{"gene":"CRISP2","run_date":"2026-04-28T17:28:53","timeline":{"discoveries":[{"year":2005,"finding":"The CRISP domain of mouse Tpx-1 (CRISP2) adopts a fold related to ion channel toxins (BgK and ShK) as determined by NMR solution structure, and this isolated CRISP domain inhibits cardiac ryanodine receptor RyR2 (IC50 0.5–1.0 µM) and activates skeletal RyR1 (AC50 1–10 µM) in bilayer assays, demonstrating that CRISP2's ion channel regulatory activity resides in its C-terminal CRISP domain.","method":"NMR solution structure; planar lipid bilayer electrophysiology with isolated recombinant CRISP domain; domain-deletion analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — NMR structure plus in vitro functional reconstitution with defined domain","pmids":["16339766"],"is_preprint":false},{"year":1998,"finding":"Rat Tpx-1 (CRISP2) acts as a spermatogenic cell adhesion molecule mediating specific binding of spermatogenic cells to Sertoli cells; a polyclonal antibody against Tpx-1 significantly inhibited this binding in primary testicular cell culture.","method":"Expression cloning; antibody-inhibition assay in primary cell culture","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — functional antibody block in a defined cell-adhesion assay, replicated by structural domain analysis in follow-up paper","pmids":["9675100"],"is_preprint":false},{"year":1999,"finding":"Structure–function analysis of rat Tpx-1 showed that the N-terminal 101 amino acids are sufficient for spermatogenic-cell–Sertoli-cell adhesion activity, whereas the C-terminal cysteine-rich region is dispensable for adhesion; the N-terminal signal peptide directs secretion of the protein.","method":"Deletion mutant expression in cultured cells; GFP-fusion secretion assay; primary testicular cell adhesion assay","journal":"Development, growth & differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — domain mapping with defined deletion mutants and functional readout","pmids":["10646801"],"is_preprint":false},{"year":2001,"finding":"Tpx-1 (CRISP2) protein is localized to two distinct sperm compartments in rat spermatids: the outer dense fibers (ODF) of the sperm tail and the acrosome, existing as 25 and 27 kDa isoforms, with translational delay of 4–5 days after mRNA expression and incorporation into ODFs consistent with their development.","method":"Immunohistochemistry; immunoelectron microscopy; Western blotting of purified sperm tail fractions","journal":"Molecular reproduction and development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal localization methods with subcellular resolution","pmids":["11144214"],"is_preprint":false},{"year":2005,"finding":"Human TPX1/CRISP2 is an intra-acrosomal protein in fresh sperm that remains associated with the equatorial segment of the acrosome after capacitation and acrosome reaction; anti-TPX1 antibody caused dose-dependent inhibition of zona-free hamster oocyte penetration without affecting motility or acrosome reaction, implicating CRISP2 in sperm–oocyte fusion.","method":"Indirect immunofluorescence; protein extraction fractionation; hamster oocyte penetration assay with antibody inhibition","journal":"Molecular human reproduction","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods; functional antibody block with defined controls","pmids":["15734896"],"is_preprint":false},{"year":2007,"finding":"Mouse CRISP2 is an intra-acrosomal component that remains on sperm after capacitation and acrosome reaction; anti-CRISP2 antibody reduced zona pellucida-intact egg penetration in IVF by accumulating perivitelline sperm, and recombinant CRISP2 bound specifically to the fusogenic area of mouse eggs, competing with CRISP1 for common egg-surface sites, establishing a role in sperm–egg fusion.","method":"Indirect immunofluorescence; protein extraction; in vitro fertilization antibody-inhibition assay; recombinant protein egg-binding competition assay","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with defined competition controls","pmids":["17202389"],"is_preprint":false},{"year":2008,"finding":"Using yeast two-hybrid screening of a mouse testis library, CRISP2 was found to interact with gametogenetin 1 (GGN1) through the ion channel regulatory region of the CRISP domain and the C-terminal 158 amino acids of GGN1; GGN1 co-localizes with CRISP2 in the principal piece of the sperm tail, and CRISP2 does not bind GGN2 or GGN3 isoforms.","method":"Yeast two-hybrid screen; co-immunoprecipitation; immunofluorescence co-localization; isoform-specificity mapping","journal":"Reproduction (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal interaction confirmed by Y2H and co-IP with domain mapping","pmids":["18502891"],"is_preprint":false},{"year":2008,"finding":"The C196R polymorphism in human CRISP2, which removes a conserved disulfide-bonding cysteine, abolishes CRISP2–GGN1 binding as demonstrated by yeast two-hybrid assay, defining the structural requirement of this residue for protein–protein interaction.","method":"Yeast two-hybrid assay with wild-type and C196R mutant CRISP2","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 — point-mutation functional test in defined interaction assay","pmids":["18550510"],"is_preprint":false},{"year":2009,"finding":"CRISP2 interacts with a novel protein SHTAP (sperm head and tail associated protein) via both the CAP and CRISP domains of CRISP2; the ~26 kDa SHTAP isoform mediates this interaction, and the SHTAP–CRISP2 complex redistributes within the sperm head during capacitation, suggesting a role in sperm functional competence.","method":"Yeast two-hybrid screen; domain-deletion mapping; immunofluorescence co-localization; immunoblotting","journal":"Biology of the cell","confidence":"Medium","confidence_rationale":"Tier 3 — Y2H plus localization; domain mapping; no Co-IP confirmation","pmids":["19686095"],"is_preprint":false},{"year":2013,"finding":"DAZAP1 promotes inclusion of CRISP2 exon 9 by binding to a regulatory region in CRISP2 intron 9; loss of DAZAP1 in mouse testes causes aberrant splicing of Crisp2, as shown by microarray exon usage analysis and splicing reporter assays.","method":"Microarray exon-usage profiling; splicing minigene reporter assay; RNA-binding domain analysis in cultured cells","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 — minigene functional validation plus in vivo exon-usage data","pmids":["23965306"],"is_preprint":false},{"year":2014,"finding":"miR-27b suppresses CRISP2 protein expression (but not mRNA) in human sperm by directly binding the 3′ UTR of CRISP2, as shown by luciferase reporter assay and transfection experiments; elevated miR-27b in asthenozoospermic sperm correlates with reduced CRISP2 protein.","method":"Luciferase 3′ UTR reporter assay; transfection with miR-27b mimic/inhibitor; Western blot; clinical correlation","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3′ UTR binding validated by reporter assay with mechanistic follow-up","pmids":["25505194"],"is_preprint":false},{"year":2016,"finding":"Crisp2-knockout mice exhibit defects in sperm