Effects of ouabain on the response to osmotic changes in dog and boar spermatozoa

Although there is much information on the response of spermatozoa from different species to osmotic changes, little has been reported about the mechanism/s by which spermatozoa react to similar changes in the osmotic pressure of the medium. In this study we examine the effect of inhibition of Na (+)K (+), ouabain-sensitive ATP-ase on the response of canine and porcine spermatozoa when they are incubated in hypoosmotic and hyperosmotic media. The presence of ouabain slightly decreased the percentages of total and progressive motility, and increased the percentages of altered acrosomes (from 13.0 +/- 0.3% to 17.2 +/- 0.4% in the presence of 10(-4) M ouabain) and, specially, swollen tails (from 0.6 +/- 0.1% to 5.9 +/- 0.2% in the presence of 10(-4) M of ouabain) in fresh dog semen, although it did not affect these parameters in boar semen samples. Moreover, ouabain increased the percentage of both altered acrosomes and swollen tails in canine spermatozoa incubated in 100 mOsm and in 900 mOsm media at concentrations higher than 10(-5) M and 10(-7) M, respectively. The percentage of viability of canine spermatozoa was not modified by ouabain after incubation in 100, 300 or 900 mOsm media. Furthermore, ouabain did not significantly affect boar spermatozoa incubated in 100, 300 or 900 mOsm media. Although ouabain induced a significant decrease in L-lactate production in canine spermatozoa in an isoosmotic medium (from 4.7 +/- 0.4 micromol mg protein x 60 min to 2.6 +/- 0.3 micromol mg protein x 60 min in the presence of 10(-4) M ouabain), there was no significant effect on L-lactate production in boar spermatozoa. These results indicate that while dog spermatozoa acted against changes in the osmotic pressure by a mechanism(s) related to Na (+)K (+), ouabain-sensitive ATP-ase, boar spermatozoa reacted to some mechanism(s) not related to ionic pumps.     

Ion-mediated resistance to osmotic changes of ram spermatozoa: the role of amiloride and ouabain

The aim of this study was to evaluate whether the Na+/K+ and Na+/H+ exchange can maintain the function of fresh ram spermatozoa. We analyzed the quality parameters of spermatozoa from fresh ram ejaculates incubated in iso- (about 300 mOsm), hypo- (about 100 mOsm) and hyperosmotic (about 900 mOsm) media in the presence of ouabain a specific inhibitor of the Na+/K+ ATP-ase or amiloride, a specific inhibitor of the Na+/H+ antiporter. Ouabain increased the percentage of morphologically altered acrosomes in isoosmotic media (from about 10% to 15% in control to about 30% with 10(-4) M ouabain) and decreased the percentage of total motility (from about 80% in control to about 50% to 55% with 10(-4) M ouabain). Ouabain decreased the mean linearity component of motility and decreased the frequency of head displacement. The addition of ouabain increased the percentage of altered acrosomes in the hypo- and hyperosmotic media, although it did not modify viability in either media. Ouabain also increased the percentage of swollen tails in the hypoosmotic medium at higher concentrations of the inhibitor. Amiloride increased the percentage of altered acrosomes in all media although its effect was less intense than that of ouabain. In isoosmotic media, total motility was decreased from about 80% in control to about 65% with 10(-4) M amiloride. Motile spermatozoa incubated with amiloride showed a clear decrease of mean velocity and mean linearity and increased frequency of head displacement. In the hyperosmotic medium, adding amiloride decreased the percentage of viability and altered tails at concentrations as low as 10(-6) to 10(-5) M. Our results indicate that the active mechanisms which control Na+ transport play a significant role in the maintenance of function in ram spermatozoa subjected to different osmotic environments. These mechanisms may be important in maintaining ram sperm function both "in vivo" and "in vitro".     

