A systematic review of interventions to prevent work-related musculoskeletal disorders in ENT surgeons.

OBJECTIVE: Work-related musculoskeletal disorders in ENT surgeons are common and detrimental, yet few are aware of preventative measures. We evaluate the evidence for interventions to prevent work-related musculoskeletal disorders in ENT surgeons. METHOD: A systematic search of databases up to 8 June 2021 was performed using Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines and predetermined inclusion criteria. RESULTS: Seven prospective cohort studies and 2 case series were identified (51 participants). Interventions included novel equipment (n = 3), patient positioning (n = 2), clinician positioning (n = 3) and operative technique (n = 1). Five studies reported Rapid Upper Limb Assessment scores as outcome measures of strain. Strain decreased when adopting a favourable operating posture, using a supportive chair and keeping patients supine for clinic procedures. CONCLUSION: A small number of low-quality studies were identified. Modifiable risk factors exist, and ergonomic education may help prevent work-related musculoskeletal disorders. Further studies with longer term follow up are required.

An unusual presentation of propylthiouracil-induced anti-MPO and PR3 positive ANCA vasculitis with associated anti-GBM antibodies, IgA nephropathy and an IgG4 interstitial infiltrate: a case report.

BACKGROUND: A number of disease processes can culminate in rapidly progressive glomerulonephritis, including pauci-immune focal segmental necrotising glomerulonephritis, usually seen with positive serum antineutrophil cytoplasmic antibodies (ANCA). Propylthiouracil (PTU) has been associated with drug-induced ANCA-associated vasculitis (AAV), with antibodies against myeloperoxidase (MPO) and proteinase 3 (PR3) present individually and together having been recognised. 'Double-positive' vasculitis with ANCA and anti-glomerular basement membrane (GBM) antibodies has also been reported in association with PTU treatment. We present a case of PTU-induced anti-MPO and PR3 positive ANCA vasculitis with associated anti-GBM antibodies, IgA nephropathy and an IgG4 interstitial infiltrate. CASE PRESENTATION: A 51-year-old man presented 2 weeks after re-commencing propylthiouracil (PTU) treatment for Graves' disease, with a severe acute kidney injury and haemato-proteinuria. He demonstrated positive titres for autoantibodies to PR3 (76.9 IU/mL), MPO (28.8 IU/mL) and GBM (94 IU/mL). Renal biopsy demonstrated numerous glomerular crescents, widespread IgG4-positive lymphoplasmacytic infiltrate and mesangial positivity for IgA. PTU was stopped and he was treated with steroids, plasma exchange and cyclophosphamide with sustained improvement in his renal function. CONCLUSIONS: This case of drug-induced AAV presented a unique and intriguing collection of serological and histological features. We propose that the PTU-induced AAV resulted in epiphenomena of anti-GBM antibody production and an IgG4-cell-rich tubulointerstitial infiltrate. It is uncertain whether the mesangial IgA deposition preceded or resulted from the AAV.

Antiparasitic efficacy of a novel plant-based functional food using an Ascaris suum model in pigs.

Ascaris lumbricoides is the most prevalent soil-transmitted helminth (STH) infection of human beings worldwide. Chemotherapy with synthetic anthelmintics such as albendazole, mebendazole, and pyrantel pamoate is the current method of treatment; however, the emergence of anthelmintic resistance could substantially decrease the efficacy of such treatments and the sustainability of STH control programs. Additionally, benzimidazoles are not recommended for pregnant women or children under age one. A blinded, controlled study was conducted to evaluate the efficacy of two microencapsulated, plant-based essential oil blends, TTN1013 (α-pinene, linalyl acetate, p-cymene, and thymol octanoate) and TTN1014 (α-pinene, linalyl acetate, p-cymene, and thymol acetate) as functional foods against Ascaris suum infection in pigs, an important pathogen that closely resembles human infections with A. lumbricoides. Four groups of 16 female, 21-24 day old, Yorkshire-cross pigs were treated daily with 0.5 or 1.0mg/kg TTN1013, 1.0mg/kg TTN1014, or 1.0mg/kg equivalent of empty capsules, delivered inside a cream-filled sandwich cookie for 14 weeks. Three days after the initiation of daily treatments, pigs were inoculated daily with A. suum eggs for four weeks. Pigs were weighed weekly and fecal egg counts (FEC) were conducted weekly starting five weeks after initial inoculation with A. suum eggs. Fourteen weeks after first infection with eggs, pigs were necropsied and worms were recovered, counted and separated according to sex. TTN1013 administered daily at a dose of 1.0mg/kg yielded a statistically significant reduction in total worm counts (76.8%), female worm counts (75.5%), FEC (68.6%), and worm volume (62.9%) when compared to control group. Reduction of total and female worm numbers and FEC were not significant for TTN1014 or at the 0.5mg/kg dose of TTN1013. All treatments were well-tolerated by all pigs and did not cause any adverse reactions. All pigs remained clinically normal and showed no signs of reduced intestinal health for the duration of treatment. Based on these results, TTN1013 shows promise as a daily supplement to reduce infection burdens of soil transmitted helminths in both pigs and human beings.

