Affinage

SPAM1

Hyaluronidase PH-20 · UniProt P38567

Length
509 aa
Mass
57.8 kDa
Annotated
2026-06-10
82 papers in source corpus 41 papers cited in narrative 41 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 8/8 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

SPAM1/PH-20 is a GPI-anchored, testis-expressed sperm surface protein that orchestrates the sperm's passage through the egg's investments during fertilization, functioning as a bifunctional molecule combining hyaluronidase enzymatic activity with adhesive activity (PMID:8195297, PMID:8793062). Its N-terminal catalytic domain hydrolyzes hyaluronic acid (minimum substrate an HA octasaccharide), enabling acrosome-intact sperm to penetrate the hyaluronan-rich cumulus cell layer surrounding the egg (PMID:8195297, PMID:17602139); this activity depends on N-linked glycosylation and disulfide bond integrity (PMID:11868814), and cryo-EM reveals a central catalytic domain together with a distinctive EGF-like domain forming a flexibly anchored disulfide-containing β-hairpin (PMID:39722545). A biochemically separable activity mediates secondary sperm binding to the zona pellucida, demonstrated by inhibitors that block one activity without the other and by the requirement for PH-20 on the inner acrosomal membrane of acrosome-reacted sperm (PMID:4066757, PMID:8793062, PMID:10369396). A third HA-binding region (aa 205–235), distinct from the catalytic site, drives HA-induced intracellular Ca²⁺ increases via aggregation of plasma-membrane PH-20, potentiating the acrosome reaction (PMID:10533704, PMID:11746965). The protein redistributes from the posterior-head plasma membrane to the inner acrosomal membrane following the acrosome reaction by a calcium-dependent translocation, and its GPI anchor constrains its lateral mobility within membrane domains (PMID:3558486, PMID:3381102, PMID:1995397). SPAM1 is acquired post-testicularly: it is delivered to maturing sperm from epididymosomes and uterosomes by a GPI-anchor-dependent vesicular mechanism and through clusterin-mediated soluble transfer, with epididymal N-glycan remodeling raising its hyaluronidase activity during maturation (PMID:9890751, PMID:18384048, PMID:19357365). Transcription is driven by a CREM-dependent promoter element (PMID:10423292), and Spam1 mRNA is cytoskeletally anchored in spermatids via 3'UTR AU-rich elements, preventing transcript sharing through intercellular bridges and underlying transmission ratio distortion (PMID:11369602, PMID:16092963). Despite these roles, Spam1-null male mice remain fertile because the testis-restricted hyaluronidase HYAL5 partially compensates for cumulus dispersal, although SPAM1 is the functionally critical enzyme for efficient cumulus entry (PMID:12065596, PMID:16330764, PMID:19605784).

Mechanistic history

Synthesis pass · year-by-year structured walk · 20 steps
  1. 1985 High

    Established that the sperm surface antigen PH-20 has an essential function in sperm adhesion to the egg zona pellucida, defining its candidacy as a fertilization protein.

    Evidence Monoclonal antibody inhibition of sperm-zona binding in guinea pig

    PMID:4066757

    Open questions at the time
    • Molecular identity and biochemical mechanism of binding undefined
    • Did not distinguish enzymatic from adhesive activity
  2. 1988 High

    Defined PH-20 as a GPI-anchored membrane protein whose mobility is constrained by ectodomain interactions, explaining how it maintains discrete surface domains.

    Evidence PI-PLC cleavage and FRAP diffusion measurements on guinea pig sperm

    PMID:3381102 PMID:3558486

    Open questions at the time
    • Identity of constraining ectodomain interactions not determined
    • Mechanism of post-acrosome-reaction redistribution unresolved
  3. 1990 High

    Molecular cloning provided the protein sequence, revealing a single-gene-encoded glycoprotein with conserved cysteines and N-glycosylation sites, enabling all subsequent domain dissection.

    Evidence cDNA cloning, Southern and Northern blots in guinea pig and across mammals

    PMID:2269661

    Open questions at the time
    • Catalytic residues not yet mapped
    • Domain boundaries for distinct functions not defined
  4. 1993 High

    Identified PH-20 as a hyaluronidase and cloned the strictly testis-specific human ortholog, linking the cloned gene to enzymatic function in humans.

    Evidence Vaccinia/recombinant expression with hyaluronidase assay and PI-PLC release; human/monkey cDNA cloning and tissue Northern blots

    PMID:8234258 PMID:8282124

    Open questions at the time
    • In vivo role in human fertilization not directly tested
    • Catalytic site residues not localized
  5. 1994 High

    Demonstrated that PH-20 hyaluronidase enables acrosome-intact sperm to penetrate the cumulus cell layer, defining its first physiological function.

    Evidence Recombinant PH-20 cumulus dispersal and antibody-blocking cumulus penetration assays in mouse

    PMID:8195297

    Open questions at the time
    • Did not establish whether cumulus penetration is the sole role
    • Relationship to zona binding activity unaddressed
  6. 1996 High

    Resolved PH-20 as a bifunctional protein with biochemically separable hyaluronidase and secondary zona-binding activities, reconciling its enzymatic and adhesive roles.

