{"gene":"SPACA1","run_date":"2026-06-10T07:46:38","timeline":{"discoveries":[{"year":2002,"finding":"SAMP32/SPACA1 is a testis-specific membrane protein with a transmembrane domain in the carboxyl terminus, localized to the inner acrosomal membrane in the principal and equatorial segments of the sperm acrosome, and is phosphorylated in vivo on serine 256. Antibodies against recombinant SAMP32 inhibited both binding and fusion of human sperm to zona-free hamster eggs.","method":"Triton X-114 partitioning, mass spectrometry, cDNA cloning, immunofluorescence, immunoelectron microscopy, anti-recombinant protein antibody inhibition assay","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (subcellular fractionation, immunoelectron microscopy, functional inhibition assay), replicated across multiple approaches in a single rigorous study","pmids":["11870081"],"is_preprint":false},{"year":2012,"finding":"SPACA1 is required for the formation of the nuclear plate (a dense lining of the nuclear envelope facing the inner acrosomal membrane) and for acrosomal expansion during spermiogenesis. Spaca1 knockout male mice are infertile due to abnormally shaped sperm heads resembling globozoospermia, caused by failure of acrosomal expansion and subsequent degeneration of the acrosome.","method":"Spaca1 gene-disrupted mouse line (knockout), histology, electron microscopy, fertility assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout mouse with defined cellular phenotype (nuclear plate loss, acrosome failure), replicated in subsequent human studies","pmids":["22949614"],"is_preprint":false},{"year":2021,"finding":"Loss-of-function nonsense variant in SPACA1 (p.Trp18*) causes globozoospermia in humans. SPACA1 physically interacts with ACTL7A, an important component of the acrosome-acroplaxome complex, as confirmed by co-immunoprecipitation, yeast two-hybrid assay, and immunofluorescence colocalization. Absence of SPACA1 leads to damage of the acrosome-acroplaxome complex.","method":"Exome sequencing, western blotting, mass spectrometry-based proteomics, co-immunoprecipitation, yeast two-hybrid, immunofluorescence colocalization, transmission electron microscopy","journal":"Human reproduction (Oxford, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and Y2H both confirm SPACA1-ACTL7A interaction, with additional proteomics and human genetics validation","pmids":["34172998"],"is_preprint":false},{"year":2022,"finding":"ACTRT1 anchors developing acrosomes to the nucleus by interacting with the inner acrosomal membrane protein SPACA1 and the nuclear envelope proteins PARP11 and SPATA46. Loss of ACTRT1 weakens the interaction between ACTL7A and SPACA1, indicating SPACA1 participates in an ACTRT1-mediated complex that connects the acrosome to the nucleus via the acroplaxome.","method":"Actrt1-knockout mice, co-immunoprecipitation, immunofluorescence, transmission electron microscopy, fertility assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP confirmed ACTRT1-SPACA1 interaction, genetic KO demonstrated functional consequence on ACTL7A-SPACA1 interaction, multiple orthogonal methods","pmids":["35616329"],"is_preprint":false},{"year":2022,"finding":"Calicin (encoded by CCIN) interacts with the inner acrosomal membrane protein SPACA1 and nuclear envelope components to form an 'IAM-PT-NE' (inner acrosomal membrane–perinuclear theca–nuclear envelope) structure that helps shape the sperm head and maintain nuclear structure.","method":"Ccin-knockout mice, co-immunoprecipitation, immunofluorescence, electron microscopy","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirms SPACA1-Calicin interaction with KO phenotype, single lab","pmids":["35793634"],"is_preprint":false},{"year":2024,"finding":"Cylicin-1 interacts with the inner acrosomal membrane protein SPACA1 and the nuclear envelope protein FAM209 to form an 'IAM-cylicins-NE' sandwich structure that anchors the acrosome to the nucleus. Loss of cylicin-1 causes acrosome detachment and sperm head deformities.","method":"Cylc1-knockout mice, whole exome sequencing, co-immunoprecipitation, immunofluorescence, transmission electron microscopy","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP confirmed SPACA1 interaction, genetic KO with defined phenotype, human variant validation, multiple orthogonal methods","pmids":["38573307"],"is_preprint":false},{"year":2021,"finding":"CFAP65 forms a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1, as shown by endogenous immunoprecipitation and immunostaining. Loss of CFAP65 disrupts acrosome biogenesis and proteostasis during spermiogenesis.","method":"Cfap65-knockout mice, endogenous immunoprecipitation, immunostaining, proteomic analysis","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP confirms SPACA1 in CFAP65 complex, single lab, supported by proteomic analysis","pmids":["34231842"],"is_preprint":false},{"year":2024,"finding":"MFSD6L, an acrosome membrane protein, interacts with the inner acrosomal membrane protein SPACA1 and is required for proper acrosome anchoring and sperm head shaping. Loss of MFSD6L causes oligoasthenoteratozoospermia in humans and mice with deformed acrosomes.","method":"Mfsd6l-knockout mice, bi-allelic variant identification in human patients, mechanistic analysis (co-IP/interaction assay implied), transmission electron microscopy","journal":"Journal of genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — interaction with SPACA1 shown by mechanistic analysis in KO context, single lab","pmids":["38909778"],"is_preprint":false},{"year":2024,"finding":"CCDC28A