hyperactivation, intracellular Ca2+ regulation during capacitation, penetration of cumulus and zona pellucida, and egg fusion, resulting in subfertility under demanding reproductive conditions, establishing CRISP2 as a regulator of Ca2+-dependent sperm function required for fertilization.","method":"Crisp2 knockout mouse; in vitro fertilization; computer-assisted sperm analysis (CASA); flow cytometry for Ca2+ levels; Western blot for tyrosine phosphorylation; acrosome reaction assay","journal":"Molecular human reproduction","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with multiple orthogonal sperm functional readouts","pmids":["26786179"],"is_preprint":false},{"year":2019,"finding":"CRISP2 binds to the CATSPER1 subunit of the CatSper ion channel (essential for sperm motility) as revealed by yeast two-hybrid screen and immunoprecipitation; Crisp2-deficient sperm have a stiff midpiece and abnormal flagellum waveform, causing subfertility and impaired acrosome reaction.","method":"Yeast two-hybrid screen; co-immunoprecipitation; Crisp2 loss-of-function mouse model; sperm motility and flagellum waveform analysis; acrosome reaction assay","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 — KO mouse phenotype plus reciprocal Co-IP identifying specific binding partner","pmids":["30759213"],"is_preprint":false},{"year":2021,"finding":"Porcine CRISP2 forms distinct oligomers in the sperm tail (insensitive to reducing conditions, dissociated by 8 M urea) and sperm head perinuclear theca (250 kDa complex dissociated by reduction of disulfide bonds), demonstrating that CRISP2 participates in structurally differentiated protein complexes in different sperm compartments.","method":"Native/non-reducing gel electrophoresis; immunogold electron microscopy; confocal immunofluorescence; sequential detergent/salt extraction; Western blot","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal biochemical and ultrastructural methods","pmids":["34309660"],"is_preprint":false},{"year":2022,"finding":"PSP94 inhibits the sterol-binding and sterol-export function of CRISP2 in a calcium-sensitive manner: coexpression of PSP94 with CRISP2 in yeast abolishes sterol export, PSP94–CRISP2 interaction inhibits sterol binding in vitro, and high calcium concentrations disrupt the PSP94–CRISP2 heteromeric complex, restoring sterol binding.","method":"Yeast sterol export assay (in vivo); in vitro sterol-binding assay; mutagenesis of interaction interface; Ca2+-dependent complex dissociation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus yeast functional assay plus mutagenesis with orthogonal readouts","pmids":["35063506"],"is_preprint":false},{"year":2022,"finding":"During in vitro capacitation, porcine sperm CRISP2 redistributes within the head (appearing on the apical ridge and equatorial segment); after acrosome reaction it is lost from the apical ridge; after fertilization, CRISP2 rapidly disperses from the perinuclear theca while the sperm nucleus is still condensed, a process likely driven by reduction of disulfide bonds within CRISP2 oligomers.","method":"Confocal immunofluorescence on sperm at defined functional stages; IVF followed by CRISP2 immunostaining of zygotes","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — direct imaging through defined functional stages with mechanistic inference from biochemical data","pmids":["36054334"],"is_preprint":false},{"year":2023,"finding":"In boar sperm, CRISP2 forms a ~150 kDa complex under non-capacitating, capacitated, and acrosome-reacted conditions; mass spectrometry of immunoprecipitated CRISP2 complexes and blue-native gel bands identified acrosin and acrosin-binding protein as the most abundant CRISP2 interaction partners under all conditions, validated by proximity ligation assay; CRISP2 interacts with pro-acrosin (~53 kDa) and acrosin-binding protein consistently, and additionally with acrosin (~35 kDa) post-capacitation.","method":"Blue-native gel electrophoresis; co-immunoprecipitation; LC-MS/MS; proximity ligation assay (in situ)","journal":"Andrology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus MS identification plus in situ PLA validation; multiple conditions tested","pmids":["36815564"],"is_preprint":false},{"year":2024,"finding":"Alpha-1-B glycoprotein (A1BG) inhibits CRISP2 sterol-binding and sterol-export function; the interaction maps to the third immunoglobulin-like domain of A1BG and requires magnesium coordination by conserved tetrad residues in the CRISP2 CAP domain, as shown by domain-mapping mutagenesis and yeast sterol-export and in vitro sterol-binding assays.","method":"Yeast sterol export assay; in vitro sterol-binding assay; A1BG domain-deletion mapping; magnesium-dependence assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus yeast functional assay plus domain/ion-dependence mutagenesis","pmids":["39433128"],"is_preprint":false},{"year":2025,"finding":"Human CRISP2 is present in the nucleus of primary spermatocytes and round/early elongated spermatids, then additionally in the cytoplasm, flagellum, and equatorial segment in elongated spermatids; in ejaculated sperm it is in the cytoplasmic droplet, flagellum, and equatorial segment. Native gel electrophoresis and mass spectrometry revealed that hCRISP2 forms stable high-molecular-weight complexes consisting exclusively of CRISP2, and the protein undergoes only limited post-translational modifications.","method":"Immunofluorescence on testis/epididymis/sperm; native and denaturing electrophoresis; Western blot; mass spectrometry","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods; MS-confirmed complex composition","pmids":["40079119"],"is_preprint":false}],"current_model":"CRISP2 is a testis-specific, two-domain secretory protein (CAP + CRISP domain) incorporated into sperm acrosome and tail structures during spermatogenesis; its CRISP domain adopts an ion-channel-toxin-like fold that directly regulates ryanodine receptor Ca2+ gating (inhibiting RyR2, activating RyR1) and binds CATSPER1 to govern flagellar waveform and hyperactivation, while the full-length protein mediates spermatogenic-cell–Sertoli-cell adhesion, participates in acrosomal protein complexes with acrosin and acrosin-binding protein, binds GGN1 and SHTAP in the sperm tail, performs Ca2+-sensitive binding to PSP94 and Mg2+-dependent binding to A1BG that modulate its sterol-binding/export activity, and engages the egg fusogenic surface to enable sperm–egg fusion, with its expression post-transcriptionally regulated by miR-27b and its pre-mRNA splicing regulated by DAZAP1."