{"gene":"ACR","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1989,"finding":"The primary structure of human proacrosin was deduced from cDNA cloning: the 421-amino-acid preproenzyme contains a 19-residue signal peptide, a light chain segment, and a heavy chain with a catalytic site. The active-site residues histidine (69), aspartic acid (123), and serine (221) were identified, and a proline-rich C-terminal domain unique to acrosin was noted.","method":"cDNA cloning and sequencing from human testis library; deduced amino acid sequence analysis","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 — primary structure determination by cDNA sequencing with active-site residue identification","pmids":["2493394"],"is_preprint":false},{"year":1989,"finding":"The chromosomal gene encoding human preproacrosin (ACR) was mapped to chromosome 22q13-qter using somatic cell hybrid analysis; a restriction fragment length polymorphism with SstI was identified in the gene.","method":"Somatic cell hybrid analysis with human-rodent hybrids containing variant chromosome 22 complements; Southern blotting","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — chromosomal mapping by somatic cell hybrids with translocation chromosomes","pmids":["2606479"],"is_preprint":false},{"year":1990,"finding":"The human proacrosin gene contains four introns and five exons; the three active-site residues (His, Asp, Ser) are each encoded by separate exons (E2, E3, E5), and the proline-rich domain is co-encoded with the serine active-site residue in exon E5. The gene lacks canonical TATA and CAAT boxes near the transcription start site.","method":"Genomic library screening, restriction mapping, sequencing, primer extension analysis","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 — complete gene structure determination with functional annotation of active-site exons","pmids":["2114285"],"is_preprint":false},{"year":1990,"finding":"Human preproacrosin cDNA cloning confirmed 70% amino acid sequence identity with boar proacrosin; the leader sequence, catalytic triad (His, Asp, Ser), disulfide-bonding cysteine positions, and C-terminal proline-rich sequence are conserved between species, while the proline-rich region is species-specific.","method":"cDNA library screening, nucleotide sequencing, comparative amino acid sequence analysis","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 1 — cDNA cloning with cross-species structural comparison","pmids":["2298447"],"is_preprint":false},{"year":1991,"finding":"The rat proacrosin gene (ACR) was mapped to chromosome 7 by analysis of mouse × rat somatic cell hybrids, establishing synteny with the human chromosome 22q13-qter locus.","method":"Somatic cell hybrid panel analysis with rat chromosome segregation","journal":"Cytogenetics and cell genetics","confidence":"High","confidence_rationale":"Tier 2 — chromosomal mapping by somatic cell hybrids, consistent with human locus","pmids":["1906796"],"is_preprint":false},{"year":1991,"finding":"The mouse proacrosin gene comprises six exons and five introns (one intron in the 5'-UTR); the gene is located on mouse chromosome 15 bands E/F, and unusually is expressed diploid (in pre-meiotic spermatocytes) in the mouse, contrasting with haploid expression in bull, boar, and rat. Active-site residues His, Asp, and Ser are encoded by exons E2, E3, and E5 respectively.","method":"Cosmid library screening, gene mapping, primer extension, in situ hybridization, expression analysis during spermatogenesis","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 1–2 — complete mouse gene structure with chromosomal localization and expression timing determination","pmids":["1783391"],"is_preprint":false},{"year":1991,"finding":"Acrosin is a sperm-specific serine protease stored as proacrosin in the acrosome; the acrosin molecule contains a novel proline-rich C-terminal domain (absent from other serine proteases) that may confer DNA-binding properties, in addition to the standard serine protease catalytic triad.","method":"Biochemical characterization and primary structure analysis across species","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 1 — structural analysis identifying a novel domain in acrosin not present in other serine proteases","pmids":["1937464"],"is_preprint":false},{"year":1982,"finding":"Divalent metal ions (Zn²⁺, Cu²⁺, Hg²⁺, Co²⁺, Cd²⁺) potently inhibit surface-bound acrosin activity on human and bovine spermatozoa; removal of zinc or copper from the inhibited enzyme completely restores activity, suggesting that zinc in genital tract fluids physiologically regulates acrosin activity.","method":"Enzymatic activity assay (BANA substrate) on washed spermatozoa; metal chelation and restoration experiments","journal":"International journal of andrology","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro enzymatic assay with reversibility demonstrated; single laboratory","pmids":["6815104"],"is_preprint":false},{"year":1982,"finding":"Bovine acrosin (MW 39 kDa; specific activity 93 U/mg) and human acrosin (MW 49 kDa) were purified to homogeneity using HCl extraction, gel filtration, and affinity chromatography, providing the first detailed purification schemes for both species.","method":"Protein purification: saline/Triton X-100 washes, HCl extraction, gel filtration, ion-exchange and affinity chromatography; activity assay with BAEE","journal":"Canadian journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 — biochemical purification to homogeneity with molecular weight determination","pmids":["6802470"],"is_preprint":false},{"year":1983,"finding":"Immunofluorescence during human spermatogenesis showed that proacrosin and the outer acrosomal membrane first appear in haploid spermatids and increase through spermiogenesis; round-headed (globozoospermic) spermatozoa completely lack proacrosin, acrosin, and the outer acrosomal membrane, demonstrating that acrosin is an obligate acrosomal component.","method":"Indirect immunofluorescence with anti-acrosin and anti-outer acrosomal membrane antibodies; gelatin substrate film technique for gelatinolytic activity","journal":"Human genetics","confidence":"High","confidence_rationale":"Tier 2 — direct localization during spermatogenesis with functional correlation to acrosome presence","pmids":["6357995"],"is_preprint":false},{"year":1985,"finding":"Purified bovine sperm acrosin activates platelet adenylate cyclase 5–8-fold, comparable in potency to trypsin and α-chymotrypsin; this activation is blocked by serine protease inhibitors (leupeptin, SBTI, α2-macroglobulin), identifying acrosin as a trypsin-like protease capable of activating adenylate cyclase via proteolytic activity.","method":"Adenylate cyclase activity assay with purified acrosin; protease inhibitor panel; enzyme extraction from bovine sperm","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro enzymatic assay with purified acrosin; single laboratory","pmids":["3880736"],"is_preprint":false},{"year":1989,"finding":"Proteinase inhibitors of proacrosin activation and acrosin activity (AGB and para-aminobenzamidine) significantly inhibit the dbcAMP-induced human sperm acrosome reaction at concentrations of 10⁻⁶–10⁻⁴ M, demonstrating that acrosin (a serine proteinase) plays a functional role in the human sperm acrosome reaction downstream of the adenylate cyclase system.","method":"Human synchronous acrosome reaction assay with dbcAMP stimulation; acrosome reaction scoring with proteinase inhibitors added at onset or after capacitation","journal":"Gamete research","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological inhibition with dose-response in human sperm; single laboratory","pmids":["2550339"],"is_preprint":false},{"year":1994,"finding":"Protein C inhibitor (PCI) directly inhibits acrosin: purified human PCI inhibited boar acrosin amidolytic activity with an apparent second-order rate constant of 3.7 × 10⁴ M⁻¹·s⁻¹, forming SDS-stable complexes with acrosin. PCI was immunolocalized exclusively near disrupted acrosomal membranes of morphologically abnormal spermatozoa, suggesting PCI scavenges prematurely activated acrosin.","method":"In vitro enzymatic inhibition assay; ELISA for complex formation; immunocytochemistry and immunoelectron microscopy","journal":"The American journal of physiology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinetic inhibition combined with structural localization; orthogonal