Changes in surface ATPase of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis were found to have a lower activity of the surface ATPase than the spermatozoa from the caput region. The enzyme from spermatozoa of both regions had the same Michaelis constant (Km) for ATP of 5 X 10(-4) M. It was partly inhibited by ouabain and fluoride, but strongly inhibited by Cu2+, Zn2+,p-chloromercuribenzoate, 8-anilino-1-naphthalenesulphonate Triton X-100, Lubrol-PX, urea, guanidine hydrochloride, sodium dodecyl sulphate and glycerylphosphorylcholine. The enzyme of the spermatozoa from the cauda epididymidis was more sensitive to inhibition by ouabain and fluoride but less sensitive to inhibition by Cu2+ than that of the cells form the caput region. The Arrhenius plot of the temperature dependence of enzymatic activity varied for the cells from the caput and cauda epididymidis. The differences in the enzyme properties of spermatozoa from the two regions of the epididymis suggested that the decline in the activity during epididymal maturation may reflect changes in the lipids and sulphydryl groups of the sperm membrane.     

Subjecting horse spermatozoa to hypoosmotic incubation: effects of ouabain

Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na (+)K (+), ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenney's medium (Time = 0). Aliquots were randomly selected to be incubated in an isoosmotic (297 mOsm) or different hypoosmotic media that were composed of citrate or of citrate w?th fructose. The osmolarity of the hypoosmotic media with citrate ranged from 18 to 96 mOsm, and the medium composed of citrate plus fructose (HOS medium) was of 153 mOsm. Moreover, aliquots of spermatozoa pretreated with ouabain were added to the isoosmotic medium and also to the HOS and the 96 mOsm citrate medium (ORT medium). Incubation of equine sperm in the hypoosmotic media resulted in a time- and osmolarity-dependent swelling of the sperm tail, reaching maximum values after incubation for 20-30 min in both the HOS and ORT media. Ouabain induced a dose-dependent effect on swollen tails and viability in fresh semen and also affected some parameters related to motility. Ouabain also increased the swelling response in a hypoosmotic medium although viability decreased. The percentage of swollen tails after incubation in ORT and HOS media snowed significant correlations to viability, altered acrosomes and total motility, but not to other parameters of horse semen analysis. Our results suggest that hypoosmotic tests could be used to improve standard horse semen analysis. Additionally, Na (+)K (+)-ATP-ase activity could be related to the response against hypoosmotic shock of horse spermatozoa.     

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.     

Season and social environment influence the membrane integrity of ejaculated boar spermatozoa as assessed by ouabain sensitivity

Eight adult Landrace boars were housed for 12 months in one of two social environments. Socially nonrestricted boars were penned near estrual females and socially restricted boars were penned behind solid walls to eliminate visual and physical contact with other pigs. All animals were subjected to natural changes in day length. The sensitivity of ejaculated spermatozoa to ouabain (in inhibitor of Na+-K+ ATPase) was determined on 4 consecutive weeks in November, March-April, and July-August. Semen was diluted in Tyrode's solution (pH 7.4) with and without 10(-3) M ouabain. Duplicate samples of control and ouabain-treated spermatozoa were incubated at 37 degrees C for 4 h, and percent motile sperm, motility type, and motility index (combination of percent and type) were determined at hourly intervals. Ouabain-induced decreases in most motility parameters varied with season (season X treatment, P less than 0.05). At hour 4, induced decreases in percent motile sperm were more pronounced in November and July-August than in March-April for socially nonrestricted boars. Decreases in motility type were greater (P less than 0.05) in November and July-August than in March-April for socially nonrestricted boars and were greater (P less than 0.01) in November than in July-August for restricted boars. In March-April motility type decreased (P less than 0.01) to a greater extent for socially restricted vs. nonrestricted boars. Similar season and social environment differences were observed for motility index values. Given the interrelationships between ouabain sensitivity, the functional integrity of sperm cells, and fertilizing capacity, season and social environment differences in ouabain-induced motility depression probably reflect qualitative changes in boar spermatozoa.     