Impact of integrated gastrointestinal nematode management training for U.S. goat and sheep producers.

The objective of this study was to determine the impact of integrated parasite management (IPM) training, including FAMACHA(©) eyelid color scoring, on the ability of U.S. sheep and goat producers to control gastrointestinal nematodes (GIN) on their farms. A survey was developed and provided to over 2000 producers trained from 2004 to 2008 in IPM with questions involving farm size (number of sheep/goats), location (U.S. state), impact of training on parasite control efforts and parasite problems on farm, and IPM practices used. Responses were divided into U.S. Census regions of the U.S. Descriptive statistics and logistic regression were used to describe results. Most of the 729 respondents were from the southern region of the U.S. (54.3%) and were small-scale producers (50 or less animals; 64.8%). Nearly all of the respondents (95.1%) agreed that IPM workshop attendance made a difference in their ability to control and monitor parasitism in their herd or flock and employed IPM practices to control GIN (96.3%). The most popular practices respondents used were rotational grazing (71.2%), genetic selection (choosing a parasite resistant breed and/or culling susceptible animals; 52.7%), grain supplementation on pasture to improve nutrition (44.0%), and increased height of plants being grazed (41.8%). Although reporting using a practice decreased (P<0.05) the likelihood of reporting fewer problems, for each 1-point increase in the number of practices which producers employed to control internal parasitism in their herd or flock, they were 16% more likely to report fewer GIN problems (P<0.05). Approximately 75% of respondents indicated an economic benefit of IPM on their farm (P<0.05), and those reporting savings of over $80 were more likely to report fewer problems (P<0.05) with parasites after the training while those reporting no economic benefit were less likely to report fewer problems with GIN (P<0.001). Overall, IPM training resulted in positive impacts for producers responding to the survey and should continue.

Magnetic resonance angiography of the circle of Willis in patients with symptomatic intracranial neuropathology: medicare LCD L25367(R4).

Patients with symptomatic intracranial neuropathology such as atherosclerotic occlusive disease or unruptured aneurysms face high risks for morbidity and mortality. Magnetic resonance angiography of the circle of Willis is an important tool used to detect and diagnose intracranial neuropathology; however, recent changes to the Medicare local coverage determinations for this procedure threaten to compromise the physician's ability to deliver this current standard of care. Physicians can assume an important role in advocating for this lifesaving procedure on behalf of this vulnerable patient population.

Urine drug testing in the treatment of chronic noncancer pain in a Kentucky private neuroscience practice: the potential effect of Medicare benefit changes in Kentucky.