},"narrative":{"teleology":[{"year":1998,"claim":"Establishing that CRISP2 is not merely a structural sperm protein but functions as a cell-adhesion molecule mediating spermatogenic cell–Sertoli cell interactions during spermatogenesis.","evidence":"Antibody inhibition of Tpx-1 blocked spermatogenic cell–Sertoli cell binding in primary rat testicular cell culture","pmids":["9675100"],"confidence":"Medium","gaps":["Identity of the Sertoli cell surface receptor is unknown","Adhesion function not confirmed in vivo or by genetic loss-of-function"]},{"year":1999,"claim":"Mapping the adhesion function to the N-terminal 101 residues (CAP domain) and showing the C-terminal cysteine-rich region is dispensable for adhesion, establishing a modular architecture with separable functions.","evidence":"Deletion mutant expression and secretion assays in cultured cells coupled with primary testicular cell adhesion assay","pmids":["10646801"],"confidence":"Medium","gaps":["Ligand on Sertoli cell surface remains unidentified","No in vivo validation of domain requirements"]},{"year":2001,"claim":"Demonstrating that CRISP2 localizes to two distinct sperm compartments—acrosome and outer dense fibers—indicating dual structural/functional roles in head and tail.","evidence":"Immunohistochemistry and immunoelectron microscopy on rat spermatids with fractionation biochemistry","pmids":["11144214"],"confidence":"High","gaps":["Functional significance of tail versus head pools not yet separated","Mechanism of targeting to ODFs unknown"]},{"year":2005,"claim":"Revealing the molecular mechanism by which CRISP2 regulates ion channels: the CRISP domain adopts a toxin-like fold and directly modulates ryanodine receptors, providing the first biochemical activity for the protein.","evidence":"NMR solution structure of the isolated CRISP domain; planar lipid bilayer electrophysiology showing inhibition of RyR2 and activation of RyR1","pmids":["16339766"],"confidence":"High","gaps":["Whether ryanodine receptors are the physiological targets in sperm is not established","In vivo relevance of differential RyR1/RyR2 modulation unclear"]},{"year":2005,"claim":"Establishing a role for CRISP2 in sperm–egg fusion: the protein persists on the equatorial segment after acrosome reaction and antibody blockade inhibits oocyte penetration without affecting motility.","evidence":"Immunofluorescence on human sperm at defined capacitation stages; hamster oocyte penetration assay with anti-TPX1 antibody inhibition","pmids":["15734896"],"confidence":"High","gaps":["Egg-surface receptor identity unknown","Heterologous (hamster egg) assay may not fully recapitulate human fusion"]},{"year":2007,"claim":"Demonstrating that CRISP2 binds the fusogenic area of the egg surface and competes with CRISP1 for shared binding sites, placing both CRISPs in a common fusion pathway.","evidence":"Recombinant mouse CRISP2 egg-binding and competition assays; IVF antibody-inhibition with zona-intact eggs","pmids":["17202389"],"confidence":"High","gaps":["Molecular identity of egg-surface binding partner remains unknown","Redundancy with CRISP1 not fully resolved"]},{"year":2008,"claim":"Identifying GGN1 as a CRISP2-interacting partner in the sperm tail principal piece, with interaction mapped to the ion-channel-regulatory region of the CRISP domain, and showing the C196R SNP abolishes this interaction.","evidence":"Yeast two-hybrid screen, co-immunoprecipitation, co-localization; point-mutant Y2H assay","pmids":["18502891","18550510"],"confidence":"Medium","gaps":["Functional consequence of CRISP2–GGN1 interaction on sperm motility not tested","C196R variant not assessed in a loss-of-function animal model"]},{"year":2009,"claim":"Identifying SHTAP as a second sperm tail/head partner that binds both CRISP2 domains and redistributes during capacitation, expanding the CRISP2 interactome.","evidence":"Yeast two-hybrid with domain deletions; immunofluorescence co-localization on sperm","pmids":["19686095"],"confidence":"Medium","gaps":["No co-IP confirmation of SHTAP–CRISP2 interaction","Functional role of this complex is undefined"]},{"year":2013,"claim":"Showing that CRISP2 pre-mRNA splicing is regulated by DAZAP1, which promotes exon 9 inclusion by binding intron 9, linking CRISP2 isoform diversity to a known RNA-binding protein.","evidence":"Microarray exon-usage profiling in Dazap1 knockout testes; splicing minigene reporter assay","pmids":["23965306"],"confidence":"Medium","gaps":["Functional difference between exon 9-included and exon 9-skipped CRISP2 isoforms not characterized","Whether altered splicing affects fertility outcomes is untested"]},{"year":2014,"claim":"Establishing post-transcriptional repression of CRISP2 by miR-27b, with clinical relevance to asthenozoospermia where elevated miR-27b correlates with reduced CRISP2 protein.","evidence":"Luciferase 3′ UTR reporter assay; miR-27b mimic/inhibitor transfection; Western blot; clinical correlation in asthenozoospermic samples","pmids":["25505194"],"confidence":"Medium","gaps":["Causal relationship between miR-27b-mediated CRISP2 reduction and motility defects not proven in vivo","Other miRNA regulators not surveyed"]},{"year":2016,"claim":"Genetic loss-of-function definitively established CRISP2 as a regulator of Ca²⁺-dependent sperm hyperactivation, zona/cumulus penetration, and egg fusion, with knockout mice showing subfertility under competitive conditions.","evidence":"Crisp2 knockout mouse with CASA, flow cytometry Ca²⁺ measurement, IVF, acrosome reaction assay","pmids":["26786179"],"confidence":"High","gaps":["Molecular targets downstream of CRISP2 that mediate Ca²⁺ regulation not identified in this study","Redundancy with CRISP1/CRISP3/CRISP4 not fully dissected"]},{"year":2019,"claim":"Identifying CATSPER1 as a direct binding partner of CRISP2, connecting its ion-channel-regulatory activity to the principal Ca²⁺ channel controlling sperm motility and explaining the stiff-midpiece/abnormal waveform phenotype of Crisp2-deficient sperm.","evidence":"Yeast two-hybrid screen; co-immunoprecipitation; flagellum waveform analysis in Crisp2 loss-of-function sperm","pmids":["30759213"],"confidence":"High","gaps":["Whether CRISP2 activates, inhibits, or allosterically modulates CatSper channel conductance is unknown","Structural basis of CRISP2–CATSPER1 interaction not resolved"]},{"year":2021,"claim":"Demonstrating that CRISP2 forms structurally distinct oligomeric assemblies in the sperm tail versus head, with different biochemical stabilities, revealing compartment-specific quaternary organization.","evidence":"Native/non-reducing gel electrophoresis; immunogold EM; sequential extraction of porcine sperm","pmids":["34309660"],"confidence":"Medium","gaps":["Stoichiometry and exact composition of oligomers not determined","Functional significance of distinct oligomeric states untested"]},{"year":2022,"claim":"Uncovering a sterol-binding/export function for CRISP2 that is inhibited by PSP94 in a Ca²⁺-sensitive manner, revealing a lipid-handling role and a physiological regulatory switch tied to the ionic milieu of the reproductive tract.","evidence":"Yeast sterol-export assay; in vitro sterol-binding assay; mutagenesis of PSP94 interaction interface; Ca²⁺-dependent complex dissociation","pmids":["35063506"],"confidence":"High","gaps":["Whether sterol binding is relevant in mammalian sperm membranes in vivo is untested","Identity of physiological sterol cargo unclear"]},{"year":2022,"claim":"Tracking CRISP2 redistribution through capacitation, acrosome reaction, and fertilization showed rapid