The PDE3-specific inhibitor milrinone improves asthenozoospermic sperm function by regulating cAMP–PKA signaling pathway and energy metabolism

CONTEXT: Inhibiting phosphodiesterase (PDE) activity to increase cyclic adenosine monophosphate (cAMP) concentrations is a core strategy in artificial sperm activation. PDE3, a key member of the PDE family, comprises two subtypes, PDE3A and PDE3B. However, their specific expression patterns and functions in human sperm remain incompletely understood. AIMS: To analyze the expression characteristics of PDE3A and PDE3B in human sperm and investigate the regulatory effects of PDE3 inhibitor milrinone on asthenozoospermic sperm function and the related mechanisms. METHODS: The expression of PDE3A and PDE3B was detected using reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescence. Changes in asthenozoospermic sperm functions after milrinone treatment were detected using computer-assisted sperm analysis (CASA), methylcellulose viscous penetration assay, and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining. Moreover, intracellular factors closely correlated with sperm function regulation were measured using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and metabolome analysis. KEY RESULTS: PDE3B expression level was greater than was the expression level of PDE3A in human sperm. Sperm function analysis showed that milrinone significantly enhanced asthenozoospermic sperm motility, mucus penetration ability, and the acrosome reaction. With respect to intracellular signals, milrinone activated the cAMP‒protein kinase A (PKA) signaling pathway, and metabolomic analysis showed that milrinone-induced differentially abundant metabolites (DAMs) were associated with the citrate cycle and fatty acid degradation pathways, suggesting that it regulates energy metabolism. CONCLUSIONS: These results demonstrated that milrinone primarily targets PDE3B to improve asthenozoospermic sperm function by regulating cAMP‒PKA signaling pathway and energy metabolism. IMPLICATIONS: The present study has gained new insight into the molecular basis of cAMP signaling regulation in human sperm and provides theoretical and experimental support for the potential application of milrinone for asthenozoospermic sperm activation.