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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH-30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.
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PMID:Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains. 222 75

The PH-20 protein is first detected in the Golgi complex at the start of differentiation of round spermatids into a polarized cell (spermiogenesis), and next appears in the membrane of the developing secretory granule (the acrosome). Thereafter, a second population of PH-20 is inserted directly into the plasma membrane. Initially, both the acrosomal membrane (PH-20AM) and the plasma membrane (PH-20PM) populations are uniformly distributed in each membrane. Subsequently, PH-20AM is restricted to the inner acrosomal membrane, and during epididymal passage PH-20PM becomes localized to the posterior head surface domain. Therefore, the PH-20 protein does not become localized to either domain by intracellular sorting and insertion into a localized domain, but by restriction following uniform insertion. When the sperm undergoes Ca2+-regulated exocytosis (the acrosome reaction), the inner acrosomal membrane becomes confluent with the plasma membrane. Consequently, the population of PH-20AM is now inserted into the plasma membrane. The PH-20 protein isolated from developing testicular cells contains a major form, approximately 66 kDa, and a minor form, approximately equal to 56 kDa, but it remains to be determined if each form enters only one or both pathways. The developmental control of surface expression of PH-20 during spermiogenesis in the guinea pig may reflect the regulation of a protein involved in sperm-egg adhesion. (Primakoff, P., Hyatt, H., and Myles, D. g. (1985), J. Cell. Biol. 101, 2239-2244).
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PMID: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. 330 12

During spermiogenesis and epididymal transit, proteins on the sperm surface become localized to specific domains. In at least one case (PH-20), the protein is initially inserted throughout the membrane and subsequently becomes restricted to a domain by some mechanism that has not yet been determined. Other proteins could become localized through localized insertion. The sperm surface is a dynamic structure that is altered even after the spermatozoon leaves the male. In the female reproductive tract the spermatozoa undergo capacitation and the acrosome reaction that enables them to fertilize the egg. Both of these processes are accompanied by alterations in protein localization: the PT-1 protein migrates during capacitation, and the PH-20 protein migrates after the acrosome reaction. In addition, an upregulation of the surface expression of PH-20 occurs during the acrosome reaction. This additional PH-20 is incorporated into the plasma membrane by the irreversible fusion of the acrosomal membrane with the plasma membrane. The acrosomal membrane contains PH-20 protein that has been stored there since the formation of the acrosome at the spermatid stage of spermiogenesis. Proteins that are freely diffusing must be maintained in a domain by a mechanism that does not involve immobilization or slowing of protein diffusion. We have suggested that barriers to membrane protein diffusion exist at the equatorial region, the posterior ring, and the annulus and that they are responsible for maintaining a localized distribution of at least some of the surface proteins. The migration of surface proteins could result from an alteration of these barriers, a change in the protein structure so that it can pass through the barrier, or active transport across the barrier. These observed changes in surface expression (localization and the level of expression) may be acting to control surface function post-testicularly.
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PMID:Rearrangement of sperm surface antigens prior to fertilization. 344 71

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.
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PMID:Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding. 891 77

The gene for the sperm adhesion molecule 1 (PH-20), SPAM1, has been known to be testis-specific and exclusively haploid expressed. We show that in mice, the 2 common isoforms of the protein (Spam1) observed in sperm are also present in the caput, corpus, and cauda epididymides. Both qualitative and quantitative variation of expression of the protein were observed in epididymis with the highest expression detected in the corpus. The endogenous production of enzymatically active (via hyaluronidase) Spam1 by epididymal cells is supported by the detection of steady-state Spam1 epididymal messenger RNA in both wild type and germ cell-deficient mice. In situ transcript hybridization shows the transcript to be localized to the principal cells of the epithelium. The protein was similarly immunolocalized to these cells, predominantly in vesicles near the apical region. The results suggest a mechanism for transportation of Spam1 from the epididymal epithelium to sperm during their transit and storage in the cauda. None of the current categories of spermatogenic-expressed genes shows the dual transcription pattern (haploid testicular/diploid epididymal) observed for Spam1. The work also confirms and extends the finding that Spam1 is expressed in the kidney.
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PMID:Mouse Spam1 (PH-20): evidence for its expression in the epididymis and for a new category of spermatogenic-expressed genes. 1110 8

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
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PMID:Posttranslational processing of PH-20 during epididymal sperm maturation in the horse. 1167 46

The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.
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PMID:Mouse epididymal Spam1 (PH-20) is released in vivo and in vitro, and Spam1 is differentially regulated in testis and epididymis. 1167 79