    Evidence Orthogonal inhibitor dissection (antibody, apigenin, zona HA removal) and soluble PH-20 characterization in guinea pig and macaque

    PMID:8608861 PMID:8724363 PMID:8793062

    Open questions at the time
    • Structural basis of the zona-binding activity not identified
    • Identity of the zona ligand undefined
  7. 1997 High

    Mapped the hyaluronidase to the N-terminal domain and identified soluble forms as proteolytically released fragments, distinguishing membrane and soluble enzyme populations.

    Evidence Peptide sequencing of purified bovine 60 kDa enzyme mapped to PH-20 cDNA; domain synthesis analysis

    PMID:9116127 PMID:9280317

    Open questions at the time
    • Protease responsible for endoproteolytic cleavage not identified
    • Catalytic residues still inferred from homology
  8. 1999 High

    Identified CREM as the transcriptional driver of Spam1 and defined epididymal N-glycan remodeling as the mechanism increasing hyaluronidase activity during sperm maturation.

    Evidence EMSA, in vitro transcription and CREM-KO Northern blots; deglycosylation and activity assays on caput vs caudal sperm in mouse

    PMID:10423292 PMID:9890751

    Open questions at the time
    • Glycan structures responsible for activity gain not resolved
    • Other transcriptional inputs beyond CREM unexplored
  9. 1999 High

    Showed that plasma-membrane PH-20 binds HA to raise intracellular Ca²⁺ and potentiate the acrosome reaction, and that PH-20 aggregation drives the signal, adding a signaling role distinct from enzymatic and adhesive functions.

    Evidence Fab blocking with Ca²⁺ fluorometry, FITC-HA binding, and aggregation experiments in human and macaque sperm

    PMID:10369396 PMID:10533704 PMID:9770775

    Open questions at the time
    • Cytoplasmic signaling partner not identified
    • Mechanism coupling aggregation to Ca²⁺ entry unresolved
  10. 2001 High

    Localized the HA-binding/signaling activity to a discrete region (aa 205–235) separate from the catalytic domain, molecularly separating PH-20's signaling from its enzymatic function.

    Evidence Recombinant deletion constructs, photoaffinity HA crosslinking, and anti-Peptide 2 Fab inhibition of Ca²⁺ in macaque

    PMID:11731269 PMID:11746965

    Open questions at the time
    • Proposed 92-kDa cytoplasmic signaling partner not directly identified
    • Structural basis of HA binding by this region undefined
  11. 2001 High

    Knockout revealed SPAM1 is dispensable for male fertility due to compensating acrosomal hyaluronidases, while haploid-expressed mRNA compartmentalization explained transmission ratio distortion.

    Evidence Spam1 homologous-recombination knockout with IVF and Western blots; RNA FISH and flow cytometry of compartmentalized transcripts in mouse

    PMID:11369602 PMID:12065596

    Open questions at the time
    • Identity of compensating hyaluronidase not yet established at this stage
    • Molecular tether anchoring mRNA not defined
  12. 2002 High

    Defined the biochemical requirements for hyaluronidase activity—N-glycosylation and disulfide bond integrity—establishing structural determinants of enzyme function.

    Evidence N-glycosidase deglycosylation, disulfide reduction, lectin and 2D analyses with activity readout in macaque

    PMID:11868814

    Open questions at the time
    • Which specific glycans/disulfides are essential not mapped
    • Catalytic mechanism at atomic level not addressed
  13. 2003 Medium

    Extended SPAM1 expression beyond sperm to the female and epididymal tracts and established that secreted SPAM1 retains its intact GPI anchor in luminal particles, setting up a post-testicular delivery model.

    Evidence RT-PCR, RNase protection, IHC, and HA zymography in female tract; PI-PLC/detergent release and 2D-PAGE of epididymal fluid in mouse

    PMID:12514083 PMID:12672666

    Open questions at the time
    • Functional role of female-tract SPAM1 not established
    • Mechanism of particle association undefined at this stage
  14. 2004 Medium

    Localized spermatid Spam1 mRNA to ER-proximal cytoskeleton and confirmed surface SPAM1 mediates synergistic HA+progesterone acrosome reactions using a Spam1 mutant, unifying the regulatory and signaling roles.

    Evidence Light/EM in situ hybridization and acrosome reaction assays comparing wild-type and Rb(6.16) Spam1 mutant sperm

    PMID:15457544

    Open questions at the time
    • Cytoskeletal anchor molecule not identified
    • Signaling effectors downstream of synergy unresolved
  15. 2005 High

    Identified the AU-rich-element-binding proteins that anchor Spam1 mRNA to the cytoskeleton, providing the molecular basis for transcript non-sharing and transmission ratio distortion.

    Evidence UV crosslinking, cytoskeletal binding, transgenic overexpression and Northern analyses in mouse testis

    PMID:16092963 PMID:16250006

    Open questions at the time
    • Individual AUBP identities not all resolved
    • Quantitative contribution to TRD in vivo not fully defined
  16. 2005 High

    Identified HYAL5 as the GPI-anchored testis hyaluronidase that compensates for SPAM1 loss, explaining why Spam1-null mice remain fertile.

    Evidence Protein purification from PH-20-null sperm, HA zymography, cumulus dispersal and apigenin inhibition in mouse

    PMID:16330764

    Open questions at the time
    • Relative in vivo contribution of each enzyme not yet quantified here
    • Regulatory interplay between SPAM1 and HYAL5 unknown
  17. 2006 High

    Demonstrated that epididymally acquired SPAM1 functionally rescues cumulus penetration of null sperm, directly linking post-testicular uptake to fertilizing capacity.