BACKGROUND: Because the symptoms of drug misuse are nonspecific and difficult to detect, pain physicians have relied heavily on the results of urine drug tests to diagnose and treat chronic noncancer pain in patients who are prescribed controlled substances. However, changes in Medicare local carrier determinations for Medicare Part B providers in Connecticut, Indiana, Kentucky, and New York went into effect on July 1, 2009, whereby qualitative drug screening was no longer recognized as medically reasonable and necessary in the treatment of patients with chronic noncancer pain unless the patient presents with suspected drug overdose. STUDY DESIGN: A retrospective review of urine drug testing services. OBJECTIVE: To determine the extent of urine drug testing in patients with chronic noncancer pain in a large, Kentucky neuroscience practice offering pain management services combined with neurologic and neurosurgical services to better understand the potential effects of recent changes to Medicare benefits. METHODS: An audit of services provided during 2007 was conducted using computer software. OUTCOME MEASURES: Outcome measures included the number of practice services, number of urine drug tests by payor, and the number of noncompliant patients by payor who self-released from care. RESULTS: Urine drug tests represented approximately 18.2% of professional medical services rendered in 2007 to patients with a diagnosis of chronic noncancer pain. Of these, UDTs represented approximately 22.2% of services provided to Medicare patients and 24.6% of services provided to Medicaid patients. In 2007, 2,081 patients with noncompliant UDTs self released from the practice against medical advice. Of these, 23.1% were enrolled in Medicare and 47.5% were enrolled in Medicaid. Approximately 40% of patients were referred to the CARE Clinic on the basis of noncompliance as indicated by UDT and/or behavioral health issues. Of these, approximately 50% remained in treatment. Urine drug tests were also instrumental in revealing that 19.6% of patients showed signs of drug abuse or addiction. Of these patients, approximately 60% were government insured. LIMITATIONS: Not a prospective, double-blinded study. We approximated the proportion of patients potentially affected by drug abuse or addiction as the percentage of patients self releasing from medical care. CONCLUSION: In 2007, UDTs were used as an effective tool in adherence monitoring in a private neuroscience practice in Kentucky that offers pain management services combined with neurologic and neurosurgical services. UDTs were instrumental in referring 40% of patients for evaluation and treatment by behavioral health and addiction medicine specialists. UDTs were also instrumental in discovering signs of drug abuse or addiction in 19.6% of patients. Of these patients, approximately 60% were government insured. Should the objective and reliable sign offered by UDTs be eliminated from the physician's toolbox, the physician's ability to accurately diagnose and treat these patients could be impaired.

Importance of urine drug testing in the treatment of chronic noncancer pain: implications of recent medicare policy changes in kentucky.

BACKGROUND: Urine drug testing has become a widely used tool in American society for deterring illicit drug use. In the practice of medicine, urine drug testing is commonly used to help diagnose substance misuse, abuse, or addiction. OBJECTIVE: This narrative review provides an informed perspective on the importance of urine drug testing in the medical treatment of chronic noncancer pain. The history and current uses of urine drug tests in the United States are reviewed, the prevalence and nature of prescription drug misuse is described as is related to chronic noncancer pain, and implications and considerations for practitioners are presented related to the noncancer pain diagnosis and treatment. DISCUSSION: Practitioners are confronted with the ethical and legal dilemma of being called to adequately treat chronic pain in a culture with a high prevalence of prescription drug abuse. Yet the symptoms of drug abuse are nonspecific and therefore of limited value to the practitioner in determining patient compliance to drug treatment regimens. In contrast, urine drug testing has a reliable history, both in and out of medicine, as an independent sign of drug misuse. This sign can be used to aid in the diagnosis and treatment of drug misuse and underlying addictions to improve patient outcomes. CONCLUSION: Regular urine drug testing should be a part of acute and chronic pain management whether or not the patient has any signs or symptoms of drug misuse.

Shape stability of sonoluminescence bubbles: comparison of theory to experiments.

Single bubble sonoluminescence (SBSL) is the brief flash of light emitted from a single, stable, acoustically forced bubble. In experiments, the maximum pressure amplitude with which a bubble may be forced is limited by considerations of spherical stability. The traditional linear stability analysis predicts a threshold for SBSL at a much lower pressure amplitude than experimental observations. This work shows that if one constructs an accurate model of the radial dynamics, the traditional linear stability analysis predicts a boundary that is in excellent agreement with experimental data.

Regulation of oxygen sensing in peripheral arterial chemoreceptors.

The carotid bodies are a small pair of highly vascularized and well perfused organs located at each carotid artery bifurcation, strategically situated to sense oxygen in arterial blood as it leaves the heart. Carotid body glomus cells are identified as the primary oxygen sensors, which respond to changes in blood P(O(2)) within milliseconds. Acute hypoxia causes a rapid increase in carotid sinus nerve (CSN) activity, providing afferent signals to the respiratory center in the brainstem. Glomus cells secrete numerous neurotransmitters that modulate CSN firing rates. This review will discuss major hypotheses that have emerged regarding acute oxygen sensing by glomus cells. In contrast, chronic responses to hypoxia are much slower, involving cytosolic reactions that take place over several minutes and nuclear reactions which occur over several hours. Converging concepts from different areas of research in oxygen sensing cells and tissues (including the carotid body) have been combined to describe molecular and biochemical changes that take place in the carotid body with chronic hypoxia. These include oxygen dependent proteolytic processes in the cytosol and gene transcription in the nucleus. In addition, cellular and nuclear responses to chronic hypoxia will be discussed.