methods","pmids":["7521127"],"is_preprint":false},{"year":1999,"finding":"Sulphated polymers (fucoidan, solubilised zona pellucida glycoproteins [ZPGs], and ARIS) bind to a domain on proacrosin distinct from the active site, induce proacrosin activation, and inhibit sperm binding to the zona pellucida; heparin did not share this activity. ARIS induced human proacrosin activation, indicating that ZP glycan chains regulate acrosomal protease activity.","method":"Monoclonal antibody competition binding assay; proacrosin activation assay; sperm-ZP binding inhibition assay","journal":"Zygote (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal binding and functional assays; single laboratory","pmids":["10418103"],"is_preprint":false},{"year":1999,"finding":"In macaque sperm, acrosin is not detectable on the inner acrosomal membrane after zona pellucida-induced acrosome reaction (only in the acrosomal shroud), and anti-acrosin antibodies do not inhibit sperm penetration of the zona pellucida; in contrast, anti-PH-20 IgG completely blocks zona penetration, indicating acrosin is not critical for secondary sperm-zona binding or zona penetration in macaques.","method":"Immunoelectron microscopy with anti-acrosin IgG/SBTI; in vitro fertilization zona penetration blocking assay","journal":"Molecular reproduction and development","confidence":"High","confidence_rationale":"Tier 1–2 — functional blocking experiment combined with ultrastructural localization; direct comparison with PH-20","pmids":["10369396"],"is_preprint":false},{"year":2001,"finding":"Mouse zona pellucida glycoprotein ZP2 binds to proacrosin/acrosin via a strong ionic interaction between polysulfate groups on mZP2 and basic residues on an internal proacrosin peptide; proacrosin-null sperm bind mZP2 considerably less effectively than wild-type. Suramin and sulphated polymers compete with mZP2 for proacrosin binding and displace sperm from eggs in an IVF system, establishing that mZP2-proacrosin interaction is required for retention of acrosome-reacted sperm on the zona during fertilization.","method":"Solid-phase binding assays; proacrosin-null mouse sperm; in vitro fertilization sperm displacement; genetic (null) and biochemical approaches","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 — reconstitution binding assay + genetic null + functional IVF assay; multiple orthogonal methods","pmids":["11739644"],"is_preprint":false},{"year":2002,"finding":"Acrosin (encoded by ACR) exhibits trypsin-like serine protease activity in the acrosome; knockout of proacrosin/acrosin in mice produces sperm that penetrate the zona pellucida with a delay but can still fertilize, indicating acrosin is not essential for zona penetration in mice but functions in limited proteolysis and/or processing of acrosomal proteins during the acrosome reaction.","method":"Review integrating gene-knockout mouse data and enzymatic studies","journal":"Human reproduction update","confidence":"High","confidence_rationale":"Tier 2 — synthesis of mouse KO phenotype with biochemical mechanism; widely replicated","pmids":["12398221"],"is_preprint":false},{"year":2002,"finding":"Human proacrosin (55 kDa) undergoes autocatalytic activation to enzymatically active intermediates of 39 and 35 kDa; activation is completely inhibited by 9 mM CaCl₂ or 100 mM benzamidine. Immunoreactive bands of 52, 43, 34, 21–26, and 16 kDa were identified during activation, mapping the sequential processing steps of proacrosin to acrosin.","method":"Zymography; Western blotting with monoclonal antibody AcrC5F10; immunocytochemistry; protease inhibitor assays","journal":"Journal of reproductive immunology","confidence":"High","confidence_rationale":"Tier 1–2 — zymography plus western blotting with inhibitors mapping autocatalytic activation intermediates","pmids":["11839395"],"is_preprint":false},{"year":2005,"finding":"The sp32 proacrosin-binding protein is tyrosine-phosphorylated during capacitation of pig sperm: sp32 appears as a phosphotyrosine-positive Mr 32,000 spot only under capacitating conditions. After the ionophore-induced acrosome reaction, both anti-phosphotyrosine and anti-sp32 labeling on the acrosome disappear, linking proacrosin's binding partner sp32 to the capacitation-dependent phosphorylation cascade.","method":"2D Western blotting; mass spectrometry/MS identification; immunoprecipitation with anti-phosphotyrosine and anti-sp32; indirect immunofluorescence","journal":"Journal of andrology","confidence":"High","confidence_rationale":"Tier 2 — mass spectrometry identification confirmed by immunoprecipitation and localization; orthogonal methods","pmids":["15955892"],"is_preprint":false},{"year":2005,"finding":"Recombinant human proacrosin/acrosin binds to ZP glycoproteins in a saturable, high-affinity manner: rec-hZPA is the primary ligand (highest binding activity), followed by ZPB and ZPC. Binding is mediated by mannosyl, fucosyl, and sulfated glycans (inhibited by dextran sulfate, fucose, and mannose). The N-terminal proenzyme fragment (Rec-40) showed 2.6–3× higher binding than mature acrosin fragments, indicating the proregion participates in ZP binding.","method":"In vitro solid-phase binding assay with recombinant ZP glycoproteins; competition with carbohydrate inhibitors; Kd determination","journal":"Fertility and sterility","confidence":"High","confidence_rationale":"Tier 1 — reconstituted binding assay with recombinant proteins, Kd quantification, and carbohydrate competition","pmids":["15950651"],"is_preprint":false},{"year":2010,"finding":"Double-knockout mice lacking both ACR (acrosin) and PRSS21 (testisin) are subfertile; the double-KO sperm are completely unable to undergo acrosomal exocytosis on the zona pellucida surface and cannot traverse the zona or penetrate cumulus matrix in vitro, demonstrating that the combined trypsin-like protease activity of ACR and PRSS21 is essential for sperm penetration through cumulus and zona in vitro, while the female reproductive tract partially compensates in vivo.","method":"Double-knockout mouse generation; in vitro fertilization assay; zona penetration assay; uterine injection of mutant sperm; recovery of 2-cell embryos","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — double KO with multiple functional readouts (IVF, zona penetration, in vivo rescue); genetic epistasis","pmids":["20484738"],"is_preprint":false},{"year":2019,"finding":"Fluoride exposure reduces sperm fertilizing ability by downregulating ACR protein expression specifically in the epididymis (not testis), along with SPAM1 and PRSS21; fluoride also reduces the ability of sperm to break down the egg cumulus cell layer, linking ACR's trypsin-like activity to cumulus matrix penetration.","method":"Animal (rat) fluoride exposure model; Western blot; immunofluorescence; RNA extraction; sperm-egg binding assay","journal":"Journal of agricultural and food chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 — in vivo model with protein localization and functional readout; single laboratory","pmids":["31008594"],"is_preprint":false},{"year":2019,"finding":"Sperm mitochondrial membrane potential (MMP) is positively correlated with acrosin activity; experimental MMP dissipation (by CCCP) causes significant declines in acrosin activity, acrosome reaction capability, and chromatin integrity, while increasing ROS and decreasing ATP, establishing that mitochondrial function is required to maintain ACR enzymatic activity in human sperm.","method":"JC-1 staining for MMP; BAPNA substrate assay for acrosin activity; CCCP treatment in vitro; chlortetracycline staining for AR; SCSA for DNA fragmentation; flow cytometry","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 — causal link established by CCCP intervention with multiple functional readouts; single laboratory","pmids":["30428044"],"is_preprint":false},{"year":2023,"finding":"A homozygous nonsense mutation in ACR (c.167G>A, p.Trp56X) identified by whole-exome sequencing in two infertile brothers causes ACROSIN deficiency, acrosomal ultrastructural defects, and total fertilization failure (TFF) in humans. Mutant sperm cannot penetrate the zona pellucida but retain normal sperm-zona binding, gamete fusion, and oocyte activation capacity (normal ACTL7A and PLCζ expression); fertilization was rescued by subzonal insemination and ICSI, confirming ACR's specific role in zona penetration.","method":"Whole-exome sequencing; protein expression analysis (Western blot/immunostaining); transmission electron microscopy of acrosome; subzonal insemination; ICSI rescue; IVF zona penetration assay","journal":"Human reproduction (Oxford, England)","confidence":"High","confidence_rationale":"Tier 1–2 — human loss-of-function mutation with structural, molecular, and functional rescue validation using multiple orthogonal methods","pmids":["37004249"],"is_preprint":false}],"current_model":"ACR encodes proacrosin/acrosin, a sperm-specific trypsin-like serine protease stored as an inactive zymogen in the acrosome; upon the acrosome reaction, proacrosin undergoes autocatalytic activation to active acrosin intermediates (39 and 35 kDa), which bind zona pellucida glycoproteins (primarily ZPA) via mannosyl/fucosyl/sulfated glycan interactions and mediate sperm penetration through the zona pellucida—a function demonstrated by total fertilization failure in humans carrying homozygous ACR nonsense mutations and by combined ACR/PRSS21 double-knockout mouse infertility—while zinc, Protein C inhibitor, and mitochondrial ATP supply regulate acrosin activity, and the proacrosin-binding protein sp32 undergoes capacitation-associated tyrosine phosphorylation at the acrosome."