    Evidence In vitro incubation of null sperm with WT luminal fluid, flow cytometry and IVF cumulus penetration assay in mouse

    PMID:16436526

    Open questions at the time
    • Vesicular vs soluble transfer routes not distinguished here
    • Stability of acquired protein on sperm not assessed
  18. 2007 High

    Defined PH-20 substrate specificity (HA octasaccharide minimum, dual hydrolysis/transglycosylation) and confirmed neutral-pH GPI-anchored activity across family members, distinguishing it enzymatically from HYALP1.

    Evidence Recombinant expression in Drosophila S2 cells and Xenopus oocytes with CZE product analysis and activity assays

    PMID:16925524 PMID:17602139

    Open questions at the time
    • Physiological relevance of transglycosylation unknown
    • Catalytic residues not yet structurally confirmed
  19. 2009 High

    Established the molecular machinery for SPAM1 transfer to sperm—GPI-dependent vesicular docking from epididymosomes/uterosomes and clusterin-mediated soluble delivery—and confirmed SPAM1 as the critical cumulus-entry enzyme.

    Evidence GPI-cleavage transfer assays, TEM vesicle docking, CLU co-IP and antibody blocking, and HYAL5/SPAM1 KO comparison in mouse and human

    PMID:18384048 PMID:19357365 PMID:19605784

    Open questions at the time
    • Receptor/docking factor on the sperm membrane not identified
    • Stoichiometry of CLU-SPAM1 complex undefined
  20. 2024 High

    Provided the first atomic structure of human PH-20, revealing the catalytic domain and a unique EGF-like β-hairpin distinguishing it from other hyaluronidases.

    Evidence Cryo-EM structure of human PH-20 with comparative structural analysis

    PMID:39722545

    Open questions at the time
    • Structures of HA-binding and zona-binding regions not resolved in complex with ligands
    • Conformational basis of aggregation-driven signaling not captured

Open questions

Synthesis pass · forward-looking unresolved questions
  • The cytoplasmic signaling partner that couples plasma-membrane PH-20 aggregation to Ca²⁺ entry, and the sperm-surface receptor mediating GPI-anchored SPAM1 docking, remain unidentified.
  • No Co-IP identification of the proposed 92-kDa signaling molecule
  • No molecular identity for the vesicle docking receptor
  • Zona pellucida ligand for secondary binding undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140098 catalytic activity, acting on RNA 5 GO:0098631 cell adhesion mediator activity 3 GO:0140299 molecular sensor activity 3 GO:0016787 hydrolase activity 2
Localization
GO:0005576 extracellular region 3 GO:0005886 plasma membrane 3 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-1474165 Reproduction 3 R-HSA-1474244 Extracellular matrix organization 3 R-HSA-162582 Signal Transduction 3
Partners
CLU