The effect of ouabain on membrane potential and flagellar wave in ejaculated bull spermatozoa.

Ejaculated bull spermatozoa were exposed to various concentrations of ouabain to ascertain the effect on membrane potential, intracellular concentrations of sodium and potassium and motility. Membrane potenital, measured by electrophysiological methods, decreased. Intracellular potassium decreased and intracellular sodium increased. Progressive motility decreased. In addition, the motility change produced by ouabain was identified as a decrease both in beat frequency and in wave amplitude.     

Na+/K+ATPase as a signaling molecule during bovine sperm capacitation

A heteromeric integral membrane protein, Na+/K+ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na+/K+ATPase, to Na+/K+ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na+/K+ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na+/K+ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na+/K+ATPase. Binding of ouabain to Na+/K+ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na+/K+ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na+/K+ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na+/K+ATPase in mammalian sperm capacitation.     

Active cation transport in dog spermatoza

1. When dog semen is stored at 5 degrees for 24hr., K(+) is lost from the spermatozoa and Na(+) accumulates in the cells. 2. If at the end of the cold-storage period the semen is incubated at 37 degrees in the presence of added glucose there is a rapid uptake of K(+) and extrusion of Na(+) from the spermatozoa, the intracellular K(+) reaching a maximum within 30min. 3. When the semen is incubated at 20 degrees after cold storage there is an uptake of K(+) by the spermatozoa over 3hr. but no change in intracellular Na(+) concentration. 4. The extrusion of Na(+) and uptake of K(+) by dog spermatozoa has been shown to be inhibited by fluoride, iodoacetate, 2,4-dinitrophenol, and cetyltrimethylammonium bromide. 5. Uptake of K(+) is inhibited by ouabain and half maximum inhibition is obtained with a concentration of 50mmum. There is a slight stimulation of K(+) uptake in the presence of ouabain at about 0.3% of the concentration required for half maximum inhibition.     

Resistance to osmotic stress of horse spermatozoa: the role of ionic pumps and their relationship to cryopreservation success

To study the resistance of horse spermatozoa against hyperosmotic stress, cells were incubated in solutions of 600 to 4000 mOsm(undisturbed media). Then, semen was immediately placed into an iso-osmotic solution (disrupted media). Incubation in undisturbed media decreased sperm viability in an osmolarity- and temperature-dependent manner. Viability was further decreased in disrupted media, with the effect dependent upon the initial osmolarity of the media and on the temperature. Treatment with ouabain or amiloride impaired the resistance of horse spermatozoa to hyperosmotic stress. Very few correlations were strong between viability after hyperosomotic stress and quality parameters of fresh and frozen-thawed horse semen. The results indicate that the usefulness of resistance to hyperosmotic stress in assessing frozen-thawed semen quality is compromised, since other factors are involved in the resistance to freezing-thawing. Both Na (+)K (+) ATP-ase and the Na (+)H (+) antiporter act in the resistance to hyperosmotic stress in horse spermatozoa.     

Short-term inhibition of the energy metabolism affects motility but not surface properties of sperm cells

Centrifugal countercurrent distribution (CCCD) in aqueous two-phase systems has been proven to be a useful method to study subtle surface properties of spermatozoa. The present work shows that a short-term inhibition of the energy metabolism of sperm cells effected by incubating bovine sperm cells with KCN or ouabain, did not account for changes in the cell surface properties, as assessed either by estimation of the cell viability or by CCCD analysis. However, the short-term inhibition of energy metabolism provoked a clear decrease of cell motility, suggesting that a drop of cellular ATP levels brings about a rapid decrease of motility followed by a very delayed effect on cell surface properties. The relevance of these results on the handling of sperm and on the understanding of the molecular events underlying asthenospermia is discussed.     

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs gamma-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with gamma-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from gamma-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.     

Assessment of Pisum sativum agglutinin in identifying acrosomal damