Protein sulfhydryl reactivity and sperm activation.
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Acrosome↗
Biomedical subjects
B M Shapiro
Publications and source records attributed to B M Shapiro.
The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs.
The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake "respiratory burst" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo.
Animals↗
pH-induced hysteretic transitions of ovoperoxidase.
Ovoperoxidase, the enzyme that catalyzes the dityrosine cross-linking of fertilization membranes of eggs from the sea urchin Stronglyocentrotus purpuratus, exhibits slow changes in catalytic activity upon alterations of pH, with attendant changes in spectral properties. For ovoperoxidase pre-equilibratated at pH 8, abrupt decreases in pH are accompanied by a slow loss in activity that is temporally associated with a change in absorbance at the Soret band. With enzyme pre-equilibrated at pH 4.5 and then shifted to higher pH, there was a slow increase in catalytic activity following a rapid change in the Soret band absorbance. These changes were reversible and led to the same equilibrium state, regardless of the direction of pH shift. The rate of approach to the equilibrium state of ovoperoxidase was independent of enzyme concentration, the presence of substrates, or temperature (from 6.5 to 39.7 degrees C). The pH-induced interconversions of catalytic and spectral properties indicate that ovoperoxidase undergoes hysteretic transitions, in which alterations in the heme environment accompany, but are not sufficient for, the expression of catalytic activity. We present a kinetic mechanism for the hysteretic relaxations and suggest how these transitions may have relevance to the assembly of the fertilization membrane in vivo.
Animals↗
Posttraumatic pseudoaneurysm of the extracranial middle meningeal artery.
Pseudoaneurysms of the extracranial arterial tree are uncommon. To our knowledge, four cases of pseudoaneurysm of the internal maxillary artery have been reported, but there have been no reports of pseudoaneurysm of the extracranial segment of the middle meningeal artery. A 15-year-old girl, who suffered a gunshot wound in the right maxillary region, suffered a pseudoaneurysm of the proximal portion of the middle meningeal artery, as shown by carotid angiography six days after injury. The lesion was successfully treated with absorbable gelatin sponge embolization. Treatment of pseudoaneurysms reduces the risk of hemorrhage from subsequent rupture. Although these lesions are amenable to surgery, transvascular embolization or mechanical interruption appears to be simpler and equally effective.
Adolescent↗
A biochemical pathway for a cellular behaviour: pHi, phosphorylcreatine shuttles, and sperm motility.
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Animals↗
Metabolite channeling: a phosphorylcreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail.
Energy utilization by the flagellum of motile sea urchin sperm is tightly coupled to the rate of energy production by the mitochondrion. This tight coupling depends upon the transport of high energy phosphate (P) from mitochondrion to axoneme, which we propose to be mediated by a phosphorylcreatine shuttle. The shuttle employs distinct mitochondrial and axonemal creatine kinase isozymes, the latter being a novel creatine kinase of 145 kd. To examine whether P is directed to the tail by such a shuttle, we inactivated creatine kinase specifically with fluorodinitrobenzene. Creatine kinase inactivation led to an inhibition of coupled, but not uncoupled, respiration and affected the pattern of sperm motility as predicted for the disruption of an obligatory link in P transport.
Animals↗
Coupled ionic and enzymatic regulation of sperm behavior.
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Acrosome↗
Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.
As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues.
Acrosome↗
Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein.
Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross-linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase-proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.
Animals↗
The use of osteosynthesis in immediate and delayed mandibular reconstruction.
Principles of osteosynthesis utilizing dynamic compression plating have been used in immediate and delayed mandibular reconstruction in five patients. Intermaxillary fixation was not used. Follow-up ranged from eight to 14 months. Four of five patients have viable bone grafts at the present time. One bone graft has been lost due to infection. We feel mandibular reconstruction based on principles of osteosynthesis helps to rapidly restore cosmesis and function with minimal morbidity.
Bone Plates↗
Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction.
The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.
Acrosome↗
Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg.