post-fertilization dispersal from the perinuclear theca, likely driven by disulfide reduction, linking CRISP2 complex disassembly to early zygote remodeling.","evidence":"Confocal immunofluorescence on porcine sperm at defined stages; IVF followed by CRISP2 immunostaining of zygotes","pmids":["36054334"],"confidence":"Medium","gaps":["Reductase responsible for disulfide reduction not identified","Functional consequence of perinuclear theca dispersal for paternal chromatin remodeling unknown"]},{"year":2023,"claim":"Identifying acrosin and acrosin-binding protein as the primary stable partners of CRISP2 in the acrosomal ~150 kDa complex under non-capacitating, capacitated, and acrosome-reacted conditions, defining the core acrosomal CRISP2 interactome.","evidence":"Blue-native gel; co-IP plus LC-MS/MS; proximity ligation assay on boar sperm","pmids":["36815564"],"confidence":"High","gaps":["Functional role of the CRISP2–acrosin–ACRBP complex in acrosome reaction or zona penetration untested","Whether this complex exists in human sperm not confirmed"]},{"year":2024,"claim":"Demonstrating that A1BG inhibits CRISP2 sterol function through Mg²⁺-dependent binding to conserved CAP domain residues, establishing a second serum protein (after PSP94) that regulates CRISP2 lipid-handling activity via divalent cation-dependent interactions.","evidence":"Yeast sterol-export assay; in vitro sterol-binding assay; A1BG domain-deletion and Mg²⁺-coordination mutagenesis","pmids":["39433128"],"confidence":"High","gaps":["Physiological context where A1BG and CRISP2 interact (seminal plasma vs. serum) not established","Whether A1BG regulation is relevant to sperm function in vivo is unknown"]},{"year":2025,"claim":"Mapping the dynamic localization of human CRISP2 from nuclear in spermatocytes through cytoplasmic/flagellar in elongated spermatids to equatorial/flagellar in ejaculated sperm, and showing that human CRISP2 forms high-MW homo-oligomeric complexes with minimal post-translational modification.","evidence":"Immunofluorescence across human spermatogenesis; native gel electrophoresis; mass spectrometry of purified complexes","pmids":["40079119"],"confidence":"Medium","gaps":["Function of nuclear CRISP2 in spermatocytes is unknown","Whether homo-oligomers are functionally equivalent to the porcine heteromeric complexes containing acrosin is unclear"]},{"year":null,"claim":"The egg-surface receptor for CRISP2, the structural basis of its CATSPER1 modulation, the in vivo relevance of its sterol-binding activity in mammalian sperm, and the function of nuclear CRISP2 in spermatocytes remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["Egg-surface receptor identity unknown","No structure of CRISP2–CATSPER1 complex","In vivo sterol-handling function not demonstrated in mammalian sperm","Role of nuclear CRISP2 in meiotic spermatocytes undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,12,14,17]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[14,17]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[1,2,4,5]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[18]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[3,6,13]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[4,5,15]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[4,5,11,12]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,14,17]}],"complexes":["CRISP2–acrosin–ACRBP acrosomal complex","CRISP2 homo-oligomeric complex (tail/head)","CRISP2–PSP94 heteromeric complex"],"partners":["CATSPER1","ACR","ACRBP","GGN1","SHTAP","PSP94","A1BG","CRISP1"],"other_free_text":[]},"mechanistic_narrative":"CRISP2 is a testis-enriched, two-domain secretory protein that functions as a multivalent regulator of sperm assembly, ion channel activity, sterol homeostasis, and gamete fusion. Its C-terminal CRISP domain adopts an ion-channel-toxin-like fold that directly modulates ryanodine receptors (inhibiting RyR2, activating RyR1) and binds the CATSPER1 subunit to govern flagellar waveform and Ca²⁺-dependent hyperactivation, while Crisp2-knockout mice exhibit defective sperm hyperactivation, impaired cumulus/zona penetration, and subfertility [PMID:16339766, PMID:30759213, PMID:26786179]. The N-terminal CAP domain mediates spermatogenic cell–Sertoli cell adhesion, Mg²⁺-dependent binding to A1BG, and sterol-binding/export activity that is inhibited by PSP94 in a Ca²⁺-sensitive manner [PMID:10646801, PMID:35063506, PMID:39433128]. During spermiogenesis CRISP2 is incorporated into distinct compartments—the acrosome (where it forms stable complexes with acrosin and acrosin-binding protein) and the sperm tail outer dense fibers—and after capacitation it redistributes to the equatorial segment to participate in sperm–egg fusion by engaging complementary sites on the egg surface [PMID:15734896, PMID:17202389, PMID:36815564, PMID:34309660]."},"prefetch_data":{"uniprot":{"accession":"P16562","full_name":"Cysteine-rich secretory protein 2","aliases":["Cancer/testis antigen 36","CT36","Testis-specific protein TPX-1"],"length_aa":243,"mass_kda":27.3,"function":"May regulate some ion channels' activity and thereby regulate calcium fluxes during sperm capacitation","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P16562/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CRISP2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CRISP2","total_profiled":1310},"omim":[{"mim_id":"618062","title":"CYSTEINE-RICH SECRETORY PROTEIN 3; CRISP3","url":"https://www.omim.org/entry/618062"},{"mim_id":"609966","title":"GAMETOGENETIN; GGN","url":"https://www.omim.org/entry/609966"},{"mim_id":"187430","title":"CYSTEINE-RICH SECRETORY PROTEIN 2; CRISP2","url":"https://www.omim.org/entry/187430"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"testis","ntpm":482.3}],"url":"https://www.proteinatlas.org/search/CRISP2"},"hgnc":{"alias_symbol":["CRISP-2","CT36"],"prev_symbol":["GAPDL5","TPX1"]},"alphafold":{"accession":"P16562","domains":[{"cath_id":"3.40.33.10","chopping":"25-169","consensus_level":"high","plddt":97.6679,"start":25,"end":169},{"cath_id":"1.10.10.740","chopping":"205-243","consensus_level":"medium","plddt":93.6318,"start":205,"end":243}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P16562","model_url":"https://alphafold.ebi.ac.uk/files/AF-P16562-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P16562-F1-predicted_aligned_error_v6.png","plddt_mean":93.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CRISP2","jax_strain_url":"https://www.jax.org/strain/search?query=CRISP2"},"sequence":{"accession":"P16562","fasta_url":"https://rest.uniprot.org/uniprotkb/P16562.