Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.     

Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors

Effects of osmolality stresses on the sperm of normospermic (>60% normal sperm/ejaculate) versus teratospermic (<40% normal sperm) domestic cats and the normospermic leopard cat and the teratospermic clouded leopard were studied. Spermatozoa were exposed to various anisotonic solutions in a single step or returned to near isotonic conditions in a single step after exposure to anisotonic solutions. The percentage of sperm motility was measured subjectively, and dual fluorescent stains were used to assess membrane integrity by flow cytometry. The percentage of sperm motility declined (P < 0.05) in domestic cat sperm exposed to osmolalities <200 and >450 mOsm. Spermatozoa from all felines underwent marked (P < 0.05) membrane disruption following a hypotonic stress, but sperm from teratospermic donors experienced greater (P < 0.05) membrane disruption in response to decreased osmolality. While feline spermatozoa appeared to be highly resistant to hypertonic (600, 1200, and 2400 mOsm) conditions, with >85% of the cells maintaining intact membranes, severe membrane disruption occurred when cells were returned to isotonicity in a single step. There was no difference (P > 0.05) between a 1- and 5-min exposure to various anisotonic solutions. Similarly, sperm from normospermic and teratospermic domestic cats responded identically after exposure to ionic or nonionic solute. Results demonstrate that: (1) spermatozoa from teratospermic males are more vulnerable to a hypotonic stress than sperm from normospermic counterparts; (2) in response to small deviations in osmolality, feline sperm experience a more rapid decline in motility than membrane integrity; and (3) an abrupt return to isotonicity after a hypertonic stress causes extensive sperm membrane damage regardless of ejaculate quality.     

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.     

Adaptation by Corneal Epithelial Cells to Chronic Hypertonic Stress Depends on Upregulation of Na:K:2Cl Cotransporter Gene and Protein Expression and Ion Transport Activity

We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 μm bumetanide, proliferation measured as ³H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive ⁸⁶Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC. Received: 7 March 2000/Revised: 18 May 2000     

ClC-3 is a main component of background chloride channels activated under isotonic conditions by autocrine ATP in nasopharyngeal carcinoma cells

In this study, the activation mechanisms of the background chloride current and the role of the current in maintaining of basal cell volume were investigated in human nasopharyngeal carcinoma CNE-2Z cells. Under isotonic conditions, a background chloride current was recorded by the patch clamp technique. The current presented the properties similar to those of the volume-activated chloride current in the same cell line and was inhibited by chloride channel blockers or by cell shrinkage induced by hypertonic challenges. Extracellular applications of reactive blue 2, a purinergic receptor antagonist, suppressed the background chloride current in a concentration-dependent manner under isotonic conditions. Depletion of extracellular ATP with apyrase or inhibition of ATP release from cells by gadolinium chloride decreased the background current. Extracellular applications of micromolar concentrations of ATP activated a chloride current which was inhibited by chloride channel blockers and hypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride. Transfection of CNE-2Z cells with ClC-3 siRNA knocked down expression of ClC-3 proteins, attenuated the background chloride current and prevented activation of the ATP-induced current. Furthermore, knockdown of ClC-3 expression or exposures of cells to ATP (10 mM), the chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, or reactive blue 2 increased cell volume under isotonic conditions. The results suggest that ClC-3 protein may be a main component of background chloride channels which can be activated under isotonic conditions by autocrine/paracrine ATP through purinergic receptor pathways; the background current is involved in maintenance of basal cell volume.     