The plasma membrane over the sperm head of several mammalian species has been shown to express a glycerolphosphatidylinositol-linked hyaluronidase known as PH-20. This protein has been associated with the sperm's interaction with the oocyte cumulus matrix and zona pellucida. The characteristics of PH-20 in equine sperm have not been clearly defined. In this study, ejaculated gel-free semen from five stallions and epididymal sperm from isolated epididymis from 10 stallions was used to characterize the PH-20 activity in equine sperm. Affinity purified anti-equine PH-20 polyclonal antibody was used to immunodetect sperm surface-associated PH-20 and immunolabel whole sperm. The intracellular calcium indicator, Fluo-3, was used to assess sperm intracellular calcium. Stallion sperm express a surface-associated hyaluronidase localized to the posterior sperm head region in ejaculated sperm. Following in vitro capacitation and acrosomal exocytosis, the inner acrosomal membrane (IAM) displays intense hyaluronidase fluorescence suggesting that the IAM and hyaluronidase plays a significant role in zona penetration by sperm. Sperm incubated in hyaluronan (HA)-containing capacitation medium display an elevated intracellular calcium concentration (P<0.01) that is associated with translocation of PH-20 antigenic sites on the sperm surface in addition to increases in protein tyrosine phosphorylation. Caput- and cauda-derived sperm display developmentally unique PH-20 immunofluorescence expression patterns. These data suggest that the differential expression of PH-20 in ejaculated and epididymal sperm could be involved in cumulus penetration, sperm-egg recognition, and oolemmal fusion in this species.
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PMID:Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase. 1174 73

Previously we demonstrated that the murine sperm adhesion molecule 1 (Spam1 or PH-20) is synthesized by the epididymal epithelium, preferentially in the distal region, and is released into the luminal fluid. We also showed that whereas testicular and epididymal Spam1 have hyaluronidase activity at neutral pH, they are under different transcriptional regulation. The aim of this study was to further compare characteristics of the two forms of this glycosyl-phosphatidylinositol-linked protein and their transcripts, and to determine whether secreted epididymal Spam1 is released with its lipid anchor. With GeneRacer amplification of the 3' end of the complementary DNA we show that the poly(A) tails are significantly (P <.05) shorter in the epididymis than in the testis. Two-dimensional polyacrylamide gel electrophoresis with immunoblotting reveals one to three isoforms for epididymal Spam1 with the isoelectric point (pl) ranging from 7.3 to 9.0, and four isoforms ranging from 6.6 to 9.0 pl for testicular Spam1. Two isoforms with a pl ranging from 7.6 to 9.0 were observed for caudal sperm. Lectin blotting analysis shows that Phaseolus vulgaris erythroagglutinin, Lycopersicon esculentum lectin (LEL), and Solanum tuberosum lectin, which all bind to N-linked chains, recognize a 67 kd band in the epididymis and caudal sperm, but not in the testis. Treatment of the protein extracts with anti-Spam1 serum prior to blotting with LEL led to the disappearance of the banding, indicating Spam1 specificity of the staining. The lectin peanut agglutinin, which preferentially binds to O-linked side chains, recognizes a 67 kd band in all three cell types. Enzymatic deglycosylation studies confirmed the presence of an O-linked glycan in all three cell types. Ultracentrifugation of the luminal fluid reveals that epididymal Spam1 is secreted predominantly as insoluble particles, which when treated with phosphatidylinositol-specific phospholipase C or Triton X-100, reveal that the majority of epididymal Spam1 is released with its lipid anchor, a form in which it can bind to sperm.
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PMID:Mouse epididymal Spam1 (pH-20) is released in the luminal fluid with its lipid anchor. 1251 83

Rat sperm surface antigen Sperm Adhesion Molecule1, SPAM1 (a.k.a. 2B1 or PH-20) is a plasma membrane-bound glycoprotein with hyaluronidase activity and putative roles during fertilization. Previously the antigen was thought to be testis-specific but recently it has been shown to be synthesized in the epididymis (mouse, macaque and human). Using the efferent ductule ligated (EDL) rat as a model to produce a sperm-free androgen-maintained epididymis, we have examined the factors regulating the expression of epididymal 2B1. RT-PCR and in situ transcript hybridization (ISH) studies showed that 2B1 mRNA is transcribed in the principal cells in all three regions of the epididymis. Its cognate protein was also detected by Western blot analysis in sperm-free cytosols from normal epididymis and found to undergo endoproteolytic cleavage into 2 subunits of similar size to the sperm-bound form. Immunohistochemistry with a monoclonal antibody to 2B1 confirmed that the protein is present in the epididymal epithelium and luminal secretions. The intensity of staining was much stronger in the sperm-free EDL epididymis than that in the normal (sperm-present) epididymis. The protein was shown to have hyaluronidase activity at neutral pH and both its quantity and activity appeared to be greater in the EDL epididymis. It is suggested that a soluble form of SPAM1 glycoprotein is synthesized and released in the epididymis and that in addition to androgens, its regulation may involve a cross-talk between the tubule epithelium and lumicrine factors, the latter possibly of testicular origin.
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PMID:Expression and secretion of rat SPAM1(2B1 or PH-20) in the epididymis: role of testicular lumicrine factors. 1506 58


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