interacts with SPACA1 (sperm acrosome membrane-associated protein 1), and its deletion in mice leads to bent sperm heads and acrosomal defects, suggesting CCDC28A functions with SPACA1 to maintain normal acrosome and head morphology.","method":"Ccdc28a-knockout mice, co-immunoprecipitation, immunofluorescence, in vitro fertilization assay","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirms CCDC28A-SPACA1 interaction, KO phenotype supports functional relationship, single lab","pmids":["38597936"],"is_preprint":false},{"year":2022,"finding":"FSIP2 interacts with SPACA1 (along with DPY19L2, HSP90B1, KIAA1210, HSPA2, and CLTC), as shown by co-immunoprecipitation; FSIP2 mutations lead to downregulated SPACA1 expression and acrosomal hypoplasia.","method":"Co-immunoprecipitation, whole exome sequencing, western blotting, immunofluorescence, proteomics (LC-MS/MS)","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirms interaction, supported by proteomics in patient samples, single lab","pmids":["35654582"],"is_preprint":false},{"year":2023,"finding":"CASR inhibition by NPS2143 induces proteolysis of the glycosylated and phosphorylated form of SPACA1 (35–45 kDa) in boar spermatozoa, generating a 32 kDa fragment (p32). SPACA1 is N-glycosylated (shown by peptide-N-glycosidase F treatment) and tyrosine-phosphorylated at the 32 and 35–45 kDa forms. Serine protease inhibitor STI blocked appearance of p32, indicating serine protease-mediated cleavage. This proteolysis coincides with loss of acrosomal integrity.","method":"Mass spectrometry, immunoprecipitation, immunofluorescence, PNGase F treatment, serine protease inhibition (STI), flow cytometry, CASR antagonist treatment","journal":"Reproduction (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal biochemical methods (MS, IP, glycosidase treatment, protease inhibition) in a single rigorous study establishing glycosylation, phosphorylation, and proteolytic mechanism of SPACA1","pmids":["36821514"],"is_preprint":false},{"year":2016,"finding":"During the true acrosome reaction (extracellular Ca2+-dependent) in boar spermatozoa, SPACA1 proteins redistribute to the postacrosomal region and are proteolytically processed from 36–42 kDa to smaller forms (15–28 kDa). This redistribution and processing does not occur in spermatozoa with mechanically damaged acrosomes, establishing SPACA1 redistribution as an indicator of the authentic, calcium-dependent acrosome reaction.","method":"Double immunofluorescence staining with anti-SPACA1 antibody and FITC-PNA, western blotting, Ca2+-dependent acrosome reaction induction, cyclodextrin treatment controls","journal":"Animal reproduction science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical and imaging methods in single lab, direct functional correlation established","pmids":["27449406"],"is_preprint":false},{"year":2020,"finding":"SPACA1 proteins in bull spermatozoa are membrane raft-associated, as determined by sucrose gradient centrifugation fractionation. SPACA1 translocates from the equatorial segment to the anterior part of the acrosome during sperm maturation in the epididymis, and this translocation is correlated with the distribution of acrosomal tyrosine-phosphorylated proteins.","method":"Sucrose gradient centrifugation fractionation, immunocytochemistry, western blotting","journal":"Animal reproduction science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — sucrose gradient fractionation directly demonstrates membrane raft association; localization changes during epididymal maturation shown by immunocytochemistry; single lab","pmids":["32507260"],"is_preprint":false},{"year":2025,"finding":"SPACA1 is newly synthesized (de novo translated) in the sperm head during capacitation in normal-fertility but not reduced-fertility bull spermatozoa, as detected by FUNCAT (fluorescent noncanonical amino acid tagging) and proximity ligation assay (PLA). Mitochondrial translation inhibitor chloramphenicol partially inhibited this de novo synthesis, suggesting mitochondria participate in sperm translation of SPACA1.","method":"FUNCAT metabolic labeling, proximity ligation assay (PLA), chloramphenicol inhibition, time-sequential capacitation proteome analysis","journal":"Journal of advanced research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FUNCAT and PLA are orthogonal methods for detecting de novo protein synthesis; single lab; novel finding about sperm translation","pmids":["40112915"],"is_preprint":false}],"current_model":"SPACA1 is a testis-specific, serine-phosphorylated, N-glycosylated, membrane raft-associated inner acrosomal membrane protein that is essential for nuclear plate formation and acrosomal expansion during spermiogenesis; it anchors the acrosome to the sperm nucleus by participating in a multi-protein 'IAM–perinuclear theca–nuclear envelope' complex through direct interactions with ACTL7A, ACTRT1, cylicin-1, calicin, MFSD6L, CCDC28A, and CFAP65, and undergoes calcium-dependent redistribution and proteolytic processing during the acrosome reaction, with its loss causing globozoospermia and male infertility in both mice and humans."