Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

In the present study, we developed a novel method to analyze the calcium (Ca2+) signal in living slices of mouse caput epididymides by applying calcium imaging on Fura-2-loaded vibratome slices. The data revealed that in epithelial cells of mouse caput epididymides, ATP induces a rapid Ca2+ signal that is sustained after 60 sec. Preincubating the sections in Ca2+-free medium in the presence of EGTA did not affect the amplitude of the ATP-induced Ca2+ signal, indicating the presence of P2Y type purinergic receptors and phospholipase C activity. Furthermore, ATP induced a similar Ca2+ signal in the different subregions of caput epididymides. The P2X type ion-gated purinergic receptors could also be responsible for the ATP-induced Ca2+ signal because immunohistochemical and reverse transcriptase-polymerase chain reaction analyses showed that P2X1, P2X2, P2X4, P2X7, P2Y1, and P2Y2 receptors were expressed in the epididymis. We propose that P2X and P2Y receptor expression is vital for the normal function of epididymal epithelium and sperm maturation. Furthermore, the method we developed allows us to analyze the activity of various G protein-coupled receptors in intact epithelial cells of mouse epididymides, and other reproductive tissues as well.
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PMID:Adenosine triphosphate induces Ca2+ signal in epithelial cells of the mouse caput epididymis through activation of P2X and P2Y purinergic receptors. 1260 41

Orexin-A (OR-A) and OR-B are derived from a common 130-amino acid precursor peptide, prepro-OR, by proteolytic cleavage. Orexins orchestrate their actions by binding and activating two types of G protein-coupled receptors, OR-1 receptor (OX1R) and OR-2 receptor (OX2R). Besides playing a role in the regulation of feeding and energy homeostasis in rats, ORs appear to increase sexual arousal as well as copulatory performance in rats. Furthermore, OR-A and -B immunoreactivity has been detected in rat testis. In view of these findings we investigated the expression of OR receptors and their signaling characteristics in the human male reproductive system. Using RT-PCR analysis, we were able to demonstrate that both OX1R and OX2R are expressed in the testis, epididymis, penis, and seminal vesicle, whereas prepro-OR was expressed only in the epididymis and penis. Protein expression of both OR receptors in human testis was confirmed by Western blotting analysis, with apparent molecular masses of 50 kDa for OX1R and 40 kDa for OX2R. Immunofluorescent studies revealed staining for both OX1R and OX2R in Leydig cells, myoid cells of the seminiferous tubules, and Sertoli cells. To test the ability of ORs to activate testicular phospholipase C, we determined the effects of OR-A and OR-B on inositol trisphosphate production. When membranes from testicular biopsies were incubated with OR-A or OR-B, there was a rapid inositol trisphosphate turnover. This effect appeared to be dose dependent. These data provide a novel insight into the expression and signaling characteristics of OR receptors in the human male reproductive system and potentially further implicate these peptides, acting in an autocrine/paracrine manner, in the regulation of arousal mechanisms in humans.
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PMID:Expression of human prepro-orexin and signaling characteristics of orexin receptors in the male reproductive system. 1507 Sep 69

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H(+)-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.
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PMID:Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H+-ATPase recycling. 1559 Oct 47

Glioma pathogenesis-related 1-like protein1 (GliPr1L1) was identified by liquid chromatography-tandem mass spectrometry analyses of proteins associated to bovine sperm lipid raft membrane domains. This protein belongs to the CAP superfamily including cysteine-rich secretory proteins, Antigen 5 and pathogenesis-related 1 protein. PCR analysis revealed that GliPr1L1 is expressed in testis and, at a much lower level, all along the epididymis. Western blotting showed a similar distribution of GliPr1L1 in testicular and epididymal tissue extracts. In the epididymal lumen, GliPr1L1 was associated with the maturing spermatozoa and epididymosomes all along the excurrent duct but was undetectable in the soluble fraction of epididymal fluid. The protein was detectable as multiple isoforms with a higher MW form in the testis and proximal caput. Treatments with PNGase F revealed that N-glycosylation was responsible of multiple bands detected on Western blots. These results suggest that the N-glycosylation moiety of GliPr1L1 is processed during the transit in the caput. Western blots demonstrated that GliPr1L1 was associated with the sperm plasma membrane preparation. GliPr1L1 is glycosyl phosphatidyl inositol (GPI) anchored to caput and cauda spermatozoa as demonstrated by the ability of phosphatidylinositol specific phospholipase C to release GliPr1L1 from intact sperm cells. Lipid raft membrane domains were separated from caput and cauda epididymal spermatozoa. GliPr1L1 was immunodetectable in the low buoyant density fractions where lipid rafts are distributed. GliPr1L1 was localized on sperm equatorial segment and neck. In vitro fertilization performed in presence of anti-GliPr1L1 showed that this protein is involved in sperm-zona pellucida interaction.
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PMID:Bovine sperm raft membrane associated Glioma Pathogenesis-Related 1-like protein 1 (GliPr1L1) is modified during the epididymal transit and is potentially involved in sperm binding to the zona pellucida. 2255 61

Bovine beta-defensin 126 (BBD126) exhibits preferential expression for the cauda epididymis of males, where it is absorbed onto the tail and postacrosomal region of the sperm. The aim of this study was to examine the role of BBD126 in bull sperm function. Fresh and frozen-thawed semen were incubated in the presence of different capacitating agents as well as with phosphatidylinositol-specific phospholipase C. These treatments, which have been successful in releasing beta-defensin 126 from macaque sperm, proved to be ineffective in bull sperm. This finding suggests that the protein behaves in a different manner in the bovine. The lack of success in removing BBD126 led us to use corpus epididymis sperm, a model in which the protein is not present, to study its functional role. Corpus sperm were incubated with cauda epididymal fluid (CEF) in the absence or presence of BBD126 antibody or with recombinant BBD126 (rBBD126). Confocal microscopy revealed that rBBD126 binds to corpus sperm with the same pattern observed for BBD126 in cauda sperm, whereas an aberrant binding pattern is observed when sperm are subject to CEF incubation. Addition of CEF increased motility as well as the number of corpus sperm migrating through cervical mucus from estrus cows. However, it decreased the ability of sperm to fertilize in vitro matured oocytes. The presence of the antibody failed to abrogate these effects. Furthermore, when rBBD126 was added in the absence of other factors and proteins from the CEF, an increase in motility was also observed and no negative effects in fertility were seen. These results suggest that BBD126 plays a key role in the acquisition of sperm motility in the epididymis.
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PMID:Cauda Epididymis-Specific Beta-Defensin 126 Promotes Sperm Motility but Not Fertilizing Ability in Cattle. 2770 13


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