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

Bovine epididymal spermatozoa incubated aerobically in vitro in the presence of 0.1 to 0.2 mM CaCl2 accumulate 25 to 50 nmol of calcium/10(8) cells. The addition of low concentrations of the ionophore A23187 (0.01 to 0.5 nmol/mg of sperm protein) induces efflux of this accumulated calcium. At high ionophore concentrations (0.5 to 5.0 nmol/mg of sperm protein), calcium release is followed by an influx of up to 25 nmol of calcium/10(8) cells that is not dependent on mitochondrial energization. A selective increase in the permeability of the sperm plasma membrane produced by treatment with the polyene antibiotic, filipin, results in the release of that calcium which is accumulated in the presence of high concentrations of A23187. Sperm first treated with filipin possess the ability to accumulate and retain calcium (in the presence of an oxidizable substrate) but release Ca2+ without subsequent reaccumulation after the addition of 3 nmol of A23187/mg of protein. These observations are explained by the existence of competing calcium pumps operating within the mitochondrial and plasma membranes of the spermatozoan. Treatment with high concentrations of A23187 allows calcium influx into a non-mitochondrial compartment of the sperm cell as a consequence of the equilibration of this cation across both mitochondrial and plasma membranes. The amount of calcium uptake and its sensitivity to filipin indicate that calcium binding to soluble, intracellular components is also involved. The ability of low concentrations of A23187 to induce calcium efflux is explained as a result of the continued operation of the plasma membrane pump coincident with ionophore-induced decay of the concentration gradient across the mitochondrial membrane. This hypothetical action of low levels of the ionophore on the mitochondria is supported by the observation of net movements of calcium with filipin-treated cells and the respiratory responses and movements of phosphate and membrane-associated calcium with intact sperm. It is suggested that the basis of this apparent selectivity of ionophore action lies in the relative activities and kinetic properties of the competing calcium pumps in the plasma and mitochondrial membranes of these cells. Ionophore-induced influx of calcium into the extramitochondrial space results in a stimulation of respiration and kinetic activity of the sperm. This activation of motility is observed also with cells made entirely dependent upon glycolysis (by treatment with respiratory inhibitors) and suggests a direct involvement of calcium in the regulation of flagellar function.
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PMID:Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. 77 78

Unlike those of many mammals, rat and hamster spermatozoa do not appear to be fully activated at ejaculation. Most rat spermatozoa transported en masse to the uterus approximately 2 minutes after coitus were vigorously motile on recovery soon thereafter in uterine fluid, whereas a majority, and sometimes all, were immotile in samples collected less than 2 minutes after coitus from the anterior vagina of normal females and up to 15 minutes after coitus from the anterior vagina in females with obstructed cervices. Many of these immotile rat vaginal spermatozoa began instant vigorous movement upon exposure to Tyrode's solution or uterine fluid. Hamster spermatozoa recovered from the vagina about 2 minutes after coitus were also immotile or displayed only slow, languid serpentine movements, but that motility profile remained very similar in spermatozoa taken 0.5 hours to 6 hours after coitus from the uterus, which is essentially fluid-free in the hamster. In the hamster, actively motile spermatozoa were evident only in the isthmus of the (transilluminated) oviduct. As in the rat, immotile vaginal and uterine hamster spermatozoa instantly began vigorous progressive motility in vitro on contact with Tyrode's solution or rat uterine fluid. Immotile spermatozoa from the rat and hamster cauda epididymidis immediately became highly motile in Tyrode's solution, and they developed a somewhat less rapid flagellar beat in 150 mmol/L NaCl or KCl, with or without 2 mmol/L CaCl2. In contrast, dilution with an isotonic sucrose solution containing no ions, or only 2 mmol/L CaCl2, evoked very slow and transient movement of rat and hamster epididymal sperm tails.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Initiation of sperm motility after mating in the rat and hamster. 142 20