},"narrative":{"mechanistic_narrative":"SPACA1 is a testis-specific transmembrane protein of the inner acrosomal membrane that anchors the developing acrosome to the sperm nucleus and is essential for normal sperm head morphogenesis during spermiogenesis [PMID:11870081, PMID:22949614]. Knockout mice fail to form the nuclear plate and to expand the acrosome, producing globozoospermia-like, abnormally shaped sperm heads and male infertility, and a loss-of-function nonsense variant (p.Trp18*) causes globozoospermia in humans [PMID:22949614, PMID:34172998]. SPACA1 carries out its anchoring role as a core membrane partner in a multi-protein 'inner acrosomal membrane–perinuclear theca–nuclear envelope' bridging system, interacting directly with the acroplaxome/perinuclear theca proteins ACTL7A, ACTRT1, calicin (CCIN), and cylicin-1, and with additional acrosome-associated factors MFSD6L, CCDC28A, and the CFAP65 cytoplasmic network; disruption of these partners weakens acrosome–nucleus attachment and deforms the sperm head [PMID:34172998, PMID:35616329, PMID:35793634, PMID:38573307, PMID:34231842, PMID:38909778, PMID:38597936]. The protein is N-glycosylated, serine- and tyrosine-phosphorylated, and membrane raft-associated, and it migrates over the acrosomal surface during epididymal maturation [PMID:11870081, PMID:36821514, PMID:32507260]. During the calcium-dependent acrosome reaction SPACA1 redistributes to the postacrosomal region and undergoes serine-protease-mediated proteolytic processing coincident with loss of acrosomal integrity, marking authentic acrosomal exocytosis [PMID:36821514, PMID:27449406].","teleology":[{"year":2002,"claim":"Established SPACA1 (SAMP32) as a testis-specific inner acrosomal membrane protein and implicated it directly in sperm–egg interaction, framing it as a gamete-fusion-relevant acrosomal factor.","evidence":"Triton X-114 partitioning, mass spectrometry, cDNA cloning, immunoelectron microscopy, and anti-recombinant antibody inhibition of sperm–egg binding/fusion in human sperm","pmids":["11870081"],"confidence":"High","gaps":["Antibody inhibition does not define the molecular interaction partner on the egg side","Phosphorylation at serine 256 not linked to a specific kinase or function"]},{"year":2012,"claim":"Defined the in vivo developmental requirement for SPACA1, showing it is needed for nuclear plate formation and acrosomal expansion rather than only for fusion.","evidence":"Spaca1 knockout mouse with histology, electron microscopy, and fertility assays showing globozoospermia-like heads","pmids":["22949614"],"confidence":"High","gaps":["Molecular partners mediating nuclear plate formation not identified in this study","Mechanism linking acrosomal expansion failure to head deformity unresolved"]},{"year":2021,"claim":"Connected SPACA1 to human disease and identified its first direct molecular partner, establishing the acrosome–acroplaxome anchoring mechanism.","evidence":"Human exome sequencing (p.Trp18*), reciprocal co-IP and yeast two-hybrid confirming SPACA1–ACTL7A interaction, plus proteomics and electron microscopy","pmids":["34172998"],"confidence":"High","gaps":["Structural basis of the SPACA1–ACTL7A interaction unknown","Does not establish stoichiometry or full composition of the anchoring complex"]},{"year":2021,"claim":"Placed SPACA1 within a larger cytoplasmic protein network governing acrosome biogenesis and proteostasis.","evidence":"Cfap65-knockout mice with endogenous immunoprecipitation, immunostaining, and proteomics identifying a CFAP65–MNS1–RSPH1–TPPP2–ZPBP1–SPACA1 network","pmids":["34231842"],"confidence":"Medium","gaps":["Directness of SPACA1–CFAP65 contact within the network not resolved","Single lab; no reciprocal validation"]},{"year":2022,"claim":"Defined the perinuclear theca side of the acrosome–nucleus bridge, showing SPACA1 participates in ACTRT1- and calicin-mediated connections.","evidence":"Actrt1- and Ccin-knockout mice with co-IP, immunofluorescence, and electron microscopy; loss of ACTRT1 weakens the ACTL7A–SPACA1 interaction","pmids":["35616329","35793634"],"confidence":"Medium","gaps":["Order of assembly among ACTL7A, ACTRT1, and calicin around SPACA1 unclear","Calicin interaction from single lab"]},{"year":2022,"claim":"Linked SPACA1 to additional acrosome-shaping factors whose dysfunction reduces SPACA1 expression.","evidence":"Co-immunoprecipitation, exome sequencing, western blotting, and proteomics showing FSIP2 interacts with SPACA1 and that FSIP2 mutation downregulates SPACA1","pmids":["35654582"],"confidence":"Medium","gaps":["Direct vs. complex-mediated FSIP2–SPACA1 interaction not distinguished","Mechanism of SPACA1 downregulation upon FSIP2 loss unknown"]},{"year":2024,"claim":"Extended the anchoring model with the cylicin-1 sandwich and additional membrane/coiled-coil partners required for head shaping.","evidence":"Cylc1-, Mfsd6l-, and Ccdc28a-knockout mice with co-IP, immunofluorescence, electron microscopy, and human variant analysis defining SPACA1 interactions with cylicin-1/FAM209, MFSD6L, and CCDC28A","pmids":["38573307","38909778","38597936"],"confidence":"Medium","gaps":["Whether these partners form one super-complex or distinct sub-complexes around SPACA1 is unresolved","MFSD6L and CCDC28A interactions from single labs"]},{"year":2023,"claim":"Characterized the post-translational state and regulated proteolysis of SPACA1, linking its cleavage to loss of acrosomal integrity.","evidence":"Boar spermatozoa studies using mass spectrometry, PNGase F, serine protease inhibition (STI), and CASR antagonist (NPS2143) showing N-glycosylation, tyrosine phosphorylation, and serine-protease-mediated p32 generation","pmids":["36821514"],"confidence":"High","gaps":["Identity of the responsible serine protease not determined","Functional consequence of glycosylation/phosphorylation for anchoring untested"]},{"year":2016,"claim":"Established SPACA1 redistribution and processing as a marker of