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P16562/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P16562"}},"corpus_meta":[{"pmid":"8665901","id":"PMC_8665901","title":"The human cysteine-rich secretory protein (CRISP) family. Primary structure and tissue distribution of CRISP-1, CRISP-2 and CRISP-3.","date":"1996","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8665901","citation_count":182,"is_preprint":false},{"pmid":"16339766","id":"PMC_16339766","title":"The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16339766","citation_count":114,"is_preprint":false},{"pmid":"9115720","id":"PMC_9115720","title":"Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family.","date":"1996","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/9115720","citation_count":85,"is_preprint":false},{"pmid":"17202389","id":"PMC_17202389","title":"Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion.","date":"2007","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/17202389","citation_count":77,"is_preprint":false},{"pmid":"11144214","id":"PMC_11144214","title":"Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa.","date":"2001","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/11144214","citation_count":69,"is_preprint":false},{"pmid":"15734896","id":"PMC_15734896","title":"Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction.","date":"2005","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/15734896","citation_count":66,"is_preprint":false},{"pmid":"9675100","id":"PMC_9675100","title":"Molecular cloning of the rat Tpx-1 responsible for the interaction between spermatogenic and Sertoli cells.","date":"1998","source":"Biochemical and 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C-terminal CRISP domain.\",\n      \"method\": \"NMR solution structure; planar lipid bilayer electrophysiology with isolated recombinant CRISP domain; domain-deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure plus in vitro functional reconstitution with defined domain\",\n      \"pmids\": [\"16339766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rat Tpx-1 (CRISP2) acts as a spermatogenic cell adhesion molecule mediating specific binding of spermatogenic cells to Sertoli cells; a polyclonal antibody against Tpx-1 significantly inhibited this binding in primary testicular cell culture.\",\n      \"method\": \"Expression cloning; antibody-inhibition assay in primary cell culture\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional antibody block in a defined cell-adhesion assay, replicated by structural domain analysis in follow-up paper\",\n      \"pmids\": [\"9675100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Structure–function analysis of rat Tpx-1 showed that the N-terminal 101 amino acids are sufficient for spermatogenic-cell–Sertoli-cell adhesion activity, whereas the C-terminal cysteine-rich region is dispensable for adhesion; the N-terminal signal peptide directs secretion of the protein.\",\n      \"method\": \"Deletion mutant expression in cultured cells; GFP-fusion secretion assay; primary testicular cell adhesion assay\",\n      \"journal\": \"Development, growth & differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with defined deletion mutants and functional readout\",\n      \"pmids\": [\"10646801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Tpx-1 (CRISP2) protein is localized to two distinct sperm compartments in rat spermatids: the outer dense fibers (ODF) of the sperm tail and the acrosome, existing as 25 and 27 kDa isoforms, with translational delay of 4–5 days after mRNA expression and incorporation into ODFs consistent with their development.\",\n      \"method\": \"Immunohistochemistry; immunoelectron microscopy; Western blotting of purified sperm tail fractions\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal localization methods with subcellular resolution\",\n      \"pmids\": [\"11144214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human TPX1/CRISP2 is an intra-acrosomal protein in fresh sperm that remains associated with the equatorial segment of the acrosome after capacitation and acrosome reaction; anti-TPX1 antibody caused dose-dependent inhibition of zona-free hamster oocyte penetration without affecting motility or acrosome reaction, implicating CRISP2 in sperm–oocyte fusion.\",\n      \"method\": \"Indirect immunofluorescence; protein extraction fractionation; hamster oocyte penetration assay with antibody inhibition\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods; functional antibody block with defined controls\",\n      \"pmids\": [\"15734896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mouse CRISP2 is an intra-acrosomal component that remains on sperm after capacitation and acrosome reaction; anti-CRISP2 antibody reduced zona pellucida-intact egg penetration in IVF by accumulating perivitelline sperm, and recombinant CRISP2 bound specifically to the fusogenic area of mouse eggs, competing with CRISP1 for common egg-surface sites, establishing a role in sperm–egg fusion.\",\n      \"method\": \"Indirect immunofluorescence; protein extraction; in vitro fertilization antibody-inhibition assay; recombinant protein egg-binding competition assay\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with defined competition controls\",\n      \"pmids\": [\"17202389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Using yeast two-hybrid screening of a mouse testis library, CRISP2 was found to interact with gametogenetin 1 (GGN1) through the ion channel regulatory region of the CRISP domain and the C-terminal 158 amino acids of GGN1; GGN1 co-localizes with CRISP2 in the principal piece of the sperm tail, and CRISP2 does not bind GGN2 or GGN3 isoforms.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation; immunofluorescence co-localization; isoform-specificity mapping\",\n      \"journal\": \"Reproduction (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction confirmed by Y2H and co-IP with domain mapping\",\n      \"pmids\": [\"18502891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The C196R polymorphism in human CRISP2, which removes a conserved disulfide-bonding cysteine, abolishes CRISP2–GGN1 binding as demonstrated by yeast two-hybrid assay, defining the structural requirement of this residue for protein–protein interaction.\",\n      \"method\": \"Yeast two-hybrid assay with wild-type and C196R mutant CRISP2\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — point-mutation functional test in defined interaction assay\",\n      \"pmids\": [\"18550510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CRISP2 interacts with a novel protein SHTAP (sperm head and tail associated protein) via both the CAP and CRISP domains of CRISP2; the ~26 kDa SHTAP isoform mediates this interaction, and the SHTAP–CRISP2 complex redistributes within the sperm head during capacitation, suggesting a role in sperm functional competence.\",\n      \"method\": \"Yeast two-hybrid screen; domain-deletion mapping; immunofluorescence co-localization; immunoblotting\",\n      \"journal\": \"Biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Y2H plus localization; domain mapping; no Co-IP confirmation\",\n      \"pmids\": [\"19686095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"DAZAP1 promotes inclusion of CRISP2 exon 9 by binding to a regulatory region in CRISP2 intron 9; loss of DAZAP1 in mouse testes causes aberrant splicing of Crisp2, as shown by microarray exon usage analysis and splicing reporter assays.