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P F Watson

Publications and source records attributed to P F Watson.

At least 109 records · Page 6Linked to original sources
PubMed1992

Predictive water loss curves for ram spermatozoa during cryopreservation: comparison with experimental observations.

Calculated curves predicting intracellular water loss during cryopreservation at different cooling rates were calculated from published equations. To compute these curves, basic cell parameters specific to ram spermatozoa were measured, i.e., the total surface area (139 microns2), the hydraulic conductivity (0.222 micron3/micron2.atm-1.min-1), and its temperature dependence (0.045/degree C). Cell surface area was derived from measurements of physical dimensions. Hydraulic conductivity was calculated from measurements of the critical medium hypotonicity on exposure of sperm to various hypotonic solutions and the time taken for membrane rupture in sperm exposed to distilled water (spermolysis time). The temperature dependence of the water permeability was derived from measurements of spermolysis time at various temperatures above zero. Several discrepancies were noted between the resulting calculated curves and experimental observations made on the effects of cooling rate on sperm cell survival. These could be due to errors in the estimates of the basic parameters, or to false assumptions in the basic equations used to compute the curves, e.g., the validity of the Boyle-van't Hoff relationship. Nevertheless, this study represents a first attempt to predict intracellular water loss from ram sperm during cooling and may provide a novel approach for the interpretation of the many empirical studies carried out to investigate optimal conditions for the cryopreservation of sperm.

Animals↗
PubMed1992

Glycerol permeability of human spermatozoa and its activation energy.

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.

Cell Death↗
PubMed1990

A new light microscopy classification of spermatogenesis in the bull and the boar applicable to tissues processed for electron microscopy.

A new light microscopy classification of spermatogenesis in the bull and the boar was formulated to be used in conjunction with electron microscopy. It uses information available from all cell types and allows for very small portions of testicular epithelium at any orientation to be evaluated. The classification devised, being simple in outline, allows for the natural overlap of cell types. Furthermore, it emphasises the direct comparability of the morphological events occurring during spermatogenesis in the bull and the boar.

Animals↗
PubMed1989

Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals.

Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis. When treated with the sterol 14 alpha-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14 alpha-methyl-erogosta-8,24(28)-dien-3 beta, 6 alpha-diol (14-methyl-3,6 diol). The stringent desaturase mutant, A2, accumulated 14 alpha-methyl-8,24(28)-dien-3 beta-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole. Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation. We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition. We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.

Antifungal Agents↗
PubMed1989

Genetic and physiological analysis of azole sensitivity in Saccharomyces cerevisiae.

Ketoconazole and fluconazole are azole antifungal agents which inhibit cytochrome P-450 mediated sterol C14 demethylation during ergosterol biosynthesis. We report on the activity of these antifungals on a variety of Saccharomyces cerevisiae strains grown under differing conditions known to affect cyt P-450 levels. Only slight increases in resistance to azoles were observed under conditions which induce the yeast cyt P-450 from undetectable levels. Strain variation was observed, with some strains exhibiting a fungicidal, and others a fungistatic response. Two cyt P-450 deficient mutants examined exhibited resistance to treatment with fluconazole and ketoconazole. This was attributed, at least in part, to an additional defect in sterol delta 5,6 desaturation and possibly to reduced cellular levels of azole drug.

Cytochrome P-450 Enzyme System↗
PubMed1988

Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa.

Ram spermatozoa were subjected to a slow rate of freezing (1 degree C/min) in various glycerol-NaCl-water solutions of known composition such that the molal concentration of NaCl (ms) and the unfrozen fraction of water (U) could be calculated at subzero temperatures from the relevant phase diagram. Sperm motility was reduced as ms increased and U correspondingly decreased with temperature. However, by freezing spermatozoa in solutions of differing initial tonicities, but with a constant weight ratio of glycerol: salt, to various subzero temperatures, the effects of ms could be separated from those of U. Motility was found to decrease dramatically at values of U less than 0.07 regardless of ms but, at higher values of U, maximum motility was dependent on the final salt concentration in that fraction, being reduced as the osmolality increased. Sperm cell concentration had no apparent effect on the influence of ms or U on viability in the range studied (3-12 x 10(8) spermatozoa/ml). In order to account for these observations, the effects of osmotic stress on spermatozoa were investigated. When subjected to sudden changes in osmolality of the suspending medium by increasing NaCl or sucrose concentration at room temperature, spermatozoa showed a decreased motility with increasing osmolality. Since no improvement in motility was found on returning the cells to isosmolar conditions cell damage appeared to be irreversible. Furthermore, when placed in solutions of increasing hypotonicity the number of swollen spermatozoa with looped tails increased with increasing hypotonicity. Since the drop in motility seen at low values of U corresponded to those spermatozoa exposed to a hypotonic starting solution, it is suggested that a hypotonic stress followed by a hypertonic stress during freezing and thawing may account for the profound loss of motility in these samples, while a hypertonic stress may account for the strong effect of ms seen at higher values of U.