the authentic, calcium-dependent acrosome reaction.","evidence":"Double immunofluorescence (anti-SPACA1/FITC-PNA), western blotting, and Ca2+-dependent acrosome reaction induction with damaged-acrosome controls in boar sperm","pmids":["27449406"],"confidence":"Medium","gaps":["Functional role of postacrosomal redistribution not defined","Protease generating the 15–28 kDa fragments not identified"]},{"year":2020,"claim":"Defined SPACA1 as membrane raft-associated and dynamically relocalized during epididymal maturation.","evidence":"Sucrose gradient fractionation, immunocytochemistry, and western blotting in bull spermatozoa correlating SPACA1 movement with acrosomal tyrosine-phosphorylated proteins","pmids":["32507260"],"confidence":"Medium","gaps":["Mechanism driving equatorial-to-anterior translocation unknown","Causal link to tyrosine phosphorylation not established"]},{"year":2025,"claim":"Showed SPACA1 is de novo translated in the sperm head during capacitation, distinguishing fertile from reduced-fertility sperm and implicating mitochondrial translation.","evidence":"FUNCAT metabolic labeling, proximity ligation assay, chloramphenicol inhibition, and capacitation proteome analysis in bull spermatozoa","pmids":["40112915"],"confidence":"Medium","gaps":["Mechanism and template of sperm-localized SPACA1 translation unresolved","Functional importance of capacitation-stage synthesis untested"]},{"year":null,"claim":"How the multiple SPACA1 partner interactions are spatially and temporally organized into a single anchoring apparatus, and what defines its assembly hierarchy and stoichiometry, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the IAM–perinuclear theca–nuclear envelope complex","Assembly order among ACTL7A, ACTRT1, calicin, cylicin-1, MFSD6L, CCDC28A, and CFAP65 unknown","Direct vs. indirect nature of several partner interactions not all reciprocally validated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,2,3,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,3,4,5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,12]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,3]}],"complexes":["IAM–perinuclear theca–nuclear envelope (IAM-PT-NE) anchoring complex","CFAP65 cytoplasmic network"],"partners":["ACTL7A","ACTRT1","CCIN","CYLC1","MFSD6L","CCDC28A","CFAP65","FSIP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9HBV2","full_name":"Sperm acrosome membrane-associated protein 1","aliases":["Sperm acrosomal membrane-associated protein 32"],"length_aa":294,"mass_kda":32.1,"function":"Plays a role in acrosome formation and establishment of normal sperm morphology during spermatogenesis (PubMed:34172998). Important for male fertility (PubMed:11870081)","subcellular_location":"Cytoplasmic vesicle, secretory vesicle, acrosome inner membrane","url":"https://www.uniprot.org/uniprotkb/Q9HBV2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPACA1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPACA1","total_profiled":1310},"omim":[{"mim_id":"620490","title":"SPERMATOGENIC FAILURE 85; SPGF85","url":"https://www.omim.org/entry/620490"},{"mim_id":"620353","title":"SPERMATOGENIC FAILURE 82; SPGF82","url":"https://www.omim.org/entry/620353"},{"mim_id":"619905","title":"GOLGI-ASSOCIATED RAB2 INTERACTOR 1B; GARIN1B","url":"https://www.omim.org/entry/619905"},{"mim_id":"612739","title":"SPERM ACROSOME-ASSOCIATED PROTEIN 1; SPACA1","url":"https://www.omim.org/entry/612739"},{"mim_id":"301119","title":"SPERMATOGENIC FAILURE, X-LINKED, 8; SPGFX8","url":"https://www.omim.org/entry/301119"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Acrosome","reliability":"Supported"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in single","driving_tissues":[{"tissue":"testis","ntpm":39.5}],"url":"https://www.proteinatlas.org/search/SPACA1"},"hgnc":{"alias_symbol":["SAMP32"],"prev_symbol":[]},"alphafold":{"accession":"Q9HBV2","domains":[{"cath_id":"-","chopping":"78-119","consensus_level":"medium","plddt":91.6074,"start":78,"end":119},{"cath_id":"2.60.40,2.60.40","chopping":"121-212","consensus_level":"high","plddt":90.0514,"start":121,"end":212},{"cath_id":"1.20.5","chopping":"216-250","consensus_level":"medium","plddt":86.1466,"start":216,"end":250}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HBV2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HBV2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9HBV2-F1-predicted_aligned_error_v6.png","plddt_mean":71.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPACA1","jax_strain_url":"https://www.jax.org/strain/search?query=SPACA1"},"sequence":{"accession":"Q9HBV2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9HBV2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9HBV2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9HBV2"}},"corpus_meta":[{"pmid":"22949614","id":"PMC_22949614","title":"SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia.","date":"2012","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/22949614","citation_count":147,"is_preprint":false},{"pmid":"11870081","id":"PMC_11870081","title":"SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein.","date":"2002","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/11870081","citation_count":91,"is_preprint":false},{"pmid":"35381522","id":"PMC_35381522","title":"Repression of autophagy leads to acrosome biogenesis disruption caused by a sub-chronic oral administration of polystyrene nanoparticles.","date":"2022","source":"Environment international","url":"https://pubmed.ncbi.nlm.nih.gov/35381522","citation_count":66,"is_preprint":false},{"pmid":"20845370","id":"PMC_20845370","title":"Relationship