CSPβ-Hsc70 partnership: Unveiling a chaperone-driven mechanism involved in acrosomal exocytosis in human sperm

Acrosomal exocytosis (AE) is a calcium-dependent membrane fusion event that enables human sperm to penetrate the zona pellucida. Defects in AE contribute to idiopathic male infertility, yet the chaperone mechanisms that regulate this process remain unclear. We sought to define the mechanistic role of Cysteine string protein β (CSPβ) and Heat shock cognate 70 (Hsc70) in AE, focusing on domain-level determinants and functional interactions with relevance to male infertility.

We integrated immunofluorescence and subcellular fractionation in human sperm with functional exocytosis assays, recombinant protein interference, GST pull-down to test binding, hydroxylamine treatment to assess palmitoylation, and in silico docking to map the CSPβ-Hsc70 interface.

CSPβ and Hsc70 localized to the acrosomal region, and both were required for AE, as isoform-specific antibodies inhibited exocytosis in a concentration dependent manner. Non-palmitoylated recombinant CSPβ impaired AE, an effect rescued by exogenous Hsc70. Pull-down assay confirmed CSPβ-Hsc70 interaction, and hydroxylamine assays showed that CSPβ is constitutively palmitoylated and remains so during capacitation. In silico modeling, indicated that the CSPβ J-domain engages the Hsc70 nucleotide-binding domain through the conserved HPD motif independently of palmitoylation, while palmitoylation promotes membrane targeting and spatial recruitment of Hsc70.

CSPβ-Hsc70 constitutes a chaperone-driven mechanism involved in sperm acrosomal exocytosis. Disruption of this partnership compromises a key step in fertilization, positioning CSPβ palmitoylation status and Hsc70 availability as candidate biomarkers and potential therapeutic targets in idiopathic male infertility.