\",\n      \"method\": \"Microarray exon-usage profiling; splicing minigene reporter assay; RNA-binding domain analysis in cultured cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — minigene functional validation plus in vivo exon-usage data\",\n      \"pmids\": [\"23965306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"miR-27b suppresses CRISP2 protein expression (but not mRNA) in human sperm by directly binding the 3′ UTR of CRISP2, as shown by luciferase reporter assay and transfection experiments; elevated miR-27b in asthenozoospermic sperm correlates with reduced CRISP2 protein.\",\n      \"method\": \"Luciferase 3′ UTR reporter assay; transfection with miR-27b mimic/inhibitor; Western blot; clinical correlation\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3′ UTR binding validated by reporter assay with mechanistic follow-up\",\n      \"pmids\": [\"25505194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crisp2-knockout mice exhibit defects in sperm hyperactivation, intracellular Ca2+ regulation during capacitation, penetration of cumulus and zona pellucida, and egg fusion, resulting in subfertility under demanding reproductive conditions, establishing CRISP2 as a regulator of Ca2+-dependent sperm function required for fertilization.\",\n      \"method\": \"Crisp2 knockout mouse; in vitro fertilization; computer-assisted sperm analysis (CASA); flow cytometry for Ca2+ levels; Western blot for tyrosine phosphorylation; acrosome reaction assay\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple orthogonal sperm functional readouts\",\n      \"pmids\": [\"26786179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRISP2 binds to the CATSPER1 subunit of the CatSper ion channel (essential for sperm motility) as revealed by yeast two-hybrid screen and immunoprecipitation; Crisp2-deficient sperm have a stiff midpiece and abnormal flagellum waveform, causing subfertility and impaired acrosome reaction.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation; Crisp2 loss-of-function mouse model; sperm motility and flagellum waveform analysis; acrosome reaction assay\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse phenotype plus reciprocal Co-IP identifying specific binding partner\",\n      \"pmids\": [\"30759213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Porcine CRISP2 forms distinct oligomers in the sperm tail (insensitive to reducing conditions, dissociated by 8 M urea) and sperm head perinuclear theca (250 kDa complex dissociated by reduction of disulfide bonds), demonstrating that CRISP2 participates in structurally differentiated protein complexes in different sperm compartments.\",\n      \"method\": \"Native/non-reducing gel electrophoresis; immunogold electron microscopy; confocal immunofluorescence; sequential detergent/salt extraction; Western blot\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical and ultrastructural methods\",\n      \"pmids\": [\"34309660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PSP94 inhibits the sterol-binding and sterol-export function of CRISP2 in a calcium-sensitive manner: coexpression of PSP94 with CRISP2 in yeast abolishes sterol export, PSP94–CRISP2 interaction inhibits sterol binding in vitro, and high calcium concentrations disrupt the PSP94–CRISP2 heteromeric complex, restoring sterol binding.\",\n      \"method\": \"Yeast sterol export assay (in vivo); in vitro sterol-binding assay; mutagenesis of interaction interface; Ca2+-dependent complex dissociation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus yeast functional assay plus mutagenesis with orthogonal readouts\",\n      \"pmids\": [\"35063506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"During in vitro capacitation, porcine sperm CRISP2 redistributes within the head (appearing on the apical ridge and equatorial segment); after acrosome reaction it is lost from the apical ridge; after fertilization, CRISP2 rapidly disperses from the perinuclear theca while the sperm nucleus is still condensed, a process likely driven by reduction of disulfide bonds within CRISP2 oligomers.\",\n      \"method\": \"Confocal immunofluorescence on sperm at defined functional stages; IVF followed by CRISP2 immunostaining of zygotes\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging through defined functional stages with mechanistic inference from biochemical data\",\n      \"pmids\": [\"36054334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In boar sperm, CRISP2 forms a ~150 kDa complex under non-capacitating, capacitated, and acrosome-reacted conditions; mass spectrometry of immunoprecipitated CRISP2 complexes and blue-native gel bands identified acrosin and acrosin-binding protein as the most abundant CRISP2 interaction partners under all conditions, validated by proximity ligation assay; CRISP2 interacts with pro-acrosin (~53 kDa) and acrosin-binding protein consistently, and additionally with acrosin (~35 kDa) post-capacitation.\",\n      \"method\": \"Blue-native gel electrophoresis; co-immunoprecipitation; LC-MS/MS; proximity ligation assay (in situ)\",\n      \"journal\": \"Andrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus MS identification plus in situ PLA validation; multiple conditions tested\",\n      \"pmids\": [\"36815564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Alpha-1-B glycoprotein (A1BG) inhibits CRISP2 sterol-binding and sterol-export function; the interaction maps to the third immunoglobulin-like domain of A1BG and requires magnesium coordination by conserved tetrad residues in the CRISP2 CAP domain, as shown by domain-mapping mutagenesis and yeast sterol-export and in vitro sterol-binding assays.\",\n      \"method\": \"Yeast sterol export assay; in vitro sterol-binding assay; A1BG domain-deletion mapping; magnesium-dependence assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus yeast functional assay plus domain/ion-dependence mutagenesis\",\n      \"pmids\": [\"39433128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Human CRISP2 is present in the nucleus of primary spermatocytes and round/early elongated spermatids, then additionally in the cytoplasm, flagellum, and equatorial segment in elongated spermatids; in ejaculated sperm it is in the cytoplasmic droplet, flagellum, and equatorial segment. Native gel electrophoresis and mass spectrometry revealed that hCRISP2 forms stable high-molecular-weight complexes consisting exclusively of CRISP2, and the protein undergoes only limited post-translational modifications.