Animals↗
PubMed1988

Isolation and analysis of ketoconazole resistant mutants of Saccharomyces cerevisiae.

Nine mutants of Saccharomyces cerevisiae which are resistant to ketoconazole, have been isolated and characterized. In each case the mutation is nuclear in origin and allelic to a previously described mutation, erg3, which gives rise to a block in the delta 5-6 desaturation step of ergosterol biosynthesis. The significance of this second site mutation to the point of inhibitory action of ketoconazole, that is the P-450-mediated C-14 demethylation of lanosterol, is discussed.

Cytochrome P-450 Enzyme System↗
PubMed1987

Cold shock injury in animal cells.

Cold shock injury (damage to cell structure and function arising from a sudden reduction in temperature) was for many years considered a phenomenon peculiar to certain cell-types. Only in recent years has it become apparent that widely different cell-types manifest cold shock injury. Thus, cold shock appears to be a more general phenomenon, differences between cell-types being quantitative (in the rate of cooling and temperature range at which injury is sustained) rather than qualitative. Loss of particular cell functions depends on cell-type, but reflects the underlying structural and biochemical damage which has been inflicted by rapid cooling. In particular, membranes lose their selective permeability with the result that many cellular components are released including lipids, proteins and ions. Additionally, sodium and calcium gain access to the interior of the cell. Consequent upon this initial disruption, metabolic activities are diminished and further secondary changes ensue. The possible mechanisms of cold shock injury include membrane thermo-tropism and protein denaturation. Susceptibility to cold shock is influenced by membrane composition, and much experimental evidence points to particular involvement of membrane lipids. One hypothesis implicates lipid phase changes in a cooling rate dependent loss of membrane integrity. Other recent hypotheses invoke biophysical concepts and cytoarchitectural features as considerations in a better understanding of cold shock.

Animals↗
PubMed1986

Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa.

Ram spermatozoa treated with the ionophore A23187, to induce the acrosome reaction, were fixed in pyroantimonate-osmium in order to demonstrate calcium at an ultrastructural level. Prior to vesiculation, granules of precipitate occurred at points of contact between plasma and outer acrosomal membranes, suggesting a discrete role for calcium during membrane fusion. The earliest stages of vesiculation always occurred in the region just anterior to the equatorial segment and it was this same site where a marked concentration of precipitate, indicating calcium binding sites, could be demonstrated.

Acrosome↗
PubMed1986

Seminal plasma composition in budgerigars (Melopsittacus undulatus).

Seminal plasma composition was studied in budgerigars. Semen was obtained from adult male budgerigars by applying gentle pressure to both sides of the cloaca. Pooled samples were centrifuged at 15,000 g for 2 min, and the seminal plasma separated for biochemical analysis. Osmolality, Na+, K+, Cl-, pH, glucose and fructose values were determined. The biochemical composition of budgerigar seminal plasma obtained in this study was: Osmolality 329.9 +/- 14.5 mOs/kg; Na+ 158.6 +/- 8.4 mEq/l; K+ 16.39 +/- 6.24 mEq/l; Cl- 109.2 +/- 7.4 mEq/l; pH 8.20 +/- 0.18 glucose 4.25 +/- 0.96 mmol/l; fructose 0.59 +/- 0.29 mmol/l. The results are discussed in relation to the values reported for the domestic fowl. This forms part of a reproductive biology study of non-domesticated avian species.

Animals↗
PubMed1986

Calcium transport in diluted or cooled ram semen.

Ram semen was subjected to various dilution rates and temperatures of dilution, and was also subjected to slow cooling and rewarming. Calcium ion movements across sperm membranes were measured using the radioisotope 45Ca2+. It was shown that even 2- to 4-fold dilution caused an increase in intracellular calcium content. An increase in intracellular calcium also occurred in proportion to a decreased temperature of dilution. After an initial increase in intracellular calcium content, spermatozoa appeared able to restore a low intracellular calcium level over a period of 2 h at 22 degrees C or above. This ability was lost at 16 degrees C or below. However, if undiluted semen was slowly cooled (0.125 degrees C/min) even to 5 degrees C and rewarmed to 22 degrees C before dilution, the spermatozoa regained this ability. In contrast, spermatozoa rapidly cooled to 5 degrees C and rewarmed to 22 degrees C before dilution were not able to restore the low intracellular calcium level.

Animals↗
PubMed1986

Occurrence, distribution and origin of peptide-containing nerves of guinea-pig and rat male genitalia and the effects of denervation on sperm characteristics.