Cell volume regulation in liver

The maintenance of liver cell volume in isotonic extracellular fluid requires the continuous supply of energy: sodium is extruded in exchange for potassium by the sodium/potassium ATPase, conductive potassium efflux creates a cell-negative membrane potential, which expelles chloride through conductive pathways. Thus, the various organic substances accumulated within the cell are osmotically counterbalanced in large part by the large difference of chloride concentration across the cell membrane. Impairment of energy supply leads to dissipation of ion gradients, depolarization and cell swelling. However, even in the presence of ouabain the liver cell can extrude ions by furosemide-sensitive transport in intracellular vesicles and subsequent exocytosis. In isotonic extracellular fluid cell swelling may follow an increase in extracellular potassium concentration, which impairs potassium efflux and depolarizes the cell membrane leading to chloride accumulation. Replacement of extracellular chloride with impermeable anions leads to cell shrinkage. During excessive sodium-coupled entry of amino acids and subsequent stimulation of sodium/potassium-ATPase by increase in intracellular sodium activity, an increase in cell volume is blunted by activation of potassium channels, which maintain cell membrane potential and allow for loss of cellular potassium. Cell swelling induced by exposure of liver cells to hypotonic extracellular fluid is followed by regulatory volume decrease (RVD), cell shrinkage induced by reexposure to isotonic perfusate is followed by regulatory volume increase (RVI). Available evidence suggests that RVD is accomplished by activation of potassium channels, hyperpolarization and subsequent extrusion of chloride along with potassium, and that RVI depends on the activation of sodium hydrogen ion exchange with subsequent activation of sodium/potassium-ATPase leading to the respective accumulation of potassium and bicarbonate. In addition, exposure of liver to anisotonic perfusates alters glycogen degradation, glycolysis and probably urea formation, which are enhanced by exposure to hypertonic perfusates and depressed by hypotonic perfusates.     

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.This work was supported in part by funding from the USDA-SBIR program, 4Cs Breeding Technologies, and the Louisiana Sea Grant College Program.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender

In the past years a series of functional assays has been developed to determine the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. Cell volume regulation is an important physiological function crucial for the success of cryopreservation. In this study, the effects induced by freezing-thawing were judged by evaluating the functional characteristics of frozen-thawed semen samples submitted to secondary stress such as osmotic challenge or incubation under capacitating conditions, following cryopreservation. Prior to freezing, dog semen samples were diluted in the presence or absence of Equex STM Paste, which contains sodium dodecyl sulphate (SDS) as the active ingredient. Cell volume regulation and capacitation and calcium ionophore-induced membrane dynamics were assessed in freshly diluted and frozen-thawed semen samples by electronic volume measurement and flow cytometry. Cryopreservation led to a disturbance of the volume regulatory function and to a rapid decrease in the proportion of acrosome-reacted live spermaotozoa. Extender containing Equex STM Paste had a protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome-reacted cells. The evaluation of the functional state of sperm submitted to secondary stress after freezing-thawing leads to a more subtle characterization of sperm function and helps improve the cryoprotective efficiency of the extender.     

Role of potassium channels, the sodium-potassium pump and the cytoskeleton in the control of dog sperm volume

Response to osmotic shock is an important aspect of mammalian sperm physiology. In this study we recorded volume changes of dog spermatozoa at 39, 33, and 25 degrees C under isotonic conditions and following hypotonic shock. Cell volume measurements were performed electronically in saline solutions of 300 and 150 mOsmol kg(-1), and Percoll-washed preparations were compared with unwashed samples. The involvement of potassium channels in volume control was tested by treatment with quinine, while the involvement of the plasma membrane Na(+)-K+ pump was tested by treatment with ouabain. The role of the cytoskeleton was investigated by treatment with colchicine and cytochalasin D. The number of cell populations observed varied with temperature and tonicity. In both types of sperm preparations, between two and three populations were present under isotonic conditions at 25 degrees C whereas at 39 and 33 degrees C only one population was detected. Hypotonic stress at the higher temperatures caused the single population to swell, whereas at 25 degrees C it resulted in a population of cells whose modal volume was similar to that of the middle isotonic sub-population. Both quinine and the cytoskeletal inhibitors markedly increased swelling both under hypotonic conditions at 39 degrees C and under isotonic conditions at 25 degrees C. However, little or no effect of ouabain was observed. We conclude that in dog spermatozoa swelling in response to hypotonic conditions is minimised through the activity of potassium channels and the presence of an intact cytoskeletal network. Under isotonic conditions at 25 degrees C, a considerable proportion of the sperm population is already swollen; this swelling varies between individual males and appears to be due to lowered cytoskeletal and potassium channel activity.     

Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg^-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg^-1, and at osmolality 300 mOsmol kg ^-1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg^-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.     

Effects of castration on thiol status in rat spermatozoa and epididymal fluid

Mammalian spermatozoa gain their fertilizing ability as they mature in the epididymis, a process which is accompanied by oxidation of sperm protein thiols. Since sperm maturation is dependent upon normal androgenic support to the epididymis, the present work was designed to study the effects of castration on thiol status. Spermatozoa and epididymal fluid were isolated from the epididymides of male rats 5 days after castration or after 11 daily injections of the antiandrogen, cyproterone acetate. Spermatozoa and epididymal fluid were labeled with the fluorescent thiol labeling agent monobromobimane. Intact spermatozoa were evaluated by fluorescence microscopy, protein thiols were analyzed by electrophoresis, and fertilizing ability was examined after insemination of sperm suspension into the uterine horns of immature superovulated female rats. We found that both treatments resulted in an increase in cauda sperm thiols as shown by increased fluorescence in the intact spermatozoa. Protamines and nonbasic proteins were found to have increased levels of reactive thiols. The protein profiles of epididymal fluid from castrated rats were different from those of the controls, and the fluorescence patterns corresponded to the protein profiles. Our results indicate that testosterone withdrawal leads to inhibition of sperm thiol oxidation. Mol. Reprod. Dev. 47:295–301, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.     

Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.     

Aquaporin isoforms involved in physiological volume regulation of murine spermatozoa

Murine epididymal spermatozoa were dispersed in a medium of native osmolality and then transferred to a hypo-osmotic medium to mimic the physiological osmotic challenge, as encountered upon ejaculation into the female tract. The addition of quinine to block sperm K(+)-channels for volume regulation resulted in a size increase of viable cells. Preincubation in 0.1 mM HgCl(2), a standard aquaporin inhibitor, prevented such cell swelling. Addition of the K(+)-ionophore valinomycin to quinine-swollen sperm reversed the swelling, but not after pretreatment of the swollen sperm by HgCl(2). Aqp7, Aqp8, and Aqp9 mRNAs were identified in spermatozoa by RT-PCR, and the entire open reading frames were sequenced and compared with the GenBank database. Western blotting demonstrated specific protein signals for sperm AQP7 and AQP8 expression but probably not AQP9. The role of Hg(2+)-insensitive AQP7, if any, in sperm volume regulation was studied in transgenic mice. Spermatozoa from Aqp7(-/-) mice were the same size as wild-type sperm in basal conditions. Quinine-swollen volume, swelling reversal by valinomycin, and inhibition by Hg(2+) were also similar, indicating efficient water transport in the absence of AQP7. However, both water influx and efflux occurred faster in Aqp7(-/-) sperm than wild-type. This faster water movement in the knockout mouse spermatozoa was explainable by an upregulation of Aqp8 expression as revealed by quantitative PCR. Therefore, the Hg(2+)-sensitive AQP8, which was localized in elongated spermatids and spermatozoa, is a likely candidate for a water channel responsible for physiological sperm volume regulation crucial to in vivo fertilization.     