},"narrative":{"teleology":[{"year":1982,"claim":"Establishing acrosin's enzymology: purification of bovine (39 kDa) and human (49 kDa) acrosin to homogeneity and demonstration that divalent metal ions reversibly inhibit surface-bound acrosin activity provided the first biochemical framework for acrosin as a regulated trypsin-like protease in sperm.","evidence":"Protein purification to homogeneity with activity assays (BAEE/BANA substrates); metal inhibition and chelation-reversal on washed spermatozoa","pmids":["6802470","6815104"],"confidence":"High","gaps":["No structural model of acrosin–zinc interaction","Physiological zinc concentration at the acrosome not determined"]},{"year":1983,"claim":"Immunofluorescence during human spermatogenesis showed that proacrosin first appears in haploid spermatids and is absent in globozoospermic (round-headed) sperm, establishing acrosin as an obligate acrosomal component whose presence is coupled to acrosome biogenesis.","evidence":"Indirect immunofluorescence with anti-acrosin and anti-outer acrosomal membrane antibodies on human testis sections and globozoospermic samples","pmids":["6357995"],"confidence":"High","gaps":["Mechanism of proacrosin trafficking to the developing acrosome unknown","Whether proacrosin is required for acrosome formation or merely co-localized was not resolved"]},{"year":1989,"claim":"cDNA cloning revealed the complete primary structure of human proacrosin—a 421-amino-acid preproenzyme with a canonical His-Asp-Ser catalytic triad and a unique proline-rich C-terminal domain—and pharmacological inhibition of acrosin activity blocked the human acrosome reaction, linking protease activity to acrosomal function.","evidence":"cDNA cloning/sequencing from human testis; deduced active-site residues; dbcAMP-induced acrosome reaction assay with protease inhibitors","pmids":["2493394","2550339"],"confidence":"High","gaps":["Function of the proline-rich C-terminal domain remained speculative","Substrate(s) of acrosin during the acrosome reaction not identified"]},{"year":1990,"claim":"Gene structure determination showed the human ACR gene contains five exons encoding the three catalytic residues on separate exons, and chromosomal mapping placed it at 22q13-qter, enabling comparative genomics with rodent orthologs.","evidence":"Genomic library screening, sequencing, and somatic cell hybrid mapping (human, rat, mouse)","pmids":["2114285","2606479","1783391"],"confidence":"High","gaps":["Promoter regulatory elements driving testis-specific expression not functionally characterized","Reason for diploid versus haploid expression differences across species unresolved"]},{"year":1994,"claim":"Identification of Protein C inhibitor (PCI) as a physiological acrosin inhibitor—forming SDS-stable complexes with a rate constant of 3.7×10⁴ M⁻¹s⁻¹ and localizing near disrupted acrosomal membranes—revealed a scavenging mechanism for prematurely activated acrosin.","evidence":"In vitro kinetic inhibition assay; ELISA complex detection; immunoelectron microscopy on human sperm","pmids":["7521127"],"confidence":"High","gaps":["In vivo relevance of PCI-acrosin inhibition during fertilization not tested genetically","Other endogenous acrosin inhibitors not systematically surveyed"]},{"year":1999,"claim":"Sulphated zona pellucida glycans were shown to bind a proacrosin domain distinct from the active site and to induce proacrosin activation, while macaque anti-acrosin antibodies failed to block zona penetration, raising the question of whether acrosin is necessary for zona penetration or instead functions in sperm–zona binding/retention.","evidence":"Monoclonal antibody competition and proacrosin activation assays; macaque anti-acrosin blocking IVF and immunoEM","pmids":["10418103","10369396"],"confidence":"High","gaps":["Species-specific differences in acrosin requirement not explained mechanistically","Identity of the proacrosin ZP-binding domain not mapped at residue resolution"]},{"year":2001,"claim":"Using proacrosin-null mouse sperm and recombinant ZP proteins, it was established that ZP2 binds proacrosin via polysulfate–basic residue ionic interactions and that this interaction retains acrosome-reacted sperm on the zona; proacrosin also undergoes autocatalytic processing through defined 55→39→35 kDa intermediates.","evidence":"Solid-phase binding assays with proacrosin-null sperm; IVF displacement assay; zymography and Western blotting of activation intermediates","pmids":["11739644","11839395"],"confidence":"High","gaps":["Whether ZP2 binding and proteolytic activity are independent or coupled functions was unclear","Mouse KO showed only delayed penetration, leaving essentiality for zona penetration unresolved in any species"]},{"year":2005,"claim":"Recombinant human proacrosin/acrosin was shown to bind ZPA with highest affinity among ZP glycoproteins (with mannose, fucose, and sulfate as key glycan determinants) and the proenzyme fragment bound ZP 2.6–3× more strongly than mature acrosin, while sp32 (a proacrosin-binding protein) was identified as a capacitation-dependent tyrosine phosphoprotein in the acrosome.","evidence":"Solid-phase binding assay with recombinant ZP glycoproteins and carbohydrate competition; 2D Western blot and mass spectrometry of sp32 phosphorylation","pmids":["15950651","15955892"],"confidence":"High","gaps":["How sp32 phosphorylation regulates proacrosin activation or release was not determined","Full glycan structural determinants on ZPA mediating acrosin binding not resolved"]},{"year":2010,"claim":"Double knockout of ACR and PRSS21 in mice produced subfertile males whose sperm completely failed to undergo acrosomal exocytosis on the zona surface and could not penetrate cumulus or zona in vitro, establishing that combined trypsin-like protease activity is essential for gamete interaction even though single knockouts retained partial function.","evidence":"ACR/PRSS21 double-KO mice; IVF, zona penetration assay, uterine injection rescue","pmids":["20484738"],"confidence":"High","gaps":["Molecular substrates cleaved by ACR versus PRSS21 not distinguished","Female tract factors that partially compensate in vivo not identified"]},{"year":2023,"claim":"A homozygous ACR nonsense mutation (p.Trp56X) in two infertile brothers caused total fertilization failure with zona penetration deficiency but preserved sperm–zona binding, gamete fusion, and oocyte activation; ICSI rescued fertility, providing definitive human genetic evidence that acrosin is specifically required for zona penetration in humans.","evidence":"Whole-exome sequencing; Western blot and immunostaining for acrosin loss; TEM of acrosomal defects; subzonal insemination and ICSI rescue","pmids":["37004249"],"confidence":"High","gaps":["Only one family reported; additional alleles needed to confirm genotype-phenotype spectrum","Whether acrosomal ultrastructural defects are secondary to acrosin loss or a direct structural role is unresolved","No crystal structure of human acrosin exists"]},{"year":null,"claim":"The precise set of acrosomal and zona pellucida substrates cleaved by acrosin during zona penetration, the structural basis for the proenzyme region's enhanced ZP binding, and how acrosin coordinates with other acrosomal proteases remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No acrosin substrate profiling by proteomics has been performed","No crystal structure of acrosin or proacrosin–ZP complex exists","Mechanism of sp32-mediated regulation of proacrosin activation is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,8,10,11,12,16,17,23]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,6,8,10,16,17]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[9,12,18,23]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[14,15]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[11,15,16,19,20,23]}],"complexes":[],"partners":["ZP2","ZP1","ZP3","SERPINA5","ACRV1","PRSS21"],"other_free_text":[]},"mechanistic_narrative":"ACR encodes acrosin, a sperm-specific trypsin-like serine protease stored as the inactive zymogen proacrosin in the acrosomal matrix, where it undergoes autocatalytic activation to 39 kDa and 35 kDa intermediates upon the acrosome reaction [PMID:11839395]. Acrosin binds zona pellucida glycoproteins—primarily ZPA—through mannosyl, fucosyl, and sulfated glycan interactions, with the proenzyme region contributing to high-affinity binding, thereby mediating secondary sperm–zona retention and zona penetration [PMID:15950651, PMID:11739644]. Combined knockout of ACR and PRSS21 in mice abolishes acrosomal exocytosis on the zona surface and zona/cumulus penetration in vitro [PMID:20484738], and a homozygous ACR nonsense mutation (p.Trp56X) in humans causes total fertilization failure due to zona penetration deficiency, rescued by ICSI [PMID:37004249]."},"prefetch_data":{"uniprot":{"accession":"P10323","full_name":"Acrosin","aliases":[],"length_aa":421,"mass_kda":45.8,"function":"Acrosin is the major protease of mammalian spermatozoa. It is a serine protease of trypsin-like cleavage specificity, it is synthesized in a zymogen form, proacrosin and stored in the acrosome","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P10323/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACR","classification":"Not Classified","n_dependent_lines":32,"n_total_lines":1208,"dependency_fraction":0.026490066225165563},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ACR","total_profiled":1310},"omim":[{"mim_id":"620500","title":"SPERMATOGENIC FAILURE 87; SPGF87","url":"https://www.omim.org/entry/620500"},{"mim_id":"620376","title":"AUTOINFLAMMATORY DISEASE, SYSTEMIC, WITH VASCULITIS; SAIDV","url":"https://www.omim.org/entry/620376"},{"mim_id":"615538","title":"CHROMOSOME 22q13 DUPLICATION SYNDROME","url":"https://www.omim.org/entry/615538"},{"mim_id":"608537","title":"VON HIPPEL-LINDAU TUMOR SUPPRESSOR; VHL","url":"https://www.omim.org/entry/608537"},{"mim_id":"606426","title":"EGL9 FAMILY HYPOXIA-INDUCIBLE FACTOR 3; EGLN3","url":"https://www.omim.org/entry/606426"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":80.9}],"url":"https://www.proteinatlas.org/search/ACR"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P10323","domains":[{"cath_id":"2.40.10.10","chopping":"58-159_283-299","consensus_level":"medium","plddt":95.821,"start":58,"end":299},{"cath_id":"2.40.10.10","chopping":"177-280","consensus_level":"medium","plddt":93.1426,"start":177,"end":280}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P10323","model_url":"https://alphafold.ebi.ac.uk/files/AF-P10323-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P10323-F1-predicted_aligned_error_v6.png","plddt_mean":79.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACR","jax_strain_url":"https://www.jax.org/strain/search?query=ACR"},"sequence":{"accession":"P10323","fasta_url":"https://rest.uniprot.org/uniprotkb/P10323.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P10323/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P10323"}},"corpus_meta":[{"pmid":"37635643","id":"PMC_37635643","title":"The 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I. Studies with recombinant human ZPA, ZPB, and ZPC.","date":"2005","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/15950651","citation_count":18,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49004,"output_tokens":869,"usd":0.080023},"stage2":{"model":"claude-opus-4-6","input_tokens":4066,"output_tokens":1074,"usd":0.07077},"total_usd":0.478492,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":63026,"output_tokens":6789,"usd":0.145456},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":9664,"output_tokens":2927,"usd":0.182243}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"The human preproacrosin gene (ACR) was mapped to chromosome 22q13-qter using human-rodent somatic cell hybrids, establishing its chromosomal location and identifying a restriction fragment length polymorphism with SstI.\",\n      \"method\": \"Somatic cell hybrid panel analysis, Southern blotting\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal mapping by somatic cell hybrids, single lab\",\n      \"pmids\": [\"2606479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The rat proacrosin gene (ACR) was assigned to chromosome 7 by analysis of mouse x rat cell hybrids that segregate rat chromosomes.\",\n      \"method\": \"Somatic cell hybrid panel analysis\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal mapping by somatic cell hybrids, single lab\",\n      \"pmids\": [\"1906796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ACR (acrosin) and PRSS21 double-knockout male mice are subfertile; mutant sperm are unable to undergo acrosomal exocytosis on the zona pellucida surface, fail to penetrate the cumulus matrix and zona pellucida in vitro, demonstrating that the trypsin-like protease activity of ACR is essential for sperm penetration through the cumulus matrix and zona pellucida.\",\n      \"method\": \"Double-knockout mouse model, in vitro fertilization assay, uterine injection rescue experiment\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean double-KO with specific fertilization phenotype, multiple orthogonal in vivo and in vitro readouts\",\n      \"pmids\": [\"20484738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fluoride exposure selectively reduces ACR protein expression in the epididymis (but not in the testis), along with SPAM1 and PRSS21, correlating with impaired sperm ability to penetrate the cumulus cell layer and reduced fertilizing capacity.\",\n      \"method\": \"In vivo fluoride exposure in rats, western blot, immunofluorescence, sperm-egg binding assay\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, multiple methods showing epididymis-specific regulation, but no direct mechanistic rescue\",\n      \"pmids\": [\"31008594\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACR (acrosin) encodes a sperm serine protease that is expressed in haploid spermatogenic cells, localizes to the acrosome, and provides trypsin-like proteolytic activity required for sperm penetration through the cumulus cell matrix and zona pellucida during fertilization; genetic knockout of ACR together with PRSS21 abolishes acrosomal exocytosis on the zona pellucida and renders sperm infertile in vitro, while the female reproductive tract can partially compensate in vivo.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll systematically classify each paper before extracting discoveries.\n\n**Classification:**\n\nThe target gene ACR here is the human gene encoding **acrosin/proacrosin** (sperm-specific serine protease involved in fertilization), mapped to chromosome 22q13-qter.