Sperm meet the elevated energy demands to attain fertilization competence by increasing flux through aldolase

Abstract Prior to ejaculation, sperm are stored in the epididymis in a ‘resting’ metabolic state. Upon ejaculation, sperm must alter their metabolism to generate the energy needed to support the motility and maturation process known as capacitation to reach and fertilize the oocyte. How sperm regulate the capacitation-induced increase in carbon flux is unknown. Here, we use 13C stable isotope labeling to follow glucose metabolism through sperm central carbon metabolic network before and after sperm activation. We identify regulatory steps which sperm use to alter their metabolic state from resting to highly active. In activated sperm, glucose flux through glycolysis is increased at the expense of the pentose phosphate pathway to increase energy yield. Increased glycolytic activity seems to be due to capacitation-induced stimulation of flux through aldolase. In the mitochondria-containing midpiece, glycolytically generated pyruvate feeds the TCA cycle to further maximize energy yield via oxidative phosphorylation. In the mitochondria-free principal piece of the tail, pyruvate produced from glycolysis is reduced to lactate by lactate dehydrogenase. Reduction to lactate regenerates oxidized NAD+ ensuring a sufficient supply to support glycolysis. The resultant lactate is at least partially secreted. Finally, we find evidence that there is an as yet unknown endogenous source of energy in sperm feeding the upregulation of TCA cycle intermediates. These studies provide the most complete picture of the metabolic shift which occurs in capacitating sperm. Significance statement A rapid switch from a quiescent to a high energy-demanding state during ejaculation is essential for sperm to reach and fertilize the oocyte. Somatic cells also undergo bioenergetic switches from low to very high energy demand. However, because metabolic processes essential for proliferation are going on in parallel, it is difficult to identify the molecular mechanisms regulating the increase in ATP production. This study represents the first complete picture of the metabolic reprogramming that happens in sperm upon ejaculation. Using stable isotope labeling, we identify rate-limiting enzymatic steps and points of regulation directing the changes in metabolic flux. Our sperm metabolic studies allow us to identify conserved mechanisms of metabolic regulation that are crucial for the survival of mammalian cells. Competing Interest Statement L.R.L. and J.B. are co-inventors of a panel of in vivo, validated sAC inhibitors and are cofounders of Sacyl Pharmaceuticals Inc., which licensed the sAC inhibitors for development into on-demand male contraceptives. M.B. is a co-inventor of the panel of in vivo, validated sAC inhibitors licensed to Sacyl Pharmaceuticals Inc. for development into on-demand male contraceptives.