Dialysis addition of trehalose/glycerol cryoprotectant allows recovery of cryopreserved mouse spermatozoa with satisfactory fertilizing ability as assessed by yield of live young.

Mouse sperm cryopreservation provides a means for storing the genetic information in genetically modified mice (mutants, transgenics, and "knockouts") in a cost- and space-effective manner. Sperm from this species are highly sensitive to cryodamage, which has impeded their cryopreservation in the past. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5% trehalose (0.22 M), which was added to a concentrated suspension of sperm from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osmotic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws and stored in liquid N2. Eggs were obtained from B6SJLF1/J superovulated females. For in vitro fertilization (IVF), 15-25 microL of sperm suspension post-thaw from one straw was added directly to each of three 1.5 mL drops of fertilization medium containing 30 eggs each, for 3 replicates per experiment. The fertilized eggs were scored for blastocyst formation, after which 12 blastocysts from each drop were implanted into pseudopregnant CD-1 females. The number of live pups were then scored at birth. Ten experiments yielded 21.7 +/- 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 +/- 0.4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of live pups was 44 per 100 eggs inseminated (44%). This yield should be satisfactory for maintaining a mouse strain through sperm cryostorage, with restart of the strain through IVF and embryo transfer. The method should also provide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm.

Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.

The metabolic hypothesis revisited.

High levels of CO are used to mimic the stimulatory response of the CSN initiated by hypoxia. Using light of different wavelengths we show that the stimulatory effects of high CO can be pinpointed to the cytochrome c oxidase in the mitochondrial respiratory chain. This supports the metabolic theory of oxygen sensing in the mitochondria.

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3.

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.

Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations.

In order to calculate the actual, rather than the relative, intracellular Ca(2+) concentration (Ca(2+))(i) in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe. Ca(2+) complex and free probe, the value of the dissociation constant for the probe. Ca(2+) complex, K(D), is required. Interaction of the probe with cellular components may change the intracellular value of K(D) from that determined in buffered solution. We had previously shown that fluo-3, whose Ca(2+) complex is highly fluorescent whereas free fluo-3 is not, could be used to monitor changes of (Ca(2+))(i) in mouse sperm. In this report, we describe a method for determining K(D) for the fluo-3. Ca(2+) complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca(2+). The method involved treating the sperm with ionomycin to provide a plasma membrane Ca(2+) carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca(2+))(i) equilibrates with medium Ca(2+) concentration (Ca(2+))(e), then titrating (Ca(2+))(e) with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca(2+), an approximation algorithm was used to calculate (Ca(2+))(e), based on the known K(D) for the EGTA. Ca(2+) complex. The fluorescence of the intracellular fluo-3. Ca(2+) complex, F, decreased with increasing additions of EGTA; (Ca(2+))(i) = (Ca(2+))(e) was plotted as a linear function of F/[F(max) - F]; the slope gives K(D). At 37 degrees C, intracellular K(D) was calculated to be 0.636 +/- 0.018 microM (+/-SEM, n = 8). At 37 degrees C and 20 degrees C, K(D) values in MJB were calculated to be 0.502 +/- 0.022 and 0.578 +/- 0.029 (+/-SEM, n =8 and n = 6), respectively. The higher intracellular K(D) value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca(2+))(i), monitored with fluo-3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 microM for K(D) of the intracellular fluo-3. Ca(2+) complex.

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12.

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.

Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation.

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; arc sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant with in vitro fertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in an in vitro fertilization system.

Human sperm glutathione reductase activity in situ reveals limitation in the glutathione antioxidant defense system due to supply of NADPH.