A systematic immunohistochemical and radio-immunological survey of the occurrence, distribution and origin of the peptidergic nerve supply in guinea-pig and rat male genitalia is presented. Neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), substance P and CGRP were detected in the genital organs of both species. The densities and distribution patterns of the peptidergic nerves were compared with those of the adrenergic nerves, as revealed by antibodies raised against dopamine-beta-hydroxylase (D beta H) and tyrosine hydroxylase (TH), and the general neuronal component, as revealed by antibodies raised against neurofilament proteins (NF). Bilateral transection of the hypogastric nerves, in the guinea-pig, resulted in a decrease of substance P-containing nerves in the vas deferens and of NPY-, PHI- and VIP-containing nerves in the seminal vesicle. Unilateral disconnection of the pelvic nerves caused a decrease of VIP, PHI, substance P and CGRP nerve supply in the ipsilateral vas deferens and cauda epididymidis in the guinea-pig. A marked reduction of noradrenergic and NPY-containing nerves was observed in the vas deferens and sexual accessory glands of rats, chemically sympathectomised by chronic injection of low doses of guanethidine. Conversely, increase of substance P and CGRP immunoreactivities were observed, particularly in the vas deferens. After guanethidine, the cauda epididymidis and vas deferens were distended with spermatozoa, suggesting paralysis of the ducts. Spermatozoa had a decreased percentage of attached cytoplasmic droplets, indicating prolonged retention in the ducts.

Animals↗
PubMed1985

The effect of buffer osmolality on the survival of cat (Felis catus ) spermatozoa at 5 degrees C.

Cat semen was diluted at 37 degrees C in Tes-Tris buffer (TesT), pH 7.5, at osmolalities ranging from 195 to 390 m0sm/kg, cooled to 5 degrees C over 90 minutes and stored for 24 hours at that temperature. Motility and percentage of spermatozoa staining with a supravital stain were estimated before cooling, after cooling and after storage for 24 hours. The osmolality of undiluted pooled ejaculates from five animals was measured, and also that of different diluents (citrate with phosphate buffer, lactose and TesT-egg yolk) used for cat semen. The osmolality measurements of cat semen suggested an osmolality of less than 320 m0sm/kg at ejaculation, increasing with time after ejaculation. Varying the egg yolk concentrations (2% to 20%) did not affect the osmolality of TesT diluent. Diluent osmolalities of less than 292 m0sm/kg were found to reduce sperm motility significantly (P <0.001 ) although there was no significant increase in the percentage of cells staining with a supravital stain, while those greater than 325 m0sm/kg increased the variation of response among animals. Cooling and storage significantly reduced motility (P <0.01 to P <0.001 ) and increased the number of stained cells (P <0.001 ). There were significant differences between ejaculates (P <0.01 ) and significant interactions between osmolality and cooling/storage (P <0.05 to P <0.001 ). The best overall results were seen with a TesT diluent of 292 to 325 m0sm/kg which supported good motility for at least 24 hours.

Journal Article↗
PubMed1985

Observations on variability in LH release and fertility during oestrus in the domestic cat (Felis catus).

Hormonal changes, behaviour, ovulation and fertility were examined in response to coitus at two different times during oestrus in the female domestic cat housed in conditions of natural light (N = 13). On Day 2 or Day 4/5 of oestrus females were allowed 1 copulation in 15 min (single matings) or 2-3 copulations in 30 min (multiple matings). Plasma LH, oestradiol-17 beta and progesterone concentrations during the 24-h period after coitus were measured by radioimmunoassay; ovulation was assumed to have occurred if progesterone values were elevated 7-30 days after coitus. With the exception of 2 out of 3 animals receiving single matings on Day 2 of oestrus, all animals showed subsequent elevated progesterone values. Females receiving multiple matings had significantly greater releases of LH as measured by the area under the curve than those receiving single matings. There was significantly greater variability in the LH response of queens on Day 2 of oestrus compared to those on Day 4/5 for peak values and area under the curve; the only failure in release of LH was in queens on Day 2. Oestradiol levels did not differ significantly between Day 2 and Day 4/5 of oestrus. Progesterone values remained less than 1 ng/ml for 24 h after coitus. Both LH peak values and area under the curve were significantly greater for animals that became pregnant. There were also significant differences in coital behaviour between queens on Day 2 and those on Day 4/5 of oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique.

Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate-osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits. In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock. Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.

Animals↗
PubMed1983

Influence of butylated hydroxytoluene (BHT) on the viability of ram spermatozoa undergoing cold shock.

Butylated hydroxytoluene (BHT) significantly reduced the degree of acrosome damage which occurred to ram spermatozoa during cold shock. A higher percentage of spermatozoa were motile after cold shock in the presence of BHT than in its absence, but it had little effect on the quality of motility or the percentage of cells which stained with eosin. A concentration of 2-4 mM-BHT provided the maximum response. No advantage was gained by using the solvent, dimethyl sulphoxide, as a vehicle to introduce BHT to the cells. An osmotic stress test after cold shock also failed to demonstrate any advantage of BHT. It is concluded that BHT provides little protection to the functional capacity of ram spermatozoa undergoing cold stress and is unlikely to be of benefit for the preservation of ram semen.

Acrosome↗