Evidence

Reading pass · 41 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1985 The PH-20 surface antigen on guinea pig sperm posterior head plasma membrane has a required function in sperm binding to the egg zona pellucida; monoclonal antibody PH-20 MAb inhibited sperm-zona binding ~90%, establishing PH-20 as essential for zona adhesion. Monoclonal antibody inhibition of sperm-zona binding assay; competitive binding with 125I-labeled MAbs The Journal of cell biology High 4066757
1986 After the acrosome reaction (exocytosis), the intracellular PH-20 population on the inner acrosomal membrane (IAM) is inserted into the plasma membrane, producing an ~3-fold increase in surface PH-20 antigen; this reveals a mechanism for rapid upregulation of a sperm surface protein via exocytosis. Indirect immunofluorescence; quantification of antigenic sites before and after acrosome reaction; permeabilization of acrosome-intact sperm The Journal of cell biology High 3771636
1987 PH-20 protein reaches the sperm plasma membrane via two transport pathways during spermiogenesis: first inserted into the acrosomal membrane, then separately into the plasma membrane; both populations are initially uniformly distributed and later become restricted to specific domains, indicating localization occurs post-insertion rather than by intracellular sorting. Developmental immunofluorescence localization during spermiogenesis; SDS-PAGE of testicular cell extracts Developmental biology Medium 3305112
1987 PH-20 on the posterior head plasma membrane of acrosome-intact sperm is mobile (D = 1.8×10⁻¹⁰ cm²/s) but restricted ~50-fold relative to lipid; after migration to the IAM of acrosome-reacted sperm it diffuses freely (D = 4.9×10⁻⁹ cm²/s, similar to lipid), supporting a barrier-to-diffusion model for domain maintenance. Fluorescence redistribution after photobleaching (FRAP) on individual sperm The Journal of cell biology High 3558486
1988 PH-20 is anchored in the sperm plasma membrane via a glycosylphosphatidylinositol (GPI/phosphatidylinositol lipid) anchor, and its lateral diffusion rate is >1000-fold slower than lipid diffusion, indicating that ectodomain interactions constrain mobility of a GPI-anchored protein. Phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage; FRAP measurement of diffusion rates Science High 3381102
1988 PH-20 purified from guinea pig sperm by monoclonal antibody affinity chromatography exists in three forms: 64 kDa, 56 kDa, and an endoproteolytically cleaved form of two disulfide-linked fragments (~41–48 kDa and ~27 kDa), indicating site-specific endoproteolysis occurs in sperm preparations. Monoclonal antibody affinity purification; SDS-PAGE; Cleveland digest analysis Biology of reproduction Medium 3042032
1990 cDNA cloning of guinea pig PH-20 revealed a novel protein of 468 amino acids with six N-linked glycosylation sites, twelve cysteines (eight clustered near the C-terminus), encoded by a single gene with a single ~2.2 kb mRNA; Southern blots confirmed a single gene and conservation across multiple mammalian species. cDNA cloning, sequencing; Southern blot; Northern blot The Journal of cell biology High 2269661
1991 PH-20 protein migration from the posterior head plasma membrane to the inner acrosomal membrane after the acrosome reaction occurs against a concentration gradient and is calcium-dependent, arguing against passive diffusion–trapping and in favor of an active translocation mechanism. Fluorescence microscopy with digital image processing of individual sperm; calcium manipulation Developmental biology Medium 1995397
1993 Human PH-20 protein expressed in RK13 cells using a vaccinia virus system has hyaluronidase activity; PI-PLC treatment releases the enzyme from the cell surface with a large increase in activity, confirming the GPI anchor and hyaluronidase function of human PH-20. Vaccinia virus expression; hyaluronidase activity assay; PI-PLC release FEBS letters High 8282124
1993 Human and cynomolgus monkey PH-20 cDNAs were isolated; human PH-20 encodes 509 residues, is 59% identical to guinea pig PH-20, and its mRNA is strictly testis-specific (single 2.4 kb transcript) with a single gene in the human genome. cDNA cloning; Southern blot; Northern blot of 16 human tissues Proceedings of the National Academy of Sciences of the United States of America High 8234258
1994 Sperm plasma membrane protein PH-20 has hyaluronidase activity enabling acrosome-intact sperm to penetrate the cumulus cell layer; purified recombinant PH-20 disperses cumulus cells from mouse eggs, and anti-PH-20 antibodies prevent acrosome-intact sperm from passing through the cumulus to reach the zona pellucida. In vitro cumulus penetration assay; treatment with purified recombinant PH-20; antibody-blocking experiments The Journal of cell biology High 8195297
1995 Human SPAM1 gene maps to chromosome 7q31, is encoded by at least 4 exons spanning ~11 kb of genomic DNA, and expression as a 2.4 kb transcript is strictly limited to testis in a panel of 16 human tissues. FISH; somatic cell hybrid PCR; Northern blot; genomic Southern blot Genomics High 8575780
1996 PH-20 is bifunctional: it has a hyaluronidase activity required for cumulus penetration and a separate, distinct activity required for secondary sperm-zona binding. Anti-PH-20 MAb that blocks zona binding (90%) has no effect on hyaluronidase activity; apigenin blocks hyaluronidase 93% without inhibiting zona binding; pretreatment of oocytes with hyaluronidase to remove zona HA does not affect secondary zona binding. Antibody inhibition assays; apigenin (hyaluronidase inhibitor) assays; zona pretreatment with hyaluronidase; sperm-zona binding assays Biology of reproduction High 8793062
1996 The soluble hyaluronidase released from guinea pig sperm at the acrosome reaction is a soluble form of PH-20 (sPH-20): anti-PH-20 antiserum recognizes it by immunoblot, completely inhibits its enzymatic activity, and 97% is removed by anti-PH-20 affinity chromatography; sPH-20 is not endoproteolytically cleaved unlike membrane PH-20, suggesting enzymatic release from its GPI anchor. Immunoblot; immunoaffinity chromatography; enzyme activity inhibition; anti-PH-20 antiserum Biology of reproduction High 8724363
