Lactate‐Responsive Glycogen Dynamics Are Associated With Bull Sperm Motility and Fertilization Under Glucose‐Limited Conditions

BACKGROUND: Mammalian sperm need to undergo hyperactivation, capacitation, and acrosome reaction to achieve fertilization competence. However, the female reproductive tract presents contrasting metabolic environments. The uterus is glucose-rich, and the oviduct is lactate-rich and glucose-limited. Furthermore, our previous study reported that lactate plays a regulatory role in glucose metabolism through suppressing glucose uptake and glycolysis. How bull sperm maintain glycolysis-associated functions when exogenous glucose is limited remains unclear. OBJECTIVES: To characterize lactate-responsive changes in sperm glycogen content and determine whether CP91149-sensitive processes are associated with glycolysis-related sperm functions and cleavage after in vitro fertilization (IVF) under glucose-limited conditions. MATERIALS AND METHODS: Fresh bull ejaculates were incubated in substrate-free HTF medium to deplete the stored energy. The energy-depleted sperm were treated with lactate, followed by lactate withdrawal and subsequent incubation with glycogen phosphorylase inhibitor (CP91149) in the presence or absence of glucose. Sperm motility, kinematics, acrosome integrity, metabolic flux, glycogen level, and cleavage were evaluated in vitro. RESULTS: Lactate pretreatment prolonged sperm motility under substrate depletion. Lactate withdrawal increased a hyperactivated-like motility subpopulation. Flow cytometry showed that the lactate withdrawal increased membrane permeability of sperm and elevated the percentage of acrosome-reacting sperm. Glycolytic activity of sperm was increased after withdrawal, but the induction was suppressed by the glycogen phosphorylase inhibitor CP91149. Lactate increased sperm glycogen content, which declined after withdrawal. Immunoblotting and immunofluorescence detected proteins associated with gluconeogenesis and glycogen metabolism in bull sperm. CP91149 reduced motility after lactate withdrawal in a dose-dependent manner, and glucose supplementation restored motility. Under glucose-free IVF conditions, CP91149 reduced the post-IVF functional outcome, which was restored by glucose supplementation. DISCUSSION AND CONCLUSION: These findings show lactate-responsive changes in sperm glycogen content and a CP91149-sensitive phenotype affecting glycolytic activity, motility, and the post-IVF functional outcome. The data are consistent with a contribution of endogenous glycogen-derived substrates to sperm function under glucose-limited conditions.