Characterization of zebra mussel (Dreissena polymorpha) sperm motility: duration of movement, effects of cations, pH and gossypol

We have examined effects of time after activation, pH, sodium and potassium, and gossypol concentrations on sperm motility of zebra mussel (Dreissena polymorpha). Zebra mussel spermatozoa appeared to have remarkable viability in the fresh water in comparison with freshwater fish sperm. Duration of sperm motility in fresh water is possibly one of the longest among freshwater animals, since it was not significantly changed 3 h after incubation at room temperature (20 °C) or 24 h of incubation at ±0 °C. High osmotic pressure suppresses sperm motility and effects of sodium and potassium are similar. Spermatozoa were inactive at acid pH and became gradually motile when exposed to pH 6.0–9.0. Gossypol appeared to be a very potent spermicidal agent and inhibited motility. This compound also inhibited fertilization. We observed some differences in gossypol effects on spermatozoa between North American and European zebra mussels. These data on zebra mussel sperm biology may be useful for better handling of gametes under laboratory conditions.     

Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders

The ability of a range of extenders to cryopreserve ram spermatozoa was tested. The extenders were modified by the inclusion of citrate, Tris buffer, trehalose, and EDTA. Ejaculates from three Pampinta rams were evaluated and pooled at 30 degrees C. The semen was diluted to contain 1 x 10(9) cells/mL, cooled to 5 degrees C, loaded into 0.25-mL straws, frozen and stored in liquid nitrogen. Evaluation was based on the hypoosmotic swelling test (HOS test), electron microscopy, and biochemical parameters such as lipid peroxidation and reduced and total glutathione levels, all measured after thawing. The HOS test indicated that the percentage of intact plasma membranes after freezing and thawing was significantly higher for the hypertonic extender containing trehalose (T), compared with an extender containing trehalose+EDTA (TE) or an isotonic Tris extender (B) (p < 0.05). Membrane evaluation by ultramicroscopy also indicated better sperm cryopreservation in extender T compared with the others, and there was a significant reduction in the number of damaged membranes (27%, p < 0.0002). The level of reduced glutathione was significantly higher after sperm cryopreservation in either hypertonic diluent (T and TE) with respect to the isotonic extender B, immediately after thawing (12%) and after a 3-h post-thawing thermotolerance test at 37 degrees C (17%, p = 0.007). Total glutathione levels did not show statistical differences among the extenders. After 3h post-thawing incubation at 37 degrees C, lipid peroxide levels in spermatozoa were statistically lower for T than TE (35%) or isotonic extender B (44%) (p = 0.002). Taken together these results indicate a reduction in the oxidative stress provoked by freezing and thawing when semen is cryopreserved in extender T. The antioxidant properties of extender T may be related to its effectiveness in membrane cryopreservation.     

Sea urchin spermatozoa generate at least two reactive oxygen species; the type of reactive oxygen species changes under different conditions

Reactive oxygen species (ROS) cause oxidative stress and act as signal transduction molecules in many cells. Spermatozoa from several mammals generate ROS, which are involved in male infertility and signaling during capacitation. In the present study, we investigated ROS generation by sea urchin spermatozoa at the initiation of motility, during dilution with seawater, and following egg jelly treatment. In seawater containing an ROS indicator, 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), fluorescence increased after the addition of spermatozoa. The ROS generation rate was dependent upon the dilution ratio and respiratory rate of the spermatozoa. Spermatozoa in sodium-free seawater did not increase fluorescence, but fluorescence did increase with the addition of NaCl. Sodium chloride also led to the initiation of sperm motility and respiration. Using the indicator MitoSOX Red, ROS generation was detected from spermatozoa exposed to egg jelly dissolved in seawater, but not in normal seawater. Moreover, the respiratory inhibitor antimycin A prevented CM-H(2)DCFDA-detectable ROS and increased MitoSox-detectable ROS at a higher concentration. These findings revealed that the ROS generated were of different species, possibly hydrogen peroxide (H(2)O(2)) and superoxide anion (O(-)(2)), and their detected levels were altered by egg jelly. We concluded that sea urchin spermatozoa generate at least two species of ROS depending on the physiological conditions to which they are exposed. It is possible that the major ROS from sea urchin spermatozoa changes during the course of fertilization.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.