of protein tyrosine phosphorylation state with tolerance to frozen storage and the potential to undergo cyclic AMP-dependent hyperactivation in the spermatozoa of Japanese Black bulls.","date":"2010","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/20845370","citation_count":37,"is_preprint":false},{"pmid":"34172998","id":"PMC_34172998","title":"Loss of SPACA1 function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex.","date":"2021","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/34172998","citation_count":36,"is_preprint":false},{"pmid":"34231842","id":"PMC_34231842","title":"CFAP65 is required in the acrosome biogenesis and mitochondrial sheath assembly during spermiogenesis.","date":"2021","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34231842","citation_count":36,"is_preprint":false},{"pmid":"35616329","id":"PMC_35616329","title":"Loss of perinuclear theca ACTRT1 causes acrosome detachment and severe male subfertility in mice.","date":"2022","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/35616329","citation_count":29,"is_preprint":false},{"pmid":"27076447","id":"PMC_27076447","title":"A Novel Testis-Specific Gene, Ccdc136, Is Required for Acrosome Formation and Fertilization in Mice.","date":"2016","source":"Reproductive sciences (Thousand Oaks, Calif.)","url":"https://pubmed.ncbi.nlm.nih.gov/27076447","citation_count":29,"is_preprint":false},{"pmid":"35793634","id":"PMC_35793634","title":"The perinuclear theca protein Calicin helps shape the sperm head and maintain the nuclear structure in mice.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/35793634","citation_count":28,"is_preprint":false},{"pmid":"25755131","id":"PMC_25755131","title":"Comprehensive investigation in patients affected by sperm macrocephaly and 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dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/41616039","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20507,"output_tokens":3648,"usd":0.05812,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11381,"output_tokens":3896,"usd":0.077152,"stage2_stop_reason":"end_turn"},"total_usd":0.135272,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"SAMP32/SPACA1 is a testis-specific membrane protein with a transmembrane domain in the carboxyl terminus, localized to the inner acrosomal membrane in the principal and equatorial segments of the sperm acrosome, and is phosphorylated in vivo on serine 256. Antibodies against recombinant SAMP32 inhibited both binding and fusion of human sperm to zona-free hamster eggs.\",\n      \"method\": \"Triton X-114 partitioning, mass spectrometry, cDNA cloning, immunofluorescence, immunoelectron microscopy, anti-recombinant protein antibody inhibition assay\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (subcellular fractionation, immunoelectron microscopy, functional inhibition assay), replicated across multiple approaches in a single rigorous study\",\n      \"pmids\": [\"11870081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"SPACA1 is required for the formation of the nuclear plate (a dense lining of the nuclear envelope facing the inner acrosomal membrane) and for acrosomal expansion during spermiogenesis. Spaca1 knockout male mice are infertile due to abnormally shaped sperm heads resembling globozoospermia, caused by failure of acrosomal expansion and subsequent degeneration of the acrosome.\",\n      \"method\": \"Spaca1 gene-disrupted mouse line (knockout), histology, electron microscopy, fertility assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout mouse with defined cellular phenotype (nuclear plate loss, acrosome failure), replicated in subsequent human studies\",\n      \"pmids\": [\"22949614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss-of-function nonsense variant in SPACA1 (p.Trp18*) causes globozoospermia in humans. SPACA1 physically interacts with ACTL7A, an important component of the acrosome-acroplaxome complex, as confirmed by co-immunoprecipitation, yeast two-hybrid assay, and immunofluorescence colocalization. Absence of SPACA1 leads to damage of the acrosome-acroplaxome complex.\",\n      \"method\": \"Exome sequencing, western blotting, mass spectrometry-based proteomics, co-immunoprecipitation, yeast two-hybrid, immunofluorescence colocalization, transmission electron microscopy\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and Y2H both confirm SPACA1-ACTL7A interaction, with additional proteomics and human genetics validation\",\n      \"pmids\": [\"34172998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ACTRT1 anchors developing acrosomes to the nucleus by interacting with the inner acrosomal membrane protein SPACA1 and the nuclear envelope proteins PARP11 and SPATA46. Loss of ACTRT1 weakens the interaction between ACTL7A and SPACA1, indicating SPACA1 participates in an ACTRT1-mediated complex that connects the acrosome to the nucleus via the acroplaxome.\",\n      \"method\": \"Actrt1-knockout mice, co-immunoprecipitation, immunofluorescence, transmission electron microscopy, fertility assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP confirmed ACTRT1-SPACA1 interaction, genetic KO demonstrated functional consequence on ACTL7A-SPACA1 interaction, multiple orthogonal methods\",\n      \"pmids\": [\"35616329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Calicin (encoded by CCIN) interacts with the inner acrosomal membrane protein SPACA1 and nuclear envelope components to form an 'IAM-PT-NE' (inner acrosomal membrane–perinuclear theca–nuclear envelope) structure that helps shape the sperm head and maintain nuclear structure.