Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa

Abstract

BACKGROUNDThere has been an ongoing debate in the reproductive field about whether mammalian spermatozoa rely on glycolysis, oxidative phosphorylation or both for their energy production. Recent studies have proposed that human spermatozoa depend mainly on glucose for motility and fertilization but the mechanism behind an efficient glycolysis in human spermatozoa is not well understood. Here, we demonstrate how human spermatozoa utilize exogenous pyruvate to enhance glycolytic ATP production, motility, hyperactivation and capacitation, events that are crucial for male fertility.METHODSPurified human spermatozoa from healthy donors were incubated under capacitating conditions (including albumin, bicarbonate and glucose) and tested for changes in ATP levels, motility, hyperactivation and tyrosine phosphorylation after treatment with pyruvate. The experiments were repeated in the presence of sodium cyanide in order to assess the contribution from mitochondrial respiration. The metabolism of 13C labeled glucose and pyruvate was traced by a combination of liquid chromatography and mass spectrometry.RESULTSThe treatment of human spermatozoa with exogenous pyruvate increased intracellular ATP levels, progressive motility and hyperactivation by 56, 21 and 130%, respectively. In addition, added pyruvate induced a significant increase in tyrosine phosphorylation levels. Blocking of the electron transport chain did not markedly affect the results, indicating that the mechanism is independent of oxidative phosphorylation. However, the observed effects could be counteracted by oxamate, an inhibitor of lactate dehydrogenase (LDH). Metabolic tracing experiments revealed that the observed rise in ATP concentration resulted from an enhanced glycolytic flux, which was increased by more than 50% in the presence of exogenous pyruvate. Moreover, all consumed 13C labeled pyruvate added was converted to lactate rather than oxidized in the tricarboxylic acid cycle.CONCLUSIONSHuman spermatozoa seem to rely mainly, if not entirely, on glycolysis as the source of ATP fueling the energy-demanding processes of motility and capacitation. The efficient glycolysis is dependent on exogenous pyruvate, which indirectly feeds the accelerated glycolysis with NAD+ through the LDH-mediated conversion of pyruvate to lactate. Pyruvate is present in the human female reproductive tract at concentrations in accordance with our results. As seen in other mammals, the motility and fertility of human spermatozoa seem to be dictated by the available energy substrates present in the conspecific female.

Mitochondria functionality and sperm quality

“…There are, however, several lines of evidence that seem to favour glycolysis as the main ATP source for sperm movement. These include, for example, the need for glucose to maintain sperm function, a need that cannot be replaced with OXPHOS substrates (Peterson & Freund 1970, Williams & Ford 2001, Hereng et al 2011. Furthermore, male mouse knock-out models for the glycolysis-associated enzymes enolase 4 (Nakamura et al 2013), phosphoglycerate kinase 2 Testis-specific cytochrome c knock-out Homozygous males were fertile, but presented testicular atrophy and their sperm were less motile, had lower levels of ATP and had a lower fertilisation ability compared with wild-types Narisawa et al (2002) Mitochondrial DNA polymerase gamma (POLG) knock-in expressing a proofreading-deficient polymerase Increased levels of mtDNA point mutations and deletions, reduced lifespan and premature onset of age-related phenotypes, including reduced fertility Trifunovic et al (2004) Transmitochondrial (carrying mtDNA deletions)…”

Mitochondrial DNA haplogroup associated with sperm motility in the Han population

“…2 The mitochondrial genome encodes 13 oxidative phosphorylation (OXPHOS) subunits and is essential for the production of adenosine triphosphate, 3 which is vital for sperm motility. 4 Male subfertility may be partially due to genetic polymorphisms in the mitochondrial genome. The geographic origin of an indigenous population can significantly impact sequence polymorphisms within their mitochondrial DNA (mtDNA).…”

Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function

“…In human spermatozoa, it may be significant that metabolism is heavily dependent on glycolysis (Hereng et al 2011) and, as a result, these cells are exquisitely sensitive to changes in their redox status. Under these circumstances, it is possible that this enzyme has, in human spermatozoa, acquired a new function as a regulator of sperm capacitation and the acrosome reaction.…”