Integrated proteomics identifies a dysfunctional acrosomal module associated with impaired acrosome reaction in asthenozoospermia

Background: Asthenozoospermia, a primary cause of male infertility, is characterized not only by reduced sperm motility, but also by functional impairments, particularly defects in the acrosome reaction. However, the molecular mechanisms underlying acrosome reaction dysfunction remain incompletely elucidated. This study aimed to identify key dysregulated proteins in asthenozoospermia and to elucidate their association with acrosome reaction defects using an integrated approach of proteomics, bioinformatics, and functional validation. Methods: Semen samples were collected from normozoospermic men and patients with asthenozoospermia. Differential expression profiling was performed using quantitative proteomics, following an initial assessment of sperm concentration and motility via a computer-assisted sperm analysis (CASA) system. From the pool of differentially expressed proteins, we identified a core set enriched in acrosome reaction-related pathways. The expression of key candidates from this set was confirmed by Western Blot, and a protein-protein interaction (PPI) network analysis was employed to delineate a core functional module. Finally, the calcium ionophore A23187 was utilized to induce the acrosome reaction in vitro, with the spontaneous (sAR) and induced (iAR) acrosome reaction rates evaluated by fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining. Results: Proteomic analysis revealed a significantly downregulated acrosomal protein module in asthenozoospermia, a finding confirmed by Western Blot. PPI network analysis indicated these proteins form a functional interactome. The asthenozoospermic group showed significantly lower sperm motility and concentration. Critically, these sperm displayed a characteristically impaired acrosomal response, with a lower induced reaction rate (iAR) and a spontaneous rate (sAR) that showed an increasing trend without statistical significance, revealing defects in both acrosomal function and stability. Conclusions: This study reveals that the coordinated downregulation of an acrosomal functional module underpins the sAR with an increasing trend without statistical significance, low iAR paradox in asthenozoospermia, thereby providing molecular insights into its pathology and identifying new targets for diagnosis and therapy.

Asthenozoospermia; Acrosome reaction; Proteomics; Protein interaction maps; Infertility; Male

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