PRSS54

PRSS54 is a testis-specific serine protease essential for acrosome biogenesis, sperm head morphogenesis, and zona pellucida penetration during mammalian spermatogenesis. During spermiogenesis, PRSS54 localizes first to the acrosomal granule and subsequently extends along the inner acrosomal membrane; its loss causes acrosomal granule detachment, unfused vacuoles, asymmetrical nuclear elongation, acrosomal instability, and male subfertility, all of which are rescued by transgenic re-expression (PMID:35863763). PRSS54 physically interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and its absence disrupts their spatial distribution without altering their protein levels, indicating a scaffolding or organizational role within the acrosome (PMID:35863763). PRSS54 undergoes proteolytic maturation from ~50 kDa in testis to ~42 kDa in mature sperm, a process that depends in part on the epididymal protein SPEM2 (PMID:35863763, PMID:38421455).

1. 2020 Medium

   An initial CRISPR knockout asked whether PRSS54 is individually required for male fertility: single-gene ablation revealed no detectable fertility or morphological defect, suggesting functional redundancy among testicular serine proteases.

   Evidence CRISPR/Cas9 knockout mice with histology, sperm morphology, and fertility assays

   PMID:31403672

   Open questions at the time

   • Discrepancy with later knockout findings unresolved — may reflect strain background or assay sensitivity differences
   • No ultrastructural analysis was performed
   • Redundancy partners not identified
2. 2022 High

   A comprehensive knockout study resolved PRSS54's cellular function, showing it is required for acrosomal granule positioning, membrane fusion during acrosome biogenesis, nuclear shaping, and sperm-zona penetration — establishing PRSS54 as a key acrosomal organizer rather than a redundant protease.

   Evidence CRISPR/Cas9 knockout with transgenic rescue, TEM ultrastructure, immunofluorescence localization, fertility and acrosome reaction assays in mice

   PMID:35863763

   Open questions at the time

   • Catalytic activity of PRSS54 not demonstrated — whether protease activity or scaffolding function underlies the phenotype is unknown
   • The basis for discrepancy with the 2020 no-phenotype knockout remains unexplained
   • Direct substrates of PRSS54 protease activity not identified
3. 2022 Medium

   Identification of ZPBP1, ZPBP2, ACRBP, and ZP3R as physical interactors whose acrosomal distribution depends on PRSS54 established the molecular network through which PRSS54 organizes the acrosome.

   Evidence Co-immunoprecipitation and immunofluorescence redistribution analysis in Prss54−/− versus control testes and sperm

   PMID:35863763

   Open questions at the time

   • Interactions demonstrated by single Co-IP without reciprocal pulldown validation
   • Whether interactions are direct or bridged by other acrosomal components is unknown
   • Functional consequence of each individual interaction not dissected
4. 2024 Low

   PRSS54 was identified as a SPEM2-interacting protein whose epididymal processing depends on SPEM2, linking PRSS54 maturation to an epididymal quality-control pathway.

   Evidence Co-immunoprecipitation of SPEM2 interactome and Western blot of Spem2−/− epididymal sperm

   PMID:38421455

   Open questions at the time

   • PRSS54 was a secondary finding in a SPEM2-focused study; awaits dedicated validation
   • Whether impaired PRSS54 processing in Spem2−/− sperm contributes to fertility defects is untested
   • Nature of SPEM2-dependent cleavage event not characterized

• It remains unknown whether PRSS54's biological role depends on its serine protease catalytic activity or on a non-enzymatic scaffolding function, and no direct proteolytic substrates have been identified.
• No catalytic-dead mutant rescue experiment performed
• No in vitro protease activity assay reported
• Human relevance for male infertility not established