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B T Storey

Publications and source records attributed to B T Storey.

At least 73 records · Page 4Linked to original sources
PubMed1982

Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effect on sperm motility.

Rabbit spermatozoa released from the cauda epididymidis into Tris phosphate medium containing KCl or NaCl and 0.4 mM EDTA underwent spontaneous lipid peroxidation during aerobic incubation at 37 degrees C. In the medium containing 130 mM K+ and O mM Na+ (KTP), the rate of lipid peroxidation, as measured by malonaldehyde production, proceeded at a linear rate of 0.045 nmol malonaldehyde/h per 10(8) cells for 22 h. The motility of these spermatozoa declined with time in medium KTP, with 40% initial forward motility decreasing to zero in 4 h and initial 60% flagellar beating ceasing after 12 h. The percent inert spermatozoa showing no flagellar motion in KTP increased linearly with production of malonaldehyde; all flagellar activity stopped at 0.5 nmol malonaldehyde/10(8) cells. In the Tris phosphate medium containing 120 mM Na+ and 10 mM K+ (NTP), the percentage of sperm showing forward motility was close to 100% and this declined to 60% after 16 h aerobic incubation. Flagellar beating was not observed. In medium NTP, the rate of lipid peroxidation was 0.0056 nmol malonaldehyde/h per 10(8) cells, eightfold lower than that observed in KTP. The same linear correlation between malonaldehyde production and percent inert sperm was found as for KTP: 0.5 nmol malonaldehyde/10(8) cells also corresponded to cessation of flagellar motion. The dependence of motility maintenance on K+ concentration in Tris phosphate medium containing (Na+ + K+)=130 mM showed maximal maintenance at 10 mM K+, with a decline at 0 mM K+ and steep decline at K+ concentrations greater than 30 mM. This strong dependence of rabbit sperm peroxidation on ionic composition of the medium is suggested to involve perturbation of the equilibrium between O2 .- and its conjugate acid species being the agent of peroxidation.

Animals↗
PubMed1982

Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa.

Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.

Animals↗
PubMed1981

Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa.

Rabbit spermatozoa from the cauda epididymis produced 0.7-0.8nmol of H(2)O(2)/min per 10(8) cells at cell concentrations below 10(7) cells/ml with linear dependence on cell concentration. Above 2 x 10(7) cells/ml, the rate again became linear with cell concentration but decreased to 0.1-0.2nmol/min per 10(8) cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H(2)O(2) production rate on cell concentration; below 10(7) cells/ml the rate was 0.3-0.4nmol/min per 10(8) cells; above 2 x 10(7) cells/ml, the rate was 0.1-0.2nmol/min per 10(8) cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta305, 557-565] produced 0.1-0.2nmol/min per 10(8) cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 10(7) to 2.2 x 10(8) cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H(2)O(2) production to 0.3nmol/min per 10(8) cells. The decreased rate of H(2)O(2) production observed with intact sperm at high cell concentrations is attributed to reaction of H(2)O(2) with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H(2)O(2) generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H(2)O(2) production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.

Amphotericin B↗
PubMed1981

Inhibition of in vitro fertilization of mouse eggs: 3-quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa.

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.

Animals↗
PubMed1981

Oxidative metabolism of spermatozoa from inbred and random bred mice.

Epididymal spermatozoa from the random-bred CFW and from the inbred C57 BL/6 strains of mice were treated either hypotonically or with the antibiotic filipin in order to study the mitochondrial oxidative activities of the two strains in the absence of permeability barriers imposed by the plasma membrane. The percentage of motile spermatozoa from C57BL/6 mice was consistently higher than that of CFW mice, but sperm from the latter fertilized a higher percentage of eggs in vitro. In vivo, there was no apparent difference in fertilizing capacity in vivo: no significant difference between strains was observed. There is a strategy of oxidative metabolism in mouse spermatozoa which is common to the two genetic strains of the species tested, but which differs from that of rabbit and bull spermatozoa. The mitochondria of mouse spermatozoa oxidize L--3-glycerolphosphate but not glutamate in the presence of malate; both activities are present in bull spermatozoa but neither are present in rabbit spermatozoa. In common wit those of the mammalian species thus far studied, the mitochondria of mouse spermatozoa readily oxidize lactate and pyruvate in the presence of malate. They also oxidize acetyl CoA, acetyl carnitine, and long-chain acyl CoA esters directly, without the intermediacy of the carnitine esters. Mouse spermatozoa, therefore, have access to endogenous acyl CoA esters as a source of metabolic energy, which is consistent with their ability to maintain motility for 4-6 hours in the absence of added energy sources. Mouse spermatozoa are self-sufficient with regard to oxidative metabolism, which suggests that energy sources are not readily available to them in the mouse female reproductive tract.