In order to characterize further the antilipoperoxidative enzyme system of human sperm, that part of the system designed to provide reducing equivalents for the reduction of highly reactive and potentially damaging lipid hydroperoxides to relatively inert hydroxylipids was examined. The substrate that provides the reducing equivalents directly to glutathione peroxidase (GPX) is reduced glutathione (GSH), which is in turn oxidized to glutathione disulfide (GSSG). The reducing equivalents needed for regeneration of GSH through the action of glutathione reductase (GRD) are provided by NADPH, produced by the action of glucose-6-phosphate dehydrogenase (G6P-DH) on substrates glucose-6-phosphate and NADP+. The kinetic properties of the enzymes GRD and G6P-DH were determined by standard enzyme activity assay at 24 and 37 degrees C. At 37 degrees C, the Vmax for GRD was found to be 36 nmol/min x 10(8) cells, with Km values for GSSG and NAPH of 150 microM and 16 microM, respectively; the Vmax for G6P-DH was 3.3 nmol/min x 10(8) cells with Km for NADP+ of 8 microM. This suggested that G6P-DH activity was limiting in this reductive pathway. The activity of GRD in situ in intact cells was estimated using the thiol-reactive fluorogenic probe ThioGlo-1, which is cell permeant and reacts rapidly with GSH to give a highly fluorescent adduct. Mixing a suspension of human sperm with the fluorogenic reagent at 37 degrees C gave an initial rapid increase in fluorescence, followed by a slower one. The rapid phase is due to reaction with intracellular GSH already present; the slow phase is due to reaction with GSH generated by the GRD-catalyzed reduction of GSSG. Both rates showed first-order kinetics. Calculation of the maximal rate as NADPH oxidation, attributable to in situ GRD activity, gave the value of 1.0 nmol/min x 10(8) cells, less than the maximum for NADPH production by the dehydrogenase. These results support the suggestion that NADPH production limits the capacity of the pathway leading to hydroperoxide reduction in human sperm. We propose that the antilipoperoxidative defense system of human sperm has just sufficient capacity to allow these cells to fulfill their function but is limited to allow their timely disposal from the female reproductive tract.

Water permeability, Lp, of the mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton.

Two parameters fundamental to cell cryobiology are the water permeability (hydraulic conductivity), Lp, and its activation energy, EA. The Lp can be calculated from two experimental determinations: the critical osmolality, Osmcrit, at which 50% of the cells lyse, and the time, tcrit, to 50% lysis in a highly hyposmotic medium, based on the assumption that the cells swell to lysis with minimal resistance to swelling. We have reported [Cryobiology 32, 220-238 (1995)] that mouse sperm in hyposmotic medium show minimal swelling and so fail to meet this assumption. The concept that resistance to swelling was due to anchoring of the plasma membrane through cytoskeletal interaction was examined by treating mouse sperm with 5 microM cytochalasin D to depolymerize the cytoskeletal filamentous actin (f-actin), whose presence was established by staining with fluorescently labeled phalloidin. Diminution of fluorescence due to loss of f-actin induced by cytochalasin D was shown by flow cytometry. Mouse sperm treated with cytochalasin D showed tail curling in hyposmotic medium, similar to that observed with bovine and human sperm, indicating that the standard swelling model was applicable to these cells. Two sets of Lp values were calculated from tcrit: one using individual means of Osmcrit and one using the mean of means of Osmcrit between 37 and 4 degrees C, as these individual means were not significantly different. Values (micron.min-1.atm-1), respectively, were 9.95, 7.15 (37 degrees C); 1.51, 0.91 (22 degrees C); 0.54, 0.78 (12 degrees C); 0.47, 0.50 (4 degrees C); 0.33 (0 degree C); and 0.36 (-3 degrees C). Arrhenius plots gave EA = 13.7 and 11.7 kcal/mol, respectively. Values of t1/2 were calculated from the first-order rate constants characterizing the kinetics of cell lysis at the higher four temperatures; Lp values calculated from these, and the two sets of Osmcrit values described were 5.70, 4.09 (37 degrees C); 1.18, 0.71 (22 degrees C); 0.62, 0.90 (12 degrees C); and 0.34, 0.37 (4 degrees C). Arrhenius plots gave EA = 14.2 and 11.0 kcal/mol, respectively. We propose that these EA values are characteristic of the plasma membrane relatively unperturbed by cytoskeletal interactions. In untreated sperm, decrease of Lp with decreasing temperature and presence of cryoprotectant and the cytoskeletal interactions all act to hamper the sperm cells' ability to respond to osmotic stress encountered during freezing and thawing, such that these cells are especially sensitive to cryodamage.