1996 Cynomolgus macaque sperm PH-20 exists as a 64 kDa form on the plasma and inner acrosomal membranes and a 53 kDa form released during acrosome reaction; the 64 kDa form has predominantly neutral pH hyaluronidase activity while the 53 kDa form has acid-active hyaluronidase activity; apigenin inhibits both. Western blot; HA substrate gel electrophoresis; ELISA hyaluronidase assay; Fab-immunolocalization; ultrastructure Developmental biology High 8608861
1997 The soluble 60 kDa hyaluronidase from bovine testes is a fragment of the membrane-bound PH-20 enzyme; peptide sequencing of 44 fragments from the purified 60 kDa protein aligned to the PH-20 cDNA sequence; it lacks the signal peptide and 56 C-terminal amino acids including the GPI-anchor site. cDNA cloning; protein purification; protease/cyanogen bromide digestion with peptide sequencing FEBS letters High 9280317
1997 PH-20 N-terminal domain contains the hyaluronidase activity (based on homology with bee venom hyaluronidase and functional data); it is GPI-anchored; its dual roles in cumulus penetration (plasma membrane form, neutral pH activity) and secondary zona binding (inner acrosomal membrane form, post-acrosome reaction) are biochemically distinct. Synthesis of review of functional data; domain analysis from cDNA sequence; functional assays cited Biology of reproduction Medium 9116127
1997 In male guinea pigs immunized with PH-20, infertility is associated with emptying of the cauda epididymis (no normal sperm) and induction of experimental autoimmune orchitis (EAO); this is the first demonstration that a purified sperm molecule of known function can induce EAO. Immunization; histopathology; antibody titer measurement; immunofluorescence of germ cells Biology of reproduction Medium 9160711
1998 Hyaluronic acid (HA) interacts with PH-20 on human sperm to increase basal intracellular calcium and potentiate the acrosome reaction induced by progesterone or zona pellucida; blocking PH-20 with anti-PH-20 Fab abolished the HA-mediated Ca²⁺ increase and enhancement of acrosome reactions. Acrosome reaction assay; intracellular calcium measurement; Fab fragment blocking of PH-20 Zygote High 9770775
1999 Murine Spam1 (PH-20) gene promoter contains a CRE (cAMP-responsive element) at position -57 that binds CREM in testicular nuclear extracts; in vitro transcription assays show CRE is necessary for promoter activity; Spam1 transcripts are absent in CREM-knockout mice, establishing CREM as a transcriptional regulator of Spam1. Gel mobility shift assay; in vitro transcription; Northern blot of CREM-KO mice; primer extension Molecular reproduction and development High 10423292
1999 Biochemical maturation of Spam1 during epididymal transit involves N-linked oligosaccharide modification: caput sperm have a larger ~74 kDa form with lower hyaluronidase activity, while caudal sperm have a ~67 kDa form with 4.3-fold higher activity; enzymatic deglycosylation of both reduces to ~56 kDa backbone, and all four N-glycosylation sites are functional. Immunofluorescence; Western blot; enzymatic deglycosylation; hyaluronidase activity assay Molecular reproduction and development High 9890751
1999 HA increases intracellular Ca²⁺ in macaque sperm through plasma membrane PH-20; anti-PH-20 Fab fragments inhibit the Ca²⁺ increase induced by HA, HA gels, and cumulus masses; FITC-HA binding to sperm is localized over the acrosome (PH-20 location) and is inhibited by anti-PH-20 Fab. PH-20 aggregation (by bivalent antibody or zona pellucida binding) is associated with increased Ca²⁺ and acrosome reaction. Fluorometry with Fluo-3 Ca²⁺ indicator; Fab fragment blocking; FITC-HA binding; video imaging; TEM Zygote High 10533704
1999 PH-20 (but not acrosin) is required for secondary sperm-zona binding and zona penetration in macaque; anti-PH-20 IgG completely blocked zona penetration while anti-acrosin and anti-CD46 IgG had no effect; PH-20 is present on the inner acrosomal membrane of zona-bound acrosome-reacted sperm whereas acrosin is not. Immunolocalization by TEM; sperm-zona penetration assay with blocking antibodies (up to 300 µg/ml) Molecular reproduction and development High 10369396
2001 A region of macaque PH-20 (Peptide 2, aa 205-235) functions as a HA-binding domain separate from the catalytic hyaluronidase domain; recombinant PH-20 protein lacking this region (E12) does not bind HA, while the full N-terminal construct (G3) binds biotinylated HA; anti-Peptide 2 Fab inhibits HA-induced Ca²⁺ increase in sperm, linking this domain to cell signaling. Recombinant protein binding assay; photoaffinity HA crosslinking; microplate HA binding assay; Fab inhibition of intracellular Ca²⁺ Molecular reproduction and development High 11746965
2001 PH-20 plasma membrane form mediates HA-induced intracellular signaling via a HA-binding domain distinct from hyaluronidase domains; signaling involves Ca²⁺ increase and aggregation of GPI-anchored PH-20 in the plasma membrane; a 92-kDa protein may be the cytoplasmic signaling molecule linked to PH-20. Review and synthesis of functional data; signaling assays; antibody-induced PH-20 aggregation; calcium measurements Matrix biology Medium 11731269
2001 Despite absence of PH-20 in Spam1 null mice produced by homologous recombination, male mice are fertile; PH-20-null sperm show reduced cumulus dispersal (delayed fertilization at early time points only) but retain fertilizing ability due to other hyaluronidases present in the acrosome detected by Western blot. Homologous recombination knockout; in vitro fertilization assay; Western blot for other hyaluronidases The Journal of biological chemistry High 12065596
2001 Spam1 is haploid expressed in mouse spermatids with both mRNA and protein compartmentalized (non-shared) among conjoined spermatids; this lack of transcript sharing via intercellular bridges leads to biochemically different sperm populations, providing a mechanism for transmission ratio distortion (TRD) in heterozygous males. RNA FISH; immunocytochemistry; flow cytometry; confocal microscopy; temporal expression analysis Biology of reproduction High 11369602
2002 Hyaluronidase activity of macaque sperm surface PH-20 requires both N-linked glycosylation (removal abolishes activity) and disulfide bond integrity (reduction with β-mercaptoethanol or DTT abolishes activity); mannose is a major sugar in the N-linked glycans; 6 isoforms with pI 5.1–6.0 were identified by 2D electrophoresis. Immunoaffinity purification; N-glycosidase F deglycosylation; β-mercaptoethanol/DTT reduction; lectin blotting; 2D electrophoresis; hyaluronidase activity assay Journal of andrology High 11868814
