 0.3 µM (180.3 ± 31.9 nM), 1 µM (220.2 ± 40.4 nM), and 3 µM (272.3 ± 70.4 nM) than at 0 µM (control; 448.6 ± 63.7 nM) and 0.1 µM (422.4 ± 41.5 nM) in the absence of glucose. However, in the groups treated with FCCP supplemented with glucose, ATP concentrations did not differ among the groups (0 µM: 577.2 ± 70.4 nM; 0.1 µM: 610.8 ± 57.8 nM; 0.3 µM: 606.2 ± 64.2 nM; 1 µM: 670.9 ± 61.9 nM; 3 µM: 696.1 ± 68.5 nM). Additionally, total motility was lower in FCCP-treated groups without glucose supplementation. On the other hand, total motility increased in the groups treated with 0.3, 0.1, 1, and 3 µM FCCP supplemented with glucose. A similar effect was verified for progressive motility. Based on these results, we can suggest that glucose supplementation is able to maintain ATP levels and motility in bull sperm undergoing FCCP-induced mitochondrial depolarisation.