The ovoperoxidase from the egg of the sea urchin, Strongylocentrotus purpuratus, has been purified to apparent homogeneity. Ovoperoxidase is secreted from the egg at fertilization and is responsible, in vivo, for hardening of the fertilization membrane by forming cross-links between protein tyrosyl residues. Purification was accomplished by activation of cortical granule exocytosis with acetic acid, followed by NH4SO4 precipitation, DEAE-Sephacel chromatography in the absence of divalent cations, and CM-Sephadex chromatography. The purified enzyme is a glycoprotein of Mr 70,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibits a UV-visible spectrum typical of heme peroxidases (epsilon 412 = 1.19 X 10(5) M-1 cm-1). Ovoperoxidase catalyzes the oxidation of tyrosine, guaiacol, iodide, and bromide, but not chloride, and can employ either H2O2 or, with 8% relative efficiency, ethyl peroxide as an oxidative substrate. Phenylhydrazine, 3-amino-1,2,4-triazole, azide, and sulfite all inhibit purified ovoperoxidase at concentrations similar to those that inhibit hardening in vivo. Inhibition by 3-amino-1,2,4-triazole is reversible, requires H2O2, and is slow relative to substrate turnover. The purified enzyme is sensitive to protease cleavage in the native state, yielding an active product of Mr approximately 50,000 which varies slightly depending upon the protease employed. Ovoperoxidase should provide a useful tool for the study of fertilization membrane formation as a paradigm of macromolecular assembly and modification.
Amino Acids↗
Hapten-mediated immunopurification of membrane proteins labeled with fluorescein derivatives.
The behavior of cell surface components labeled with fluorochromes can be studied by fluorescence microscopy and spectroscopy; further structural analyses would be facilitated by purification of the labeled components. We have developed a protocol for identifying the targets for labeling with fluorescein derivatives, by using 125I- diiodofluorescein isothiocyanate ( 125IFC ) and for isolating the labeled components with anti-IFC immunoadsorbents. Anti-IFC antibodies obtained from rabbits immunized with IFC-hemocyanin were purified by affinity chromatography and coupled to CNBr-activated Sepharose 4B. The anti-IFC immunoadsorbents could then be used to isolate the entire set of 125IFC -proteins from crude detergent extracts of labeled sea urchin sperm, with a 70% yield and a purification of more than 250 fold. Nonspecific binding of unlabeled proteins to the immunoadsorbent was insignificant. When the immunoadsorbent IFC-protein complex was used directly as an immunogen, antibodies were obtained that reacted with the underivatized proteins that were targets for IFC labeling, as indicated by immunoblotting after gel electrophoresis. The antibodies also reacted with the surface of unlabeled sperm as shown by immunofluorescence. Thus, by treating the IFC-sperm proteins as a class, we obtained antibodies that recognized the unlabeled proteins in situ or in cell extracts. This approach should be generally useful in obtaining reagents directed against specific cell surface components.
Animals↗
Molecular aspects of sperm-egg fusion.
Fertilization, as one of the few well-studied physiological cell fusion systems, provides a glimpse of hierarchies of control that may exist in other membrane fusions as well. Sperm become fusogenic only after undergoing exocytosis from an apical vesicle; this acrosome reaction, induced by an egg surface component, confers upon the sperm the capacity to bind to and fuse with an egg. The acrosome reaction requires Ca2+ and Na+ and is mediated by a complex series of ionic alterations in sperm, including plasma membrane potential depolarization, Ca2+ influx, and increased intracellular pH. These changes take their toll of the sperm, which dies soon thereafter if it does not fertilize an egg. Sperm-egg fusion itself is rapid, with a negligible requirement for extracellular Ca2+, and is inhibited by depolarization of the egg plasma membrane potential. Gamete membrane fusion is followed by dramatic changes in egg physiology, including those that inhibit subsequent sperm-egg fusions. These blocks to polyspermy include a partial decrease in egg surface receptivity caused by egg membrane depolarization immediately after gamete fusion, followed by a complete inhibition of sperm entry due to a massive exocytosis from vesicles beneath the egg plasma membrane, with a concomitant change in the egg plasma membrane and cell coat. The sperm that has successfully fused with an egg contributes not only its genome, but cytoplasmic components as well. Some cytoplasmic constituents from the sperm, including several proteins, persist without degradation throughout early development; several remain localized in a single region. Whether these cytoplasmic components transferred from the sperm play a role in subsequent morphogenesis of the embryo is not clear.
Acrosome↗
Sperm surface proteins persist after fertilization.