\n\n**EXCLUDE (alias collisions / off-target):**\n- Papers about ACR/EULAR/American College of Rheumatology criteria → EXCLUDE (acronym collision)\n- Paper [4] circRNA ACR (autophagy-related circular RNA) → EXCLUDE (alt-locus product, circRNA)\n- Paper [3] Mycobacterium tuberculosis Acr (alpha-crystallin homolog, Rv2031c) → EXCLUDE (symbol collision, bacterial protein)\n- Papers [12],[14],[42],[43],[63],[71] M. tuberculosis acr gene → EXCLUDE\n- Papers [8],[9],[24],[26],[38],[39],[48],[50],[55],[59],[68],[70],[72],[81] C. elegans acr genes (nicotinic AChR subunits) → EXCLUDE (symbol collision, nematode ion channels unrelated to mammalian acrosin)\n- Paper [13] 5-ACR (5-azacytidine) → EXCLUDE\n- Paper [17] ACR-toxin (Alternaria alternata) → EXCLUDE\n- Paper [29] NeuAcR linkages in CHO cells → EXCLUDE\n- Paper [37] F4ab/acR- pigs (F4 receptor) → EXCLUDE\n- Paper [46] acrAB efflux pump (Salmonella) → EXCLUDE\n- Paper [47] ACRTS2 polyketide synthase → EXCLUDE\n- Paper [52] Retracted circRNA ACR paper → EXCLUDE\n- Paper [67] PD1-ACR chimeric receptor → EXCLUDE\n- Paper [78],[100] Acr-sP(EO-stat-PO) hydrogels → EXCLUDE (material science acronym)\n- Paper [94] ACR gene family in Oryza sativa (plant ACT domain proteins) → EXCLUDE\n\n**KEEP (human/mammalian acrosin/proacrosin gene ACR):**\n- [56] Chromosomal assignment of ACR to 22q13-qter\n- [61] Rat ACR mapped to chromosome 7\n- [14-curated=8] Acrosin review (klemm1991)\n- [9-curated] Role of acrosomal matrix proteases\n- [10-curated] Primary structure of human proacrosin cDNA\n- [11-curated] Mitochondrial functionality and acrosin activity\n- [12-curated] mZP2-proacrosin interactions\n- [13-curated] sp32 tyrosine phosphorylation\n- [14-curated] Mouse proacrosin gene structure\n- [15-curated] Inhibition of acrosome reaction by proteinase inhibitors\n- [16-curated] PCI inhibits acrosin\n- [17-curated] PH-20 vs acrosin in macaque zona penetration\n- [19-curated] Bovine sperm acrosin activates adenylate cyclase\n- [21-curated] Human proacrosin gene exon-intron organization\n- [22-curated] Human preproacrosin cDNA cloning\n- [23-curated] Acrosin in human spermatogenesis\n- [24-curated] Proacrosin/acrosin activation mechanism\n- [25-curated] Zinc inhibition of acrosin\n- [27-curated] ACROSIN deficiency causes TFF in humans\n- [28-curated] Purification of bovine and human acrosin\n- [29-curated] Sulphated polysaccharides and proacrosin\n- [30-curated] Proacrosin binding to ZP glycoproteins\n- [15] Double-knockout ACR and PRSS21 mice\n- [44] Fluoride effects on ACR/PRSS21/SPAM1 in epididymis\n- [69] ACR gene expression in NOA testicular biopsies\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"The primary structure of human proacrosin was deduced from cDNA cloning: the 421-amino-acid preproenzyme contains a 19-residue signal peptide, a light chain segment, and a heavy chain with a catalytic site. The active-site residues histidine (69), aspartic acid (123), and serine (221) were identified, and a proline-rich C-terminal domain unique to acrosin was noted.\",\n      \"method\": \"cDNA cloning and sequencing from human testis library; deduced amino acid sequence analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — primary structure determination by cDNA sequencing with active-site residue identification\",\n      \"pmids\": [\"2493394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"The chromosomal gene encoding human preproacrosin (ACR) was mapped to chromosome 22q13-qter using somatic cell hybrid analysis; a restriction fragment length polymorphism with SstI was identified in the gene.\",\n      \"method\": \"Somatic cell hybrid analysis with human-rodent hybrids containing variant chromosome 22 complements; Southern blotting\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chromosomal mapping by somatic cell hybrids with translocation chromosomes\",\n      \"pmids\": [\"2606479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"The human proacrosin gene contains four introns and five exons; the three active-site residues (His, Asp, Ser) are each encoded by separate exons (E2, E3, E5), and the proline-rich domain is co-encoded with the serine active-site residue in exon E5. The gene lacks canonical TATA and CAAT boxes near the transcription start site.\",\n      \"method\": \"Genomic library screening, restriction mapping, sequencing, primer extension analysis\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — complete gene structure determination with functional annotation of active-site exons\",\n      \"pmids\": [\"2114285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Human preproacrosin cDNA cloning confirmed 70% amino acid sequence identity with boar proacrosin; the leader sequence, catalytic triad (His, Asp, Ser), disulfide-bonding cysteine positions, and C-terminal proline-rich sequence are conserved between species, while the proline-rich region is species-specific.\",\n      \"method\": \"cDNA library screening, nucleotide sequencing, comparative amino acid sequence analysis\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cDNA cloning with cross-species structural comparison\",\n      \"pmids\": [\"2298447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The rat proacrosin gene (ACR) was mapped to chromosome 7 by analysis of mouse × rat somatic cell hybrids, establishing synteny with the human chromosome 22q13-qter locus.\",\n      \"method\": \"Somatic cell hybrid panel analysis with rat chromosome segregation\",\n      \"journal\": \"Cytogenetics and cell genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chromosomal mapping by somatic cell hybrids, consistent with human locus\",\n      \"pmids\": [\"1906796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The mouse proacrosin gene comprises six exons and five introns (one intron in the 5'-UTR); the gene is located on mouse chromosome 15 bands E/F, and unusually is expressed diploid (in pre-meiotic spermatocytes) in the mouse, contrasting with haploid expression in bull, boar, and rat. Active-site residues His, Asp, and Ser are encoded by exons E2, E3, and E5 respectively.\",\n      \"method\": \"Cosmid library screening, gene mapping, primer extension, in situ hybridization, expression analysis during spermatogenesis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — complete mouse gene structure with chromosomal localization and expression timing determination\",\n      \"pmids\": [\"1783391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Acrosin is a sperm-specific serine protease stored as proacrosin in the acrosome; the acrosin molecule contains a novel proline-rich C-terminal domain (absent from other serine proteases) that may confer DNA-binding properties, in addition to the standard serine protease catalytic triad.\",\n      \"method\": \"Biochemical characterization and primary structure analysis across species\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural analysis identifying a novel domain in acrosin not present in other serine proteases\",\n      \"pmids\": [\"1937464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1982,\n      \"finding\": \"Divalent metal ions (Zn²⁺, Cu²⁺, Hg²⁺, Co²⁺, Cd²⁺) potently inhibit surface-bound acrosin activity on human and bovine spermatozoa; removal of zinc or copper from the inhibited enzyme completely restores activity, suggesting that zinc in genital tract fluids physiologically regulates acrosin activity.\",\n      \"method\": \"Enzymatic activity assay (BANA substrate) on washed spermatozoa; metal chelation and restoration experiments\",\n      \"journal\": \"International journal of andrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with reversibility demonstrated; single laboratory\",\n      \"pmids\": [\"6815104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1982,\n      \"finding\": \"Bovine acrosin (MW 39 kDa; specific activity 93 U/mg) and human acrosin (MW 49 kDa) were purified to homogeneity using HCl extraction, gel filtration, and affinity chromatography, providing the first detailed purification schemes for both species.\",\n      \"method\": \"Protein purification: saline/Triton X-100 washes, HCl extraction, gel filtration, ion-exchange and affinity chromatography; activity assay with BAEE\",\n      \"journal\": \"Canadian journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical purification to homogeneity with molecular weight determination\",\n      \"pmids\": [\"6802470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1983,\n      \"finding\": \"Immunofluorescence during human spermatogenesis showed that proacrosin and the outer acrosomal membrane first appear in haploid spermatids and increase through spermiogenesis; round-headed (globozoospermic) spermatozoa completely lack proacrosin, acrosin, and the outer acrosomal membrane, demonstrating that acrosin is an obligate acrosomal component.\",\n      \"method\": \"Indirect immunofluorescence with anti-acrosin and anti-outer acrosomal membrane antibodies; gelatin substrate film technique for gelatinolytic activity\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization during spermatogenesis with functional correlation to acrosome presence\",\n      \"pmids\": [\"6357995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1985,\n      \"finding\": \"Purified bovine sperm acrosin activates platelet adenylate cyclase 5–8-fold, comparable in potency to trypsin and α-chymotrypsin; this activation is blocked by serine protease inhibitors (leupeptin, SBTI, α2-macroglobulin), identifying acrosin as a trypsin-like protease capable of activating adenylate cyclase via proteolytic activity.\",\n      \"method\": \"Adenylate cyclase activity assay with purified acrosin; protease inhibitor panel; enzyme extraction from bovine sperm\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with purified acrosin; single laboratory\",\n      \"pmids\": [\"3880736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"Proteinase inhibitors of proacrosin activation and acrosin activity (AGB and para-aminobenzamidine) significantly inhibit the dbcAMP-induced human sperm acrosome reaction at concentrations of 10⁻⁶–10⁻⁴ M, demonstrating that acrosin (a serine proteinase) plays a functional role in the human sperm acrosome reaction downstream of the adenylate cyclase system.\",\n      \"method\": \"Human synchronous acrosome reaction assay with dbcAMP stimulation; acrosome reaction scoring with proteinase inhibitors added at onset or after capacitation\",\n      \"journal\": \"Gamete research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological inhibition with dose-response in human sperm; single laboratory\",\n      \"pmids\": [\"2550339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Protein C inhibitor (PCI) directly inhibits acrosin: purified human PCI inhibited boar acrosin amidolytic activity with an apparent second-order rate constant of 3.7 × 10⁴ M⁻¹·s⁻¹, forming SDS-stable complexes with acrosin. PCI was immunolocalized exclusively near disrupted acrosomal membranes of morphologically abnormal spermatozoa, suggesting PCI scavenges prematurely activated acrosin.\",\n      \"method\": \"In vitro enzymatic inhibition assay; ELISA for complex formation; immunocytochemistry and immunoelectron microscopy\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinetic inhibition combined with structural localization; orthogonal methods\",\n      \"pmids\": [\"7521127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Sulphated polymers (fucoidan, solubilised zona pellucida glycoproteins [ZPGs], and ARIS) bind to a domain on proacrosin distinct from the active site, induce proacrosin activation, and inhibit sperm binding to the zona pellucida; heparin did not share this activity. ARIS induced human proacrosin activation, indicating that ZP glycan chains regulate acrosomal protease activity.\",\n      \"method\": \"Monoclonal antibody competition binding assay; proacrosin activation assay; sperm-ZP binding inhibition assay\",\n      \"journal\": \"Zygote (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal binding and functional assays; single laboratory\",\n      \"pmids\": [\"10418103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"In macaque sperm, acrosin is not detectable on the inner acrosomal membrane after zona pellucida-induced acrosome reaction (only in the acrosomal shroud), and anti-acrosin antibodies do not inhibit sperm penetration of the zona pellucida; in contrast, anti-PH-20 IgG completely blocks zona penetration, indicating acrosin is not critical for secondary sperm-zona binding or zona penetration in macaques.\",\n      \"method\": \"Immunoelectron microscopy with anti-acrosin IgG/SBTI; in vitro fertilization zona penetration blocking assay\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — functional blocking experiment combined with ultrastructural localization; direct comparison with PH-20\",\n      \"pmids\": [\"10369396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mouse zona pellucida glycoprotein ZP2 binds to proacrosin/acrosin via a strong ionic interaction between polysulfate groups on mZP2 and basic residues on an internal proacrosin peptide; proacrosin-null sperm bind mZP2 considerably less effectively than wild-type. Suramin and sulphated polymers compete with mZP2 for proacrosin binding and displace sperm from eggs in an IVF system, establishing that mZP2-proacrosin interaction is required for retention of acrosome-reacted sperm on the zona during fertilization.\",\n      \"method\": \"Solid-phase binding assays; proacrosin-null mouse sperm; in vitro fertilization sperm displacement; genetic (null) and biochemical approaches\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution binding assay + genetic null + functional IVF assay; multiple orthogonal methods\",\n      \"pmids\": [\"11739644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Acrosin (encoded by ACR) exhibits trypsin-like serine protease activity in the acrosome; knockout of proacrosin/acrosin in mice produces sperm that penetrate the zona pellucida with a delay but can still fertilize, indicating acrosin is not essential for zona penetration in mice but functions in limited proteolysis and/or processing of acrosomal proteins during the acrosome reaction.\",\n      \"method\": \"Review integrating gene-knockout mouse data and enzymatic studies\",\n      \"journal\": \"Human reproduction update\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — synthesis of mouse KO phenotype with biochemical mechanism; widely replicated\",\n      \"pmids\": [\"12398221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human proacrosin (55 kDa) undergoes autocatalytic activation to enzymatically active intermediates of 39 and 35 kDa; activation is completely inhibited by 9 mM CaCl₂ or 100 mM benzamidine. Immunoreactive bands of 52, 43, 34, 21–26, and 16 kDa were identified during activation, mapping the sequential processing steps of proacrosin to acrosin.\",\n      \"method\": \"Zymography; Western blotting with monoclonal antibody AcrC5F10; immunocytochemistry; protease inhibitor assays\",\n      \"journal\": \"Journal of reproductive immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — zymography plus western blotting with inhibitors mapping autocatalytic activation intermediates\",\n      \"pmids\": [\"11839395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The sp32 proacrosin-binding protein is tyrosine-phosphorylated during capacitation of pig sperm: sp32 appears as a phosphotyrosine-positive Mr 32,000 spot only under capacitating conditions. After the ionophore-induced acrosome reaction, both anti-phosphotyrosine and anti-sp32 labeling on the acrosome disappear, linking proacrosin's binding partner sp32 to the capacitation-dependent phosphorylation cascade.\",\n      \"method\": \"2D Western blotting; mass spectrometry/MS identification; immunoprecipitation with anti-phosphotyrosine and anti-sp32; indirect immunofluorescence\",\n      \"journal\": \"Journal of andrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mass spectrometry identification confirmed by immunoprecipitation and localization; orthogonal methods\",\n      \"pmids\": [\"15955892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Recombinant human proacrosin/acrosin binds to ZP glycoproteins in a saturable, high-affinity manner: rec-hZPA is the primary ligand (highest binding activity), followed by ZPB and ZPC. Binding is mediated by mannosyl, fucosyl, and sulfated glycans (inhibited by dextran sulfate, fucose, and mannose). The N-terminal proenzyme fragment (Rec-40) showed 2.6–3× higher binding than mature acrosin fragments, indicating the proregion participates in ZP binding.\",\n      \"method\": \"In vitro solid-phase binding assay with recombinant ZP glycoproteins; competition with carbohydrate inhibitors; Kd determination\",\n      \"journal\": \"Fertility and sterility\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted binding assay with recombinant proteins, Kd quantification, and carbohydrate competition\",\n      \"pmids\": [\"15950651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Double-knockout mice lacking both ACR (acrosin) and PRSS21 (testisin) are subfertile; the double-KO sperm are completely unable to undergo acrosomal exocytosis on the zona pellucida surface and cannot traverse the zona or penetrate cumulus matrix in vitro, demonstrating that the combined trypsin-like protease activity of ACR and PRSS21 is essential for sperm penetration through cumulus and zona in vitro, while the female reproductive tract partially compensates in vivo.\",\n      \"method\": \"Double-knockout mouse generation; in vitro fertilization assay; zona penetration assay; uterine injection of mutant sperm; recovery of 2-cell embryos\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — double KO with multiple functional readouts (IVF, zona penetration, in vivo rescue); genetic epistasis\",\n      \"pmids\": [\"20484738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fluoride exposure reduces sperm fertilizing ability by downregulating ACR protein expression specifically in the epididymis (not testis), along with SPAM1 and PRSS21; fluoride also reduces the ability of sperm to break down the egg cumulus cell layer, linking ACR's trypsin-like activity to cumulus matrix penetration.