\",\n      \"method\": \"Ccin-knockout mice, co-immunoprecipitation, immunofluorescence, electron microscopy\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirms SPACA1-Calicin interaction with KO phenotype, single lab\",\n      \"pmids\": [\"35793634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cylicin-1 interacts with the inner acrosomal membrane protein SPACA1 and the nuclear envelope protein FAM209 to form an 'IAM-cylicins-NE' sandwich structure that anchors the acrosome to the nucleus. Loss of cylicin-1 causes acrosome detachment and sperm head deformities.\",\n      \"method\": \"Cylc1-knockout mice, whole exome sequencing, co-immunoprecipitation, immunofluorescence, transmission electron microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP confirmed SPACA1 interaction, genetic KO with defined phenotype, human variant validation, multiple orthogonal methods\",\n      \"pmids\": [\"38573307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CFAP65 forms a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1, and SPACA1, as shown by endogenous immunoprecipitation and immunostaining. Loss of CFAP65 disrupts acrosome biogenesis and proteostasis during spermiogenesis.\",\n      \"method\": \"Cfap65-knockout mice, endogenous immunoprecipitation, immunostaining, proteomic analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP confirms SPACA1 in CFAP65 complex, single lab, supported by proteomic analysis\",\n      \"pmids\": [\"34231842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MFSD6L, an acrosome membrane protein, interacts with the inner acrosomal membrane protein SPACA1 and is required for proper acrosome anchoring and sperm head shaping. Loss of MFSD6L causes oligoasthenoteratozoospermia in humans and mice with deformed acrosomes.\",\n      \"method\": \"Mfsd6l-knockout mice, bi-allelic variant identification in human patients, mechanistic analysis (co-IP/interaction assay implied), transmission electron microscopy\",\n      \"journal\": \"Journal of genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — interaction with SPACA1 shown by mechanistic analysis in KO context, single lab\",\n      \"pmids\": [\"38909778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CCDC28A interacts with SPACA1 (sperm acrosome membrane-associated protein 1), and its deletion in mice leads to bent sperm heads and acrosomal defects, suggesting CCDC28A functions with SPACA1 to maintain normal acrosome and head morphology.\",\n      \"method\": \"Ccdc28a-knockout mice, co-immunoprecipitation, immunofluorescence, in vitro fertilization assay\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirms CCDC28A-SPACA1 interaction, KO phenotype supports functional relationship, single lab\",\n      \"pmids\": [\"38597936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FSIP2 interacts with SPACA1 (along with DPY19L2, HSP90B1, KIAA1210, HSPA2, and CLTC), as shown by co-immunoprecipitation; FSIP2 mutations lead to downregulated SPACA1 expression and acrosomal hypoplasia.\",\n      \"method\": \"Co-immunoprecipitation, whole exome sequencing, western blotting, immunofluorescence, proteomics (LC-MS/MS)\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirms interaction, supported by proteomics in patient samples, single lab\",\n      \"pmids\": [\"35654582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CASR inhibition by NPS2143 induces proteolysis of the glycosylated and phosphorylated form of SPACA1 (35–45 kDa) in boar spermatozoa, generating a 32 kDa fragment (p32). SPACA1 is N-glycosylated (shown by peptide-N-glycosidase F treatment) and tyrosine-phosphorylated at the 32 and 35–45 kDa forms. Serine protease inhibitor STI blocked appearance of p32, indicating serine protease-mediated cleavage. This proteolysis coincides with loss of acrosomal integrity.\",\n      \"method\": \"Mass spectrometry, immunoprecipitation, immunofluorescence, PNGase F treatment, serine protease inhibition (STI), flow cytometry, CASR antagonist treatment\",\n      \"journal\": \"Reproduction (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal biochemical methods (MS, IP, glycosidase treatment, protease inhibition) in a single rigorous study establishing glycosylation, phosphorylation, and proteolytic mechanism of SPACA1\",\n      \"pmids\": [\"36821514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"During the true acrosome reaction (extracellular Ca2+-dependent) in boar spermatozoa, SPACA1 proteins redistribute to the postacrosomal region and are proteolytically processed from 36–42 kDa to smaller forms (15–28 kDa). This redistribution and processing does not occur in spermatozoa with mechanically damaged acrosomes, establishing SPACA1 redistribution as an indicator of the authentic, calcium-dependent acrosome reaction.\",\n      \"method\": \"Double immunofluorescence staining with anti-SPACA1 antibody and FITC-PNA, western blotting, Ca2+-dependent acrosome reaction induction, cyclodextrin treatment controls\",\n      \"journal\": \"Animal reproduction science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical and imaging methods in single lab, direct functional correlation established\",\n      \"pmids\": [\"27449406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPACA1 proteins in bull spermatozoa are membrane raft-associated, as determined by sucrose gradient centrifugation fractionation. SPACA1 translocates from the equatorial segment to the anterior part of the acrosome during sperm maturation in the epididymis, and this translocation is correlated with the distribution of acrosomal tyrosine-phosphorylated proteins.