Animals↗
PubMed1981

The role of calcium in maintaining motility in mouse spermatozoa.

Maintenance of mouse sperm motility requires exogenous Ca+2 in most, but not all, samples of epididymal spermatozoa. In these samples, the loss of motility with time is the same in Tris/NaCl buffer containing 1.7 mM Ca+2 (medium TNC) as it is in complete culture medium used for in vitro fertilization (medium CM) over the first 2 hours; spermatozoa in medium TNC lose motility at more rapid rate thereafter. The cation specificity for maintenance of motility is unusual in that both Sr+2 and Mg+2 substitute for Ca+2, with Mg+2 being the more effective. In the absence of Ca+2, these samples of mouse epididymal spermatozoa in Tris/NaCl buffer (medium TN) lose motility in about 30 minutes. If Ca+2 is added after incubation in TN for 15 minutes, motility is maintained as well as it is in medium TNC. If Ca+2 is added at 30 minutes, motility is partially restored. But if Ca+2 is added after 60 minutes, there is no restoration of motility. Spermatozoa suspended in medium TNC lose motility rapidly on addition of EGTA in excess of the Ca+2 present. Attempts to show uptake of Ca+2 by spectrophotometric assay, by effects of Ca+2 on oxidative metabolism, and by electron probe X-ray microanalysis were unsuccessful; motility is not maintained by entry of Ca+2 into the cells. Our results are consistent with a Ca+2 site on the plasma membrane and suggest that these sites function as oligomers of ordered subunits whose structure requires Ca+2. In the absence of Ca+2, the ability to form the oligomer is lost with time, possibly due to an irreversible conformational change.

Animals↗
PubMed1981

Carotid body O2 chemoreception and mitochondrial oxidative phosphorylation.

The effect on carotid chemoreceptor afferents of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation that does not affect energy conservation, was studied in 20 cats that were anesthetized, paralyzed, and artificially ventilated. Responses of single or a few chemoreceptor afferents to changes in arterial O2 tension (PaO2) at constant arterial CO2 tension were recorded. In addition, responses to nicotine, cyanide, and antimycin A or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP) were tested in normoxia. Oligomycin (50-500 microgram) was administered by close intra-arterial injection, and the same tests were repeated at timed intervals. Initially, oligomycin caused vigorous stimulation of carotid chemoreceptor activity. Subsequently, although the afferent fibers were still active and could be vigorously stimulated by nicotine, they no longer responded to changes in PaO2 or to doses of cyanide, antimycin A, or FCCP. These results separate stimulation of chemoreceptor afferents by hypoxia and metabolic inhibitors and uncouplers from that by nicotine and suggest that intact oxidative phosphorylation, required for maintenance of the intracellular high-energy phosphate levels, forms the basis of O2 chemoreception in the carotid body.

Animals↗
PubMed1980

Energy-linked quinacrine fluorescence changes in submitochondrial particles from skeletal muscle mitochondria. Evidence for intramembrane H+ transfer as a primary reaction of energy coupling.

Submitochondrial particles obtained from skeletal muscle are open membrane fragments which show energy coupling yet cannot sustain transmembrane ion gradients. An energy-linked decrease in fluorescence emission of the fluorescent probe quinacrine is observed with skeletal muscle particles which is enhanced by the anion SCN-. This is essentially the same reaction observed with beef heart submitochondrial particles, in which the reaction is known to be a monitor of intramembrane H+ transfer. The results with skeletal muscle particles show that this intramembrane H+ transfer occurs in the absence of any electrochemical potential across the membrane. Further, it occurs independently of the direction of H+ exchange with the suspendingly medium, since energization of skeletal muscle submitochondrial particles is known to give H+ efflux into the medium, while energization of beef heart particles gives H+ uptake from the medium. We conclude that a primary reaction of energy coupling is intramembrane H+ transfer linked to electron transport but shielded from the suspending medium. Movements of H+ between membrane and medium are secondary reactions derived from the primary one, possibly linked by a series of intramembrane conformational changes comprising a sequential membrane Bohr effect.

Animals↗
PubMed1980

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell.

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.

Adenosine Triphosphatases↗
PubMed1980

Mitochondrial carbonic anhydrase.

We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and bicarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16O18O. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCO3-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.

Animals↗
PubMed1979

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction.

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.

Acrosome↗