Certain sperm components labeled with fluorescein isothiocyanate or its radioactive derivative, 125I-diiodofluorescein isothiocyanate (125IFC), are transferred at fertilization to the egg, where they persist throughout early cleavage stages at a localized site in the embryo cytoplasm (Gabel, C. A., E. M. Eddy, and B. M. Shapiro, 1979, Cell, 18:207-215; Gundersen, G. G., C. A. Gabel, and B. M. Shapiro, 1982, Dev. Biol., 93:59-72). By using image intensification we have extended these observations in the sea urchin to the pluteus larval stage, in which greater than 60% of the embryos have localized fluorescent sperm components. Because of the unusual persistence of the sperm components in the embryo, a characterization of the nature of the labeled species in sea urchin sperm was undertaken. Approximately 10% of the 125IFC was in sperm polypeptides of Mr greater than 15,000. These proteins were on the sperm surface as shown by their sensitivity to externally added proteases. The remainder of the 125IFC in sperm was in several low-molecular-weight species, none of which was 125IFC-derivatized phospholipid. To determine if any labeled sperm polypeptides remained intact in the embryo after fertilization, 125IFC-labeled sperm proteins were recovered from one-cell and late gastrula stage embryos by using an anti-IFC immunoadsorbent. Most of the labeled sperm proteins were degraded shortly after fertilization; however, distinct sets of labeled polypeptides were recovered from both one-cell and gastrula stage embryos. Six of the labeled polypeptides recovered from both embryonic stages had identical SDS gel mobilities as labeled sperm polypeptides. Other polypeptides in the embryos appeared to arise from limited proteolysis of sperm proteins. Thus, in this physiological cell fusion system, individual sperm proteins are transferred to the egg at fertilization, and some persist intact or after specific, limited degradation long after gamete fusion, until at least the late gastrula stage.
Animals↗
Metabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration.
Mitochondria of sperm of the sea urchin Strongylo-centrotus purpuratus are tightly coupled before induction of the acrosomal reaction. When the sperm are diluted at low external pH (pH 5.5) or in high potassium (200 mM) or in the absence of sodium, their internal pH is acidic (6.2-7.0) as measured by amine accumulation. Under these conditions the internal ATPase activity (primarily the dynein ATPase) is inhibited, sperm are immotile, and mitochondria are in respiratory state 4 (ATP concentration is maximal). When the internal pH is alkalinized, the internal ATPase activity is increased, as estimated either in vivo by measuring the decrease in ATP concentration after addition of oligomycin to prevent ATP synthesis, or in vitro using Triton X-100 permeabilized cells. This increase in ATPase activity correlates with an increase of up to 50-fold in respiratory rates and a mitochondrial transition to state 3. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone-uncoupled respiration is also sensitive to the internal pH for it is inhibited at acidic internal pH. However, since nonmotile, nonrespiring sperm that are obtained when the internal pH is acidic have high concentrations of ATP, we conclude that in vivo the internal pH controls the rate of dynein ATPase and that this ATPase activity is limiting for the respiration of tightly coupled mitochondria. The redox state of the respiratory chain may also be under the direct influence of the internal pH.
Adenosine Triphosphatases↗
Interactions between sperm and sea urchin egg jelly.
The addition of egg jelly to sea urchin sperm induces multiple changes in morphology and behavior. When jelly is added to sperm diluted in seawater, the acrosome reaction is triggered, the mitochondrion rounds up, the internal pH is transiently alkalinized and then reacidified, and respiration becomes uncoupled and rapidly decreases. Sperm also become unable to fertilize eggs within a few minutes after jelly addition. In order to explore in more detail the effect of egg jelly on sperm, we have studied the response to jelly in the presence of inhibitors of the acrosome reaction. When jelly is added to sperm under conditions which are inhibitory for the acrosome reaction, an alkalinization takes place without the subsequent reacidification, the mitochondria remain coupled, and respiration and intracellular ATP levels remain high. Sperm viability is prolonged by some of these conditions, but not others. The addition of jelly to sperm in the absence of calcium elicits an internal alkalinization but no other rapid change in sperm physiology. The capacity of egg jelly to alter sperm physiology even when the overall acrosome reaction is inhibited indicates that some of the physiological changes either are early events in the triggering pathway that happen before the inhibitory step or are unrelated to the acrosomal reaction itself. The reacidification of the internal pH, the uncoupling and decrease of the respiration, and the decrease of the ATP levels might be linked together by the large influx of calcium that occurs after the acrosome reaction.
Acrosome↗
31P-NMR analysis of sea urchin sperm activation. Reversible formation of high energy phosphate compounds by changes in intracellular pH.
31P-NMR has been used to estimate the internal pH (pHi) of sperm from the sea urchin Strongylocentrotus purpuratus. The values for pHi obtained from the chemical shift of inorganic phosphate agree well with those obtained from amine accumulation. At low pHi, when sperm are quiescent (immotile and non-respiring), they accumulate phosphocreatine (PCr), but have a low level of inorganic phosphate (Pi). Conversely, when the pHi is elevated, sperm respiration and motility are activated, PCr is decreased and Pi is increased. This change is reversible upon decrease of the pHi, whereupon respiration and motility are arrested, Pi disappears and PCr increases. We conclude that the overall balance of energy metabolism, and thus the phosphate potential, of sea urchin sperm are under the control of the pHi.
Adenosine Triphosphate↗