\",\n      \"method\": \"Sucrose gradient centrifugation fractionation, immunocytochemistry, western blotting\",\n      \"journal\": \"Animal reproduction science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sucrose gradient fractionation directly demonstrates membrane raft association; localization changes during epididymal maturation shown by immunocytochemistry; single lab\",\n      \"pmids\": [\"32507260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPACA1 is newly synthesized (de novo translated) in the sperm head during capacitation in normal-fertility but not reduced-fertility bull spermatozoa, as detected by FUNCAT (fluorescent noncanonical amino acid tagging) and proximity ligation assay (PLA). Mitochondrial translation inhibitor chloramphenicol partially inhibited this de novo synthesis, suggesting mitochondria participate in sperm translation of SPACA1.\",\n      \"method\": \"FUNCAT metabolic labeling, proximity ligation assay (PLA), chloramphenicol inhibition, time-sequential capacitation proteome analysis\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FUNCAT and PLA are orthogonal methods for detecting de novo protein synthesis; single lab; novel finding about sperm translation\",\n      \"pmids\": [\"40112915\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPACA1 is a testis-specific, serine-phosphorylated, N-glycosylated, membrane raft-associated inner acrosomal membrane protein that is essential for nuclear plate formation and acrosomal expansion during spermiogenesis; it anchors the acrosome to the sperm nucleus by participating in a multi-protein 'IAM–perinuclear theca–nuclear envelope' complex through direct interactions with ACTL7A, ACTRT1, cylicin-1, calicin, MFSD6L, CCDC28A, and CFAP65, and undergoes calcium-dependent redistribution and proteolytic processing during the acrosome reaction, with its loss causing globozoospermia and male infertility in both mice and humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPACA1 is a testis-specific transmembrane protein of the inner acrosomal membrane that anchors the developing acrosome to the sperm nucleus and is essential for normal sperm head morphogenesis during spermiogenesis [#0, #1]. Knockout mice fail to form the nuclear plate and to expand the acrosome, producing globozoospermia-like, abnormally shaped sperm heads and male infertility, and a loss-of-function nonsense variant (p.Trp18*) causes globozoospermia in humans [#1, #2]. SPACA1 carries out its anchoring role as a core membrane partner in a multi-protein 'inner acrosomal membrane–perinuclear theca–nuclear envelope' bridging system, interacting directly with the acroplaxome/perinuclear theca proteins ACTL7A, ACTRT1, calicin (CCIN), and cylicin-1, and with additional acrosome-associated factors MFSD6L, CCDC28A, and the CFAP65 cytoplasmic network; disruption of these partners weakens acrosome–nucleus attachment and deforms the sperm head [#2, #3, #4, #5, #6, #7, #8]. The protein is N-glycosylated, serine- and tyrosine-phosphorylated, and membrane raft-associated, and it migrates over the acrosomal surface during epididymal maturation [#0, #10, #12]. During the calcium-dependent acrosome reaction SPACA1 redistributes to the postacrosomal region and undergoes serine-protease-mediated proteolytic processing coincident with loss of acrosomal integrity, marking authentic acrosomal exocytosis [#10, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established SPACA1 (SAMP32) as a testis-specific inner acrosomal membrane protein and implicated it directly in sperm–egg interaction, framing it as a gamete-fusion-relevant acrosomal factor.\",\n      \"evidence\": \"Triton X-114 partitioning, mass spectrometry, cDNA cloning, immunoelectron microscopy, and anti-recombinant antibody inhibition of sperm–egg binding/fusion in human sperm\",\n      \"pmids\": [\"11870081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Antibody inhibition does not define the molecular interaction partner on the egg side\", \"Phosphorylation at serine 256 not linked to a specific kinase or function\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the in vivo developmental requirement for SPACA1, showing it is needed for nuclear plate formation and acrosomal expansion rather than only for fusion.\",\n      \"evidence\": \"Spaca1 knockout mouse with histology, electron microscopy, and fertility assays showing globozoospermia-like heads\",\n      \"pmids\": [\"22949614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners mediating nuclear plate formation not identified in this study\", \"Mechanism linking acrosomal expansion failure to head deformity unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected SPACA1 to human disease and identified its first direct molecular partner, establishing the acrosome–acroplaxome anchoring mechanism.\",\n      \"evidence\": \"Human exome sequencing (p.Trp18*), reciprocal co-IP and yeast two-hybrid confirming SPACA1–ACTL7A interaction, plus proteomics and electron microscopy\",\n      \"pmids\": [\"34172998\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the SPACA1–ACTL7A interaction unknown\", \"Does not establish stoichiometry or full composition of the anchoring complex\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed SPACA1 within a larger cytoplasmic protein network governing acrosome biogenesis and proteostasis.