\",\n      \"method\": \"Animal (rat) fluoride exposure model; Western blot; immunofluorescence; RNA extraction; sperm-egg binding assay\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — in vivo model with protein localization and functional readout; single laboratory\",\n      \"pmids\": [\"31008594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Sperm mitochondrial membrane potential (MMP) is positively correlated with acrosin activity; experimental MMP dissipation (by CCCP) causes significant declines in acrosin activity, acrosome reaction capability, and chromatin integrity, while increasing ROS and decreasing ATP, establishing that mitochondrial function is required to maintain ACR enzymatic activity in human sperm.\",\n      \"method\": \"JC-1 staining for MMP; BAPNA substrate assay for acrosin activity; CCCP treatment in vitro; chlortetracycline staining for AR; SCSA for DNA fragmentation; flow cytometry\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — causal link established by CCCP intervention with multiple functional readouts; single laboratory\",\n      \"pmids\": [\"30428044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A homozygous nonsense mutation in ACR (c.167G>A, p.Trp56X) identified by whole-exome sequencing in two infertile brothers causes ACROSIN deficiency, acrosomal ultrastructural defects, and total fertilization failure (TFF) in humans. Mutant sperm cannot penetrate the zona pellucida but retain normal sperm-zona binding, gamete fusion, and oocyte activation capacity (normal ACTL7A and PLCζ expression); fertilization was rescued by subzonal insemination and ICSI, confirming ACR's specific role in zona penetration.\",\n      \"method\": \"Whole-exome sequencing; protein expression analysis (Western blot/immunostaining); transmission electron microscopy of acrosome; subzonal insemination; ICSI rescue; IVF zona penetration assay\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — human loss-of-function mutation with structural, molecular, and functional rescue validation using multiple orthogonal methods\",\n      \"pmids\": [\"37004249\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACR encodes proacrosin/acrosin, a sperm-specific trypsin-like serine protease stored as an inactive zymogen in the acrosome; upon the acrosome reaction, proacrosin undergoes autocatalytic activation to active acrosin intermediates (39 and 35 kDa), which bind zona pellucida glycoproteins (primarily ZPA) via mannosyl/fucosyl/sulfated glycan interactions and mediate sperm penetration through the zona pellucida—a function demonstrated by total fertilization failure in humans carrying homozygous ACR nonsense mutations and by combined ACR/PRSS21 double-knockout mouse infertility—while zinc, Protein C inhibitor, and mitochondrial ATP supply regulate acrosin activity, and the proacrosin-binding protein sp32 undergoes capacitation-associated tyrosine phosphorylation at the acrosome.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ACR encodes acrosin, a sperm-specific trypsin-like serine protease that localizes to the acrosome and is essential for sperm penetration through the cumulus cell matrix and zona pellucida during fertilization. Double knockout of ACR together with PRSS21 in mice abolishes acrosomal exocytosis on the zona pellucida surface and renders sperm unable to penetrate the cumulus matrix or zona pellucida in vitro, though the female reproductive tract can partially compensate in vivo [PMID:20484738]. ACR protein expression in the epididymis is selectively reduced by environmental insults such as fluoride exposure, correlating with impaired cumulus penetration and reduced fertilizing capacity [PMID:31008594].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Before a functional framework could be built, the chromosomal location of ACR was established by mapping the human preproacrosin gene to chromosome 22q13-qter, providing the first genomic anchor for the locus.\",\n      \"evidence\": \"Somatic cell hybrid panel analysis with Southern blotting in human–rodent hybrids\",\n      \"pmids\": [\"2606479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab mapping without independent confirmation by FISH or linkage analysis\",\n        \"No functional data on gene product activity or localization\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The central mechanistic question — whether ACR protease activity is required for fertilization — was answered by showing that ACR/PRSS21 double-knockout sperm fail to undergo acrosomal exocytosis on the zona pellucida and cannot penetrate the cumulus matrix or zona pellucida in vitro, establishing ACR as essential for sperm-egg barrier traversal.\",\n      \"evidence\": \"Double-knockout mouse model with in vitro fertilization assays and uterine injection rescue experiments\",\n      \"pmids\": [\"20484738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Individual contribution of ACR versus PRSS21 not fully dissected in the double-KO design\",\n        \"In vivo partial compensation by the female tract obscures the absolute requirement for ACR under physiological conditions\",\n        \"No structural or biochemical characterization of ACR's substrate specificity on zona pellucida glycoproteins\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Post-testicular regulation of ACR was demonstrated when fluoride exposure selectively reduced ACR protein in the epididymis without affecting testicular expression, linking epididymal ACR levels to cumulus penetration capacity.\",\n      \"evidence\": \"In vivo fluoride exposure in rats with western blot, immunofluorescence, and sperm-egg binding assays\",\n      \"pmids\": [\"31008594\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct rescue experiment to confirm ACR reduction as the causal mediator of impaired penetration\",\n        \"Mechanism of epididymis-specific ACR downregulation by fluoride is unknown\",\n        \"Single-lab observation without independent replication\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions remain: the individual contribution of ACR versus PRSS21 to acrosomal exocytosis and zona penetration, the specific zona pellucida substrates cleaved by ACR, the structural basis of ACR's trypsin-like activity, and whether ACR loss alone causes human male infertility.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No ACR single-knockout phenotype cleanly separated from PRSS21\",\n        \"No human genetic study linking ACR mutations to infertility\",\n        \"No crystal structure or substrate identification for ACR\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PRSS21\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ACR encodes acrosin, a sperm-specific trypsin-like serine protease stored as the inactive zymogen proacrosin in the acrosomal matrix, where it undergoes autocatalytic activation to 39 kDa and 35 kDa intermediates upon the acrosome reaction [PMID:11839395]. Acrosin binds zona pellucida glycoproteins—primarily ZPA—through mannosyl, fucosyl, and sulfated glycan interactions, with the proenzyme region contributing to high-affinity binding, thereby mediating secondary sperm–zona retention and zona penetration [PMID:15950651, PMID:11739644]. Combined knockout of ACR and PRSS21 in mice abolishes acrosomal exocytosis on the zona surface and zona/cumulus penetration in vitro [PMID:20484738], and a homozygous ACR nonsense mutation (p.Trp56X) in humans causes total fertilization failure due to zona penetration deficiency, rescued by ICSI [PMID:37004249].\",\n  \"teleology\": [\n    {\n      \"year\": 1982,\n      \"claim\": \"Establishing acrosin's enzymology: purification of bovine (39 kDa) and human (49 kDa) acrosin to homogeneity and demonstration that divalent metal ions reversibly inhibit surface-bound acrosin activity provided the first biochemical framework for acrosin as a regulated trypsin-like protease in sperm.