Pyruvate enhances stallion sperm function increasing glycolysis efficiency through NAD+

Pyruvate entry into the mitochondrial matrix is a critical step for the efficient generation of reducing equivalents and ATP (Yiew and Finck, 2022). Improvements in sperm function using metabolites readily metabolizable through the tricarboxylic acid cycle (TCA), have been attributed to their ability to readily feed the electron transport chain (ETC) in the mitochondria; 8 NADH and 2 FADH2 are produced in the TCA cycle. However, noncanonical mechanisms may explain improvements in sperm functions. For example, the direct use of lactate in the TCA (Martinez-Reyes and Chandel, 2017) or a more efficient glycolysis (Hereng et al., 2011); in both mechanisms lactate dehydrogenase (LDH) plays a major role. If pyruvate improves the efficiency of glycolysis, the detrimental effects of higher glucose concentrations could be counteracted by increasing pyruvate concentration in the media. To test this hypothesis stallion spermatozoa were incubated in presence of high glucose concentration (67mM) in presence of 1- or 10-mM pyruvate. To investigate the role of LDH, we used the specific inhibitor oxamate. If our hypothesis is correct improved functionality will be observed in high pyruvate media. Flow cytometry, CASA, and metabolomics were used to disclose the potential mechanisms behind this. Stallion spermatozoa (n=4, ANOVA and Dunnett´s test) incubated for 3 h at 37°C in 67mM Tyrode´s media supplemented with 10 mM pyruvate showed increased total motility in comparison with aliquots incubated in Tyrode's 5mM glucose and Tyrode´s67mM glucose, from 57.1 ± 3.5 and 58.1 ± 1.9 to 73.0± 1.1 % (P<0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose and 10mM pyruvate maintained viability along the incubation period (64.03 ± 15.4% vs 61.3 ± 10.2%), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P<0.01). The presence of 40 mM oxamate (LDH inhibitor) reduced sperm viability (P<0.05, from 76± 5 % in the media containing 67 mM Glucose and 10 mM pyruvate to 68.0 ± 4.3 % in presence of oxamate, P<0.05). Apoptotic markers also increased when the LDH inhibitor oxamate was present (P<0.01). The incorporation of glucose was reduced in the presence of 10mM pyruvate, suggesting that the stallion spermatozoa have mechanisms to regulate the incorporation of this hexose. Furthermore, UHPLC/MS/MS showed that in presence of 10 mM pyruvate, the relative amounts of lactate, ATP, NAD+, and NADH increased. Our findings indicate that the mechanisms that explain the improvement of sperm function in presence of 10 mM pyruvate may involve the conversion of pyruvate to lactate producing NAD+ that improves the efficiency of glycolysis. Acknowledgements: Ministerio de Ciencia-European Fund for Regional Development (EFRD) Spain, grants PID2021-122351OB-I00 and PRE2018-083354 Junta de Extremadura-EFDR (IB 20008).