2003 Mouse Spam1 is transcribed in the female genital tract (uterus, oviduct, vagina) in a region-dependent manner; the protein has hyaluronidase activity at neutral pH but not acidic pH in female tissues, and its expression in the uterus fluctuates with the estrous cycle. RT-PCR; RNase protection assay; in situ transcript hybridization; Western blot; immunohistochemistry; HA substrate gel electrophoresis Biology of reproduction Medium 12672666
2003 Epididymal SPAM1 is released with its intact GPI lipid anchor predominantly in insoluble particles in luminal fluid; PI-PLC treatment or Triton X-100 releases the majority, confirming lipid anchor retention on secreted protein; two-dimensional gel shows distinct isoforms in epididymis vs. testis, with N-linked and O-linked glycosylation differences. Ultracentrifugation; PI-PLC treatment; 2D-PAGE with immunoblotting; lectin blotting; enzymatic deglycosylation Journal of andrology High 12514083
2004 Spam1 mRNA in mouse spermatids is localized near the ER and anchored to the cytoskeleton, absent from intercellular bridges; this compartmentalization mediates non-sharing of transcripts. Spam1 on caudal sperm surface mediates the synergistic increase in acrosome reactions induced by HA and progesterone, confirmed by reduced response in Rb(6.16) Spam1 mutant sperm. In situ hybridization by light and electron microscopy; immunocytochemistry; acrosome reaction assay with HA+progesterone; comparison to Spam1 mutant mice Molecular reproduction and development Medium 15457544
2005 HYAL5 (a GPI-anchored hyaluronidase active at pH 5–7) is expressed exclusively in testis and is present on sperm plasma and acrosomal membranes; it can disperse cumulus cells and is the principal cumulus matrix depolymerase in mouse sperm when PH-20 is absent, while PH-20 compensates for Hyal5's role. Protein purification from PH-20-null sperm; hyaluronan zymography; cumulus dispersal assay; apigenin inhibition; GPI-anchor characterization Proceedings of the National Academy of Sciences of the United States of America High 16330764
2005 Spam1 mRNA 3'UTR AU-rich elements (AREs) bind six testicular cytoplasmic proteins (AU-binding proteins, AUBPs) that interact with the cytoskeleton; these interactions mediate post-transcriptional regulation of Spam1 in spermatids; antisense RNA in testis can modulate ARE function. Transgenic overexpression attempts failed, indicating tight post-transcriptional control. UV cross-linking assay; transgenic overexpression; Northern analysis; cytoskeletal binding experiments Molecular reproduction and development Medium 16250006
2005 Spam1 RNA is anchored to the cytoskeleton in spermatids and interacts via 3' UTR AU-rich sequences with cytoskeletal-associated AU-binding proteins, preventing transcript sharing through intercellular bridges; this mechanism drives TRD. Spam1 overexpression (transgene) causes TRD by producing sperm with cytoplasmic droplets containing excess Spam1, reducing fertilizing ability. EM in situ hybridization; Northern analysis of fractionated testicular RNA; UV cross-linking; flow cytometry; FISH Reproductive biology and endocrinology High 16092963
2006 Epididymal SPAM1 acquired by Spam1-null sperm from wild-type epididymal luminal fluid in vitro significantly increases cumulus penetration ability, directly linking epididymal SPAM1 uptake with fertilizing ability and establishing it as a marker of sperm maturation. In vitro incubation of null sperm with WT epididymal luminal fluid; flow cytometry; immunocytochemistry; IVF cumulus penetration assay Biology of reproduction High 16436526
2007 Recombinant mouse SPAM1 and HYAL5 expressed in Xenopus oocytes both exhibit hyaluronidase activity at neutral pH and are GPI-anchored; HYALP1 lacks hyaluronidase activity despite sequence similarity. Recombinant expression in Xenopus oocytes; hyaluronidase activity assay; GPI-anchor characterization The Biochemical journal High 16925524
2007 Human recombinant PH-20 expressed in Drosophila S2 cells uses an HA octasaccharide as its minimum substrate (not hexasaccharide as for bovine testicular hyaluronidase); PH-20 catalyzes both hydrolysis and transglycosylation of HA octasaccharide. Recombinant expression; capillary zone electrophoresis analysis of reaction products; minimum substrate determination Glycobiology High 17602139
2008 SPAM1 is transferred to sperm from murine epididymosomes (male) and newly identified uterosomes (female) via a vesicular docking mechanism; uptake requires the intact GPI anchor (abolished by enzymatic anchor cleavage); fluorescently labeled vesicles are observed juxtaposed to sperm plasma membranes by TEM. Ultracentrifugation; immunogold labeling; confocal and TEM microscopy; GPI anchor cleavage experiments; fluorescent vesicle tracking Molecular reproduction and development High 18384048
2009 SPAM1 is required for efficient sperm entry into and penetration through the cumulus matrix; SPAM1-null sperm accumulate at the surface/outer edge of the cumulus, while HYAL5-null sperm show no such defect; double comparison demonstrates SPAM1 but not HYAL5 is the functionally critical hyaluronidase for cumulus entry. HYAL5 knockout mouse generation; in vitro fertilization assay; comparative analysis of WT, HYAL5-null, and SPAM1-null sperm Biology of reproduction High 19605784
2009 Clusterin (CLU) in epididymal/uterine luminal fluid stabilizes monomeric GPI-linked SPAM1 and mediates its transfer to human and mouse sperm membranes; anti-CLU antibody reduces SPAM1 delivery; co-immunoprecipitation reveals direct CLU-SPAM1 association; high CLU concentrations reduce and low concentrations enhance SPAM1 transfer. Co-immunoprecipitation; native Western blot; anti-CLU antibody blocking; dose-response transfer assay; apolipoprotein-enhanced transfer Biology of reproduction High 19357365
2024 Cryo-EM structure of human PH-20 shows: a central catalytic domain with conserved catalytically essential residues; a unique EGF-like domain with a longer sequence forming a flexibly anchored β-hairpin containing a disulfide bond, distinguishing it from other hyaluronidase family members. Cryogenic electron microscopy (cryo-EM); structural comparison with other hyaluronidase structures Proteins High 39722545