\",\n      \"method\": \"Immunofluorescence on testis/epididymis/sperm; native and denaturing electrophoresis; Western blot; mass spectrometry\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods; MS-confirmed complex composition\",\n      \"pmids\": [\"40079119\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CRISP2 is a testis-specific, two-domain secretory protein (CAP + CRISP domain) incorporated into sperm acrosome and tail structures during spermatogenesis; its CRISP domain adopts an ion-channel-toxin-like fold that directly regulates ryanodine receptor Ca2+ gating (inhibiting RyR2, activating RyR1) and binds CATSPER1 to govern flagellar waveform and hyperactivation, while the full-length protein mediates spermatogenic-cell–Sertoli-cell adhesion, participates in acrosomal protein complexes with acrosin and acrosin-binding protein, binds GGN1 and SHTAP in the sperm tail, performs Ca2+-sensitive binding to PSP94 and Mg2+-dependent binding to A1BG that modulate its sterol-binding/export activity, and engages the egg fusogenic surface to enable sperm–egg fusion, with its expression post-transcriptionally regulated by miR-27b and its pre-mRNA splicing regulated by DAZAP1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CRISP2 is a testis-enriched, two-domain secretory protein that functions as a multivalent regulator of sperm assembly, ion channel activity, sterol homeostasis, and gamete fusion. Its C-terminal CRISP domain adopts an ion-channel-toxin-like fold that directly modulates ryanodine receptors (inhibiting RyR2, activating RyR1) and binds the CATSPER1 subunit to govern flagellar waveform and Ca²⁺-dependent hyperactivation, while Crisp2-knockout mice exhibit defective sperm hyperactivation, impaired cumulus/zona penetration, and subfertility [PMID:16339766, PMID:30759213, PMID:26786179]. The N-terminal CAP domain mediates spermatogenic cell–Sertoli cell adhesion, Mg²⁺-dependent binding to A1BG, and sterol-binding/export activity that is inhibited by PSP94 in a Ca²⁺-sensitive manner [PMID:10646801, PMID:35063506, PMID:39433128]. During spermiogenesis CRISP2 is incorporated into distinct compartments—the acrosome (where it forms stable complexes with acrosin and acrosin-binding protein) and the sperm tail outer dense fibers—and after capacitation it redistributes to the equatorial segment to participate in sperm–egg fusion by engaging complementary sites on the egg surface [PMID:15734896, PMID:17202389, PMID:36815564, PMID:34309660].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that CRISP2 is not merely a structural sperm protein but functions as a cell-adhesion molecule mediating spermatogenic cell–Sertoli cell interactions during spermatogenesis.\",\n      \"evidence\": \"Antibody inhibition of Tpx-1 blocked spermatogenic cell–Sertoli cell binding in primary rat testicular cell culture\",\n      \"pmids\": [\"9675100\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the Sertoli cell surface receptor is unknown\", \"Adhesion function not confirmed in vivo or by genetic loss-of-function\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping the adhesion function to the N-terminal 101 residues (CAP domain) and showing the C-terminal cysteine-rich region is dispensable for adhesion, establishing a modular architecture with separable functions.\",\n      \"evidence\": \"Deletion mutant expression and secretion assays in cultured cells coupled with primary testicular cell adhesion assay\",\n      \"pmids\": [\"10646801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ligand on Sertoli cell surface remains unidentified\", \"No in vivo validation of domain requirements\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that CRISP2 localizes to two distinct sperm compartments—acrosome and outer dense fibers—indicating dual structural/functional roles in head and tail.\",\n      \"evidence\": \"Immunohistochemistry and immunoelectron microscopy on rat spermatids with fractionation biochemistry\",\n      \"pmids\": [\"11144214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of tail versus head pools not yet separated\", \"Mechanism of targeting to ODFs unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealing the molecular mechanism by which CRISP2 regulates ion channels: the CRISP domain adopts a toxin-like fold and directly modulates ryanodine receptors, providing the first biochemical activity for the protein.\",\n      \"evidence\": \"NMR solution structure of the isolated CRISP domain; planar lipid bilayer electrophysiology showing inhibition of RyR2 and activation of RyR1\",\n      \"pmids\": [\"16339766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ryanodine receptors are the physiological targets in sperm is not established\", \"In vivo relevance of differential RyR1/RyR2 modulation unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing a role for CRISP2 in sperm–egg fusion: the protein persists on the equatorial segment after acrosome reaction and antibody blockade inhibits oocyte penetration without affecting motility.\",\n      \"evidence\": \"Immunofluorescence on human sperm at defined capacitation stages; hamster oocyte penetration assay with anti-TPX1 antibody inhibition\",\n      \"pmids\": [\"15734896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Egg-surface receptor identity unknown\", \"Heterologous (hamster egg) assay may not fully recapitulate human fusion\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that CRISP2 binds the fusogenic area of the egg surface and competes with CRISP1 for shared binding sites, placing both CRISPs in a common fusion pathway.\",\n      \"evidence\": \"Recombinant mouse CRISP2 egg-binding and competition assays; IVF antibody-inhibition with zona-intact eggs\",\n      \"pmids\": [\"17202389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of egg-surface binding partner remains unknown\", \"Redundancy with CRISP1 not fully resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying GGN1 as a CRISP2-interacting partner in the sperm tail principal piece, with interaction mapped to the ion-channel-regulatory region of the CRISP domain, and showing the C196R SNP abolishes this interaction.\",\n      \"evidence\": \"Yeast two-hybrid screen, co-immunoprecipitation, co-localization; point-mutant Y2H assay\",\n      \"pmids\": [\"18502891\", \"18550510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of CRISP2–GGN1 interaction on sperm motility not tested\", \"C196R variant not assessed in a loss-of-function animal model\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying SHTAP as a second sperm tail/head partner that binds both CRISP2 domains and redistributes during capacitation, expanding the CRISP2 interactome.\",\n      \"evidence\": \"Yeast two-hybrid with domain deletions; immunofluorescence co-localization on sperm\",\n      \"pmids\": [\"19686095\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No co-IP confirmation of SHTAP–CRISP2 interaction\", \"Functional role of this complex is undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Showing that CRISP2 pre-mRNA splicing is regulated by DAZAP1, which promotes exon 9 inclusion by binding intron 9, linking CRISP2 isoform diversity to a known RNA-binding protein.\",\n      \"evidence\": \"Microarray exon-usage profiling in Dazap1 knockout testes; splicing minigene reporter assay\",\n      \"pmids\": [\"23965306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional difference between exon 9-included and exon 9-skipped CRISP2 isoforms not characterized\", \"Whether altered splicing affects fertility outcomes is untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing post-transcriptional repression of CRISP2 by miR-27b, with clinical relevance to asthenozoospermia where elevated miR-27b correlates with reduced CRISP2 protein.