\",\n      \"evidence\": \"Protein purification to homogeneity with activity assays (BAEE/BANA substrates); metal inhibition and chelation-reversal on washed spermatozoa\",\n      \"pmids\": [\"6802470\", \"6815104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of acrosin–zinc interaction\",\n        \"Physiological zinc concentration at the acrosome not determined\"\n      ]\n    },\n    {\n      \"year\": 1983,\n      \"claim\": \"Immunofluorescence during human spermatogenesis showed that proacrosin first appears in haploid spermatids and is absent in globozoospermic (round-headed) sperm, establishing acrosin as an obligate acrosomal component whose presence is coupled to acrosome biogenesis.\",\n      \"evidence\": \"Indirect immunofluorescence with anti-acrosin and anti-outer acrosomal membrane antibodies on human testis sections and globozoospermic samples\",\n      \"pmids\": [\"6357995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism of proacrosin trafficking to the developing acrosome unknown\",\n        \"Whether proacrosin is required for acrosome formation or merely co-localized was not resolved\"\n      ]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"cDNA cloning revealed the complete primary structure of human proacrosin—a 421-amino-acid preproenzyme with a canonical His-Asp-Ser catalytic triad and a unique proline-rich C-terminal domain—and pharmacological inhibition of acrosin activity blocked the human acrosome reaction, linking protease activity to acrosomal function.\",\n      \"evidence\": \"cDNA cloning/sequencing from human testis; deduced active-site residues; dbcAMP-induced acrosome reaction assay with protease inhibitors\",\n      \"pmids\": [\"2493394\", \"2550339\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Function of the proline-rich C-terminal domain remained speculative\",\n        \"Substrate(s) of acrosin during the acrosome reaction not identified\"\n      ]\n    },\n    {\n      \"year\": 1990,\n      \"claim\": \"Gene structure determination showed the human ACR gene contains five exons encoding the three catalytic residues on separate exons, and chromosomal mapping placed it at 22q13-qter, enabling comparative genomics with rodent orthologs.\",\n      \"evidence\": \"Genomic library screening, sequencing, and somatic cell hybrid mapping (human, rat, mouse)\",\n      \"pmids\": [\"2114285\", \"2606479\", \"1783391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Promoter regulatory elements driving testis-specific expression not functionally characterized\",\n        \"Reason for diploid versus haploid expression differences across species unresolved\"\n      ]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Identification of Protein C inhibitor (PCI) as a physiological acrosin inhibitor—forming SDS-stable complexes with a rate constant of 3.7×10⁴ M⁻¹s⁻¹ and localizing near disrupted acrosomal membranes—revealed a scavenging mechanism for prematurely activated acrosin.\",\n      \"evidence\": \"In vitro kinetic inhibition assay; ELISA complex detection; immunoelectron microscopy on human sperm\",\n      \"pmids\": [\"7521127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"In vivo relevance of PCI-acrosin inhibition during fertilization not tested genetically\",\n        \"Other endogenous acrosin inhibitors not systematically surveyed\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Sulphated zona pellucida glycans were shown to bind a proacrosin domain distinct from the active site and to induce proacrosin activation, while macaque anti-acrosin antibodies failed to block zona penetration, raising the question of whether acrosin is necessary for zona penetration or instead functions in sperm–zona binding/retention.\",\n      \"evidence\": \"Monoclonal antibody competition and proacrosin activation assays; macaque anti-acrosin blocking IVF and immunoEM\",\n      \"pmids\": [\"10418103\", \"10369396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Species-specific differences in acrosin requirement not explained mechanistically\",\n        \"Identity of the proacrosin ZP-binding domain not mapped at residue resolution\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Using proacrosin-null mouse sperm and recombinant ZP proteins, it was established that ZP2 binds proacrosin via polysulfate–basic residue ionic interactions and that this interaction retains acrosome-reacted sperm on the zona; proacrosin also undergoes autocatalytic processing through defined 55→39→35 kDa intermediates.\",\n      \"evidence\": \"Solid-phase binding assays with proacrosin-null sperm; IVF displacement assay; zymography and Western blotting of activation intermediates\",\n      \"pmids\": [\"11739644\", \"11839395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ZP2 binding and proteolytic activity are independent or coupled functions was unclear\",\n        \"Mouse KO showed only delayed penetration, leaving essentiality for zona penetration unresolved in any species\"\n      ]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Recombinant human proacrosin/acrosin was shown to bind ZPA with highest affinity among ZP glycoproteins (with mannose, fucose, and sulfate as key glycan determinants) and the proenzyme fragment bound ZP 2.6–3× more strongly than mature acrosin, while sp32 (a proacrosin-binding protein) was identified as a capacitation-dependent tyrosine phosphoprotein in the acrosome.\",\n      \"evidence\": \"Solid-phase binding assay with recombinant ZP glycoproteins and carbohydrate competition; 2D Western blot and mass spectrometry of sp32 phosphorylation\",\n      \"pmids\": [\"15950651\", \"15955892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How sp32 phosphorylation regulates proacrosin activation or release was not determined\",\n        \"Full glycan structural determinants on ZPA mediating acrosin binding not resolved\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Double knockout of ACR and PRSS21 in mice produced subfertile males whose sperm completely failed to undergo acrosomal exocytosis on the zona surface and could not penetrate cumulus or zona in vitro, establishing that combined trypsin-like protease activity is essential for gamete interaction even though single knockouts retained partial function.\",\n      \"evidence\": \"ACR/PRSS21 double-KO mice; IVF, zona penetration assay, uterine injection rescue\",\n      \"pmids\": [\"20484738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular substrates cleaved by ACR versus PRSS21 not distinguished\",\n        \"Female tract factors that partially compensate in vivo not identified\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A homozygous ACR nonsense mutation (p.Trp56X) in two infertile brothers caused total fertilization failure with zona penetration deficiency but preserved sperm–zona binding, gamete fusion, and oocyte activation; ICSI rescued fertility, providing definitive human genetic evidence that acrosin is specifically required for zona penetration in humans.\",\n      \"evidence\": \"Whole-exome sequencing; Western blot and immunostaining for acrosin loss; TEM of acrosomal defects; subzonal insemination and ICSI rescue\",\n      \"pmids\": [\"37004249\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Only one family reported; additional alleles needed to confirm genotype-phenotype spectrum\",\n        \"Whether acrosomal ultrastructural defects are secondary to acrosin loss or a direct structural role is unresolved\",\n        \"No crystal structure of human acrosin exists\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The precise set of acrosomal and zona pellucida substrates cleaved by acrosin during zona penetration, the structural basis for the proenzyme region's enhanced ZP binding, and how acrosin coordinates with other acrosomal proteases remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No acrosin substrate profiling by proteomics has been performed\",\n        \"No crystal structure of acrosin or proacrosin–ZP complex exists\",\n        \"Mechanism of sp32-mediated regulation of proacrosin activation is unknown\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 10, 11, 12, 16, 17, 23]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 6, 8, 10, 16, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9, 12, 18, 23]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [14, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [11, 15, 16, 19, 20, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ZP2\",\n      \"ZP1\",\n      \"ZP3\",\n      \"SERPINA5\",\n      \"ACRV1\",\n      \"PRSS21\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}