Source papers

Stage 0 corpus · 82 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1988 Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 218 3419530
1994 A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. The Journal of cell biology 213 8195297
1985 A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida. The Journal of cell biology 201 4066757
2002 Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg. The Journal of biological chemistry 144 12065596
2001 The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling. Matrix biology : journal of the International Society for Matrix Biology 144 11731269
1997 Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Biology of reproduction 133 9116127
1990 cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals. The Journal of cell biology 127 2269661
1988 Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein. Science (New York, N.Y.) 124 3381102
1993 The human sperm protein PH-20 has hyaluronidase activity. FEBS letters 122 8282124
2005 Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass. Proceedings of the National Academy of Sciences of the United States of America 111 16330764
1996 Sperm surface protein PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct activity is required in secondary sperm-zona binding. Biology of reproduction 100 8793062
1996 The PH-20 protein in cynomolgus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity. Developmental biology 93 8608861
2008 Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model. Molecular reproduction and development 85 18384048
1993 Molecular cloning of the human and monkey sperm surface protein PH-20. Proceedings of the National Academy of Sciences of the United States of America 78 8234258
1987 Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. The Journal of cell biology 76 3558486
2006 Epididymal SPAM1 and its impact on sperm function. Molecular and cellular endocrinology 74 16420970
2009 Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. Biology of reproduction 73 19605784
1986 Sperm exocytosis increases the amount of PH-20 antigen on the surface of guinea pig sperm. The Journal of cell biology 70 3771636
1987 The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution. Developmental biology 69 3305112
1988 Purification of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulfide-linked fragments. Biology of reproduction 66 3042032
1997 The PH-20 protein in human spermatozoa. Journal of andrology 64 9154509
1995 Location of the PH-20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomolgus macaques. Biology of reproduction 62 7711169
1997 Mechanism of infertility in male guinea pigs immunized with sperm PH-20. Biology of reproduction 60 9160711
2002 Expression of PH-20 in normal and neoplastic breast tissue. The Journal of surgical research 54 11922735
2001 Lack of sharing of Spam1 (Ph-20) among mouse spermatids and transmission ratio distortion. Biology of reproduction 53 11369602
2000 Mouse Spam1 (PH-20): evidence for its expression in the epididymis and for a new category of spermatogenic-expressed genes. Journal of andrology 52 11105908
1998 Hyaluronic acid enhances induction of the acrosome reaction of human sperm through interaction with the PH-20 protein. Zygote (Cambridge, England) 51 9770775
2003 Mouse epididymal Spam1 (pH-20) is released in the luminal fluid with its lipid anchor. Journal of andrology 48 12514083
1999 Biochemical maturation of Spam1 (PH-20) during epididymal transit of mouse sperm involves modifications of N-linked oligosaccharides. Molecular reproduction and development 48 9890751
1996 Structural relationship of sperm soluble hyaluronidase to the sperm membrane protein PH-20. Biology of reproduction 48 8724363
2003 Mouse Spam1 (PH-20) is a multifunctional protein: evidence for its expression in the female reproductive tract. Biology of reproduction 46 12672666
1999 Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20. Zygote (Cambridge, England) 43 10533704
1997 The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme. FEBS letters 42 9280317
1995 Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAM1. Genomics 42 8575780
1999 PH-20 but not acrosin is involved in sperm penetration of the macaque zona pellucida. Molecular reproduction and development 40 10369396
2007 Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology 39 17602139
2006 Epididymal SPAM1 is a marker for sperm maturation in the mouse. Biology of reproduction 37 16436526
2009 Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes. Biology of reproduction 36 19357365
2001 Identification of a hyaluronic acid (HA) binding domain in the PH-20 protein that may function in cell signaling. Molecular reproduction and development 36 11746965
1997 Reversible contraceptive effect of PH-20 immunization in male guinea pigs. Biology of reproduction 35 9160712
2003 SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR. Reproductive biology and endocrinology : RB&E 33 12932297
2001 Mouse epididymal Spam1 (PH-20) is released in vivo and in vitro, and Spam1 is differentially regulated in testis and epididymis. Biology of reproduction 33 11673279
1991 Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism. Developmental biology 33 1995397
2007 Mouse testicular hyaluronidase-like proteins SPAM1 and HYAL5 but not HYALP1 degrade hyaluronan. The Biochemical journal 31 16925524
2019 Fluoride Interferes with the Sperm Fertilizing Ability via Downregulated SPAM1, ACR, and PRSS21 Expression in Rat Epididymis. Journal of agricultural and food chemistry 29 31008594
2004 Expression and secretion of rat SPAM1(2B1 or PH-20) in the epididymis: role of testicular lumicrine factors. Matrix biology : journal of the International Society for Matrix Biology 29 15062858