\",\n      \"evidence\": \"Luciferase 3′ UTR reporter assay; miR-27b mimic/inhibitor transfection; Western blot; clinical correlation in asthenozoospermic samples\",\n      \"pmids\": [\"25505194\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal relationship between miR-27b-mediated CRISP2 reduction and motility defects not proven in vivo\", \"Other miRNA regulators not surveyed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic loss-of-function definitively established CRISP2 as a regulator of Ca²⁺-dependent sperm hyperactivation, zona/cumulus penetration, and egg fusion, with knockout mice showing subfertility under competitive conditions.\",\n      \"evidence\": \"Crisp2 knockout mouse with CASA, flow cytometry Ca²⁺ measurement, IVF, acrosome reaction assay\",\n      \"pmids\": [\"26786179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets downstream of CRISP2 that mediate Ca²⁺ regulation not identified in this study\", \"Redundancy with CRISP1/CRISP3/CRISP4 not fully dissected\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying CATSPER1 as a direct binding partner of CRISP2, connecting its ion-channel-regulatory activity to the principal Ca²⁺ channel controlling sperm motility and explaining the stiff-midpiece/abnormal waveform phenotype of Crisp2-deficient sperm.\",\n      \"evidence\": \"Yeast two-hybrid screen; co-immunoprecipitation; flagellum waveform analysis in Crisp2 loss-of-function sperm\",\n      \"pmids\": [\"30759213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CRISP2 activates, inhibits, or allosterically modulates CatSper channel conductance is unknown\", \"Structural basis of CRISP2–CATSPER1 interaction not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that CRISP2 forms structurally distinct oligomeric assemblies in the sperm tail versus head, with different biochemical stabilities, revealing compartment-specific quaternary organization.\",\n      \"evidence\": \"Native/non-reducing gel electrophoresis; immunogold EM; sequential extraction of porcine sperm\",\n      \"pmids\": [\"34309660\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and exact composition of oligomers not determined\", \"Functional significance of distinct oligomeric states untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Uncovering a sterol-binding/export function for CRISP2 that is inhibited by PSP94 in a Ca²⁺-sensitive manner, revealing a lipid-handling role and a physiological regulatory switch tied to the ionic milieu of the reproductive tract.\",\n      \"evidence\": \"Yeast sterol-export assay; in vitro sterol-binding assay; mutagenesis of PSP94 interaction interface; Ca²⁺-dependent complex dissociation\",\n      \"pmids\": [\"35063506\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether sterol binding is relevant in mammalian sperm membranes in vivo is untested\", \"Identity of physiological sterol cargo unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Tracking CRISP2 redistribution through capacitation, acrosome reaction, and fertilization showed rapid post-fertilization dispersal from the perinuclear theca, likely driven by disulfide reduction, linking CRISP2 complex disassembly to early zygote remodeling.\",\n      \"evidence\": \"Confocal immunofluorescence on porcine sperm at defined stages; IVF followed by CRISP2 immunostaining of zygotes\",\n      \"pmids\": [\"36054334\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reductase responsible for disulfide reduction not identified\", \"Functional consequence of perinuclear theca dispersal for paternal chromatin remodeling unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying acrosin and acrosin-binding protein as the primary stable partners of CRISP2 in the acrosomal ~150 kDa complex under non-capacitating, capacitated, and acrosome-reacted conditions, defining the core acrosomal CRISP2 interactome.\",\n      \"evidence\": \"Blue-native gel; co-IP plus LC-MS/MS; proximity ligation assay on boar sperm\",\n      \"pmids\": [\"36815564\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the CRISP2–acrosin–ACRBP complex in acrosome reaction or zona penetration untested\", \"Whether this complex exists in human sperm not confirmed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that A1BG inhibits CRISP2 sterol function through Mg²⁺-dependent binding to conserved CAP domain residues, establishing a second serum protein (after PSP94) that regulates CRISP2 lipid-handling activity via divalent cation-dependent interactions.\",\n      \"evidence\": \"Yeast sterol-export assay; in vitro sterol-binding assay; A1BG domain-deletion and Mg²⁺-coordination mutagenesis\",\n      \"pmids\": [\"39433128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context where A1BG and CRISP2 interact (seminal plasma vs. serum) not established\", \"Whether A1BG regulation is relevant to sperm function in vivo is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapping the dynamic localization of human CRISP2 from nuclear in spermatocytes through cytoplasmic/flagellar in elongated spermatids to equatorial/flagellar in ejaculated sperm, and showing that human CRISP2 forms high-MW homo-oligomeric complexes with minimal post-translational modification.\",\n      \"evidence\": \"Immunofluorescence across human spermatogenesis; native gel electrophoresis; mass spectrometry of purified complexes\",\n      \"pmids\": [\"40079119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Function of nuclear CRISP2 in spermatocytes is unknown\", \"Whether homo-oligomers are functionally equivalent to the porcine heteromeric complexes containing acrosin is unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The egg-surface receptor for CRISP2, the structural basis of its CATSPER1 modulation, the in vivo relevance of its sterol-binding activity in mammalian sperm, and the function of nuclear CRISP2 in spermatocytes remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Egg-surface receptor identity unknown\", \"No structure of CRISP2–CATSPER1 complex\", \"In vivo sterol-handling function not demonstrated in mammalian sperm\", \"Role of nuclear CRISP2 in meiotic spermatocytes undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 12, 14, 17]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [1, 2, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [3, 6, 13]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [4, 5, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [4, 5, 11, 12]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 14, 17]}\n    ],\n    \"complexes\": [\n      \"CRISP2–acrosin–ACRBP acrosomal complex\",\n      \"CRISP2 homo-oligomeric complex (tail/head)\",\n      \"CRISP2–PSP94 heteromeric complex\"\n    ],\n    \"partners\": [\n      \"CATSPER1\",\n      \"ACR\",\n      \"ACRBP\",\n      \"GGN1\",\n      \"SHTAP\",\n      \"PSP94\",\n      \"A1BG\",\n      \"CRISP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}