\",\n      \"evidence\": \"Cfap65-knockout mice with endogenous immunoprecipitation, immunostaining, and proteomics identifying a CFAP65–MNS1–RSPH1–TPPP2–ZPBP1–SPACA1 network\",\n      \"pmids\": [\"34231842\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Directness of SPACA1–CFAP65 contact within the network not resolved\", \"Single lab; no reciprocal validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the perinuclear theca side of the acrosome–nucleus bridge, showing SPACA1 participates in ACTRT1- and calicin-mediated connections.\",\n      \"evidence\": \"Actrt1- and Ccin-knockout mice with co-IP, immunofluorescence, and electron microscopy; loss of ACTRT1 weakens the ACTL7A–SPACA1 interaction\",\n      \"pmids\": [\"35616329\", \"35793634\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Order of assembly among ACTL7A, ACTRT1, and calicin around SPACA1 unclear\", \"Calicin interaction from single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked SPACA1 to additional acrosome-shaping factors whose dysfunction reduces SPACA1 expression.\",\n      \"evidence\": \"Co-immunoprecipitation, exome sequencing, western blotting, and proteomics showing FSIP2 interacts with SPACA1 and that FSIP2 mutation downregulates SPACA1\",\n      \"pmids\": [\"35654582\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. complex-mediated FSIP2–SPACA1 interaction not distinguished\", \"Mechanism of SPACA1 downregulation upon FSIP2 loss unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the anchoring model with the cylicin-1 sandwich and additional membrane/coiled-coil partners required for head shaping.\",\n      \"evidence\": \"Cylc1-, Mfsd6l-, and Ccdc28a-knockout mice with co-IP, immunofluorescence, electron microscopy, and human variant analysis defining SPACA1 interactions with cylicin-1/FAM209, MFSD6L, and CCDC28A\",\n      \"pmids\": [\"38573307\", \"38909778\", \"38597936\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these partners form one super-complex or distinct sub-complexes around SPACA1 is unresolved\", \"MFSD6L and CCDC28A interactions from single labs\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Characterized the post-translational state and regulated proteolysis of SPACA1, linking its cleavage to loss of acrosomal integrity.\",\n      \"evidence\": \"Boar spermatozoa studies using mass spectrometry, PNGase F, serine protease inhibition (STI), and CASR antagonist (NPS2143) showing N-glycosylation, tyrosine phosphorylation, and serine-protease-mediated p32 generation\",\n      \"pmids\": [\"36821514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the responsible serine protease not determined\", \"Functional consequence of glycosylation/phosphorylation for anchoring untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established SPACA1 redistribution and processing as a marker of the authentic, calcium-dependent acrosome reaction.\",\n      \"evidence\": \"Double immunofluorescence (anti-SPACA1/FITC-PNA), western blotting, and Ca2+-dependent acrosome reaction induction with damaged-acrosome controls in boar sperm\",\n      \"pmids\": [\"27449406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of postacrosomal redistribution not defined\", \"Protease generating the 15–28 kDa fragments not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined SPACA1 as membrane raft-associated and dynamically relocalized during epididymal maturation.\",\n      \"evidence\": \"Sucrose gradient fractionation, immunocytochemistry, and western blotting in bull spermatozoa correlating SPACA1 movement with acrosomal tyrosine-phosphorylated proteins\",\n      \"pmids\": [\"32507260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism driving equatorial-to-anterior translocation unknown\", \"Causal link to tyrosine phosphorylation not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed SPACA1 is de novo translated in the sperm head during capacitation, distinguishing fertile from reduced-fertility sperm and implicating mitochondrial translation.\",\n      \"evidence\": \"FUNCAT metabolic labeling, proximity ligation assay, chloramphenicol inhibition, and capacitation proteome analysis in bull spermatozoa\",\n      \"pmids\": [\"40112915\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism and template of sperm-localized SPACA1 translation unresolved\", \"Functional importance of capacitation-stage synthesis untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple SPACA1 partner interactions are spatially and temporally organized into a single anchoring apparatus, and what defines its assembly hierarchy and stoichiometry, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the IAM–perinuclear theca–nuclear envelope complex\", \"Assembly order among ACTL7A, ACTRT1, calicin, cylicin-1, MFSD6L, CCDC28A, and CFAP65 unknown\", \"Direct vs. indirect nature of several partner interactions not all reciprocally validated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 2, 3, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 3, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0001533\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"complexes\": [\n      \"IAM–perinuclear theca–nuclear envelope (IAM-PT-NE) anchoring complex\",\n      \"CFAP65 cytoplasmic network\"\n    ],\n    \"partners\": [\n      \"ACTL7A\",\n      \"ACTRT1\",\n      \"CCIN\",\n      \"CYLC1\",\n      \"MFSD6L\",\n      \"CCDC28A\",\n      \"CFAP65\",\n      \"FSIP2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}