1997 Biochemical characterization of the PH-20 protein on the plasma membrane and inner acrosomal membrane of cynomolgus macaque spermatozoa. Molecular reproduction and development 28 9322248
2016 Isolating Sperm from Cell Mixtures Using Magnetic Beads Coupled with an Anti-PH-20 Antibody for Forensic DNA Analysis. PloS one 26 27442128
2005 Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit. Molecular reproduction and development 26 15892045
2001 Spam1 (PH-20) mutations and sperm dysfunction in mice with the Rb(6.16) or Rb(6.15) translocation. Mammalian genome : official journal of the International Mammalian Genome Society 25 11845284
1997 The murine Spam1 gene: RNA expression pattern and lower steady-state levels associated with the Rb(6.16) translocation. Molecular reproduction and development 25 9041127
2002 Restricted entry of IgG into male and female rabbit reproductive ducts following immunization with recombinant rabbit PH-20. American journal of reproductive immunology (New York, N.Y. : 1989) 24 12069203
2005 Sperm dysfunction in the Rb(6.16)- and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5. Molecular reproduction and development 22 16078272
2005 Spam1-associated transmission ratio distortion in mice: elucidating the mechanism. Reproductive biology and endocrinology : RB&E 22 16092963
1997 The mouse Spam1 maps to proximal chromosome 6 and is a candidate for the sperm dysfunction in Rb(6.16)24Lub and Rb(6.15)1Ald heterozygotes. Mammalian genome : official journal of the International Mammalian Genome Society 22 9060406
2005 Chondrocytes, synoviocytes and dermal fibroblasts all express PH-20, a hyaluronidase active at neutral pH. Arthritis research & therapy 21 15987477
2004 Spam1 (PH-20) expression in the extratesticular duct and accessory organs of the mouse: a possible role in sperm fluid reabsorption. Biology of reproduction 19 15175239
2002 Importance of glycosylation and disulfide bonds in hyaluronidase activity of macaque sperm surface PH-20. Journal of andrology 19 11868814
2001 Characterization of an 80-kilodalton bull sperm protein identified as PH-20. Biology of reproduction 19 11466235
1998 Rearrangement of the PH-20 protein on the surface of macaque spermatozoa following exposure to anti-PH-20 antibodies or binding to zona pellucida. Molecular reproduction and development 18 9590538
2004 Cytoplasmic localization during testicular biogenesis of the murine mRNA for Spam1 (PH-20), a protein involved in acrosomal exocytosis. Molecular reproduction and development 17 15457544
1999 Characterization of the genomic structure of the murine Spam1 gene and its promoter: evidence for transcriptional regulation by a cAMP-responsive element. Molecular reproduction and development 17 10423292
2004 Effects of scrotal heating on sperm surface protein PH-20 expression in sheep. Molecular reproduction and development 16 15039954
2001 Posttranslational processing of PH-20 during epididymal sperm maturation in the horse. Biology of reproduction 16 11673246
2002 Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides. Journal of reproductive immunology 14 11839398
2005 Transcription of the human and rodent SPAM1 / PH-20 genes initiates within an ancient endogenous retrovirus. BMC genomics 12 15804358
2011 Sexual size dimorphism predicts rates of sequence evolution of SPerm Adhesion Molecule 1 (SPAM1, also PH-20) in monkeys, but not in hominoids (apes including humans). Molecular phylogenetics and evolution 11 22197807
2002 Characterization of the porcine sperm adhesion molecule gene SPAM1- expression analysis, genomic structure, and chromosomal mapping. Animal genetics 11 12030925
2023 Transgenic viral expression of PH-20, IL-12, and sPD1-Fc enhances immune cell infiltration and anti-tumor efficacy of an oncolytic virus. Molecular therapy oncolytics 10 37727704
2024 Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris. Applied microbiology and biotechnology 9 38175240
2023 Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells. Cell and tissue research 9 37833433
2022 Sperm Adhesion Molecule 1 (SPAM1) Distribution in Selected Human Sperm by Hyaluronic Acid Test. Biomedicines 8 36289815
2018 SPAM1 and PH-20 are two gene products expressed in bovine testis and present in sperm. Reproduction (Cambridge, England) 8 30328344
2007 Expression of SPAM1 (PH-20) in the murine kidney is not accompanied by hyaluronidase activity: evidence for potential roles in fluid and water reabsorption. Kidney & blood pressure research 6 17446714
2006 Murine Spam1 mRNA: involvement of AU-rich elements in the 3'UTR and antisense RNA in its tight post-transcriptional regulation in spermatids. Molecular reproduction and development 6 16250006
2004 Cloning and characterization of the bovine testicular PH-20 hyaluronidase core domain. Biotechnology letters 6 15289682
2002 Characterization of PH-20 in canine spermatozoa and testis. Theriogenology 6 11991398
2022 Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor. International journal of molecular sciences 4 36555637
1999 Identification of linear surface epitopes on the guinea pig sperm membrane protein PH-20. Life sciences 4 10374924
2024 Cryo-EM Structure of Human Hyaluronidase PH-20. Proteins 3 39722545
2020 Knockout of hyaluronidase Spam1 reduces age-related bone and cartilage changes in mouse knee. Morphologie : bulletin de l'Association des anatomistes 1 32224028
2026 Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/SPAM1-armed oncolytic adenovirus Ad5Δ24RGD. Molecular therapy. Oncology 0 41716467

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