The aim of this study was to determine whether methoxamine and barium stimulate 45Ca2+ uptake or efflux in the rat vas deferens in a manner that correlates with their contractile activity, and whether 45Ca2+ movements are inhibited by verapamil or nifedipine. Basal La(3+)-resistant (cellular) 45Ca2+ uptake was significantly greater in the epididymal half (791 +/- 27 nmol g-1) than in the prostatic half (654 +/- 14 nmol g-1) of the rat vas deferens and was unaffected by verapamil (61 microM) or nifedipine (14 microM). Methoxamine (8 microM) was without effect on 45Ca2+ uptake in either half but BaCl2 (1 mM) increased 45Ca2+ uptake by 31% in the prostatic half and by 22% in the epididymal half. The barium-induced increases in 45Ca2+ uptake were markedly reduced or abolished by verapamil (2 microM) or nifedipine (0.3 microM), which at these concentrations have no effect on the rhythmic contractions but abolish the initial small phasic contraction induced by barium. The basal rate of 45Ca2+ efflux from the intact vas deferens (into Ca2+ containing Krebs-Henseleit solution or into Ca-free Krebs-Henseleit solution +/- EGTA 0.05 mM) was not affected by verapamil (61 microM) or nifedipine (14 microM). Methoxamine (8 microM) produced a marked, transient and reversible increase in 45Ca2+ efflux into 2.5 mM CaCl2 Krebs-Henseleit in 50% of the intact vasa deferentia examined which was augmented by verapamil (61 microM). BaCl2 (1 mM) produced a small increase in 45Ca2+ efflux into Ca(2+)-containing and Ca(2+)-free Krebs-Henseleit solutions from some intact vasa deferentia and this was not inhibited by nifedipine (14 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of methoxamine and barium on 45Ca2+ fluxes in the rat vas deferens. 149 62

In this report we describe and partially characterize a preparation of digitonin-permeabilized guinea pig spermatozoa that undergo a rapid and synchronous modification of the acrosomal matrix in response to calcium. Permeabilization of cauda epididymal spermatozoa by digitonin was monitored by using adenylate cyclase activity as an indicator. Spermatozoa (5 x 10(7) cells) treated with 0.005% digitonin for 15 s exhibited maximal adenylate cyclase activity but generally retained their structural morphology, as examined by phase-contrast and transmission electron microscopy. The ratio fo cell number to detergent concentration was the critical factor for determining both the efficiency of permeabilization and the maintenance of structural integrity. When permeabilized spermatozoa were treated with 2 mM CaCl2, the cells underwent a rapid and synchronous modification of the acrosomal matrix (AM). As observed by phase-contrast microscopy, the response to CaCl2 was characterized by events that occurred in the following temporal sequence: disruption of the sperm rouleaux, the loss of refractility by the apical segment of the sperm acrosome, and detachment of the apical segment from the spermatozoa. Transmission electron microscopy indicated that the loss of refractility from the sperm apical segment was coincident with a calcium-induced dispersion of the AM. Analysis of the proteins released during this response, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that a specific subset of sperm proteins was released from the spermatozoa, including a major = staining, 45,000 Mr protein apparently generated from a higher molecular weight precursor during the acrosome reaction.
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PMID:Calcium-induced modification of the acrosomal matrix in digitonin-permeabilized guinea pig spermatozoa. 250 1

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.
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PMID:Crosslinking of uteroglobin by transglutaminase. 614 14

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.
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PMID:Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts. 624 91

The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.
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PMID:Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin. 632 51

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.
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PMID:Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. 753 69

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. 774 26

A trypsin-like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted sperm protease (CSP) was purified by ion-exchange chromatography on CM-23, gel filtration on Sephadex G-100, affinity chromatography on benzamidine-CH-Sepharose 4B, and HPLC on CM-5PW. CSP is a two chain protein composed of M(r) 2.6K and M(r) 37K chains, which are covalently cross-linked by disulfide bonds. CSP exhibited a pH optimum between pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50 mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kcat/Km) of CSP toward Bz-L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the presence of 50 mM CaCl2 differed from that of acrosin by factors of 0.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested peptides of CSP, and its L- and H-chains showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.
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PMID:Purification and characterization of a novel acrosin-like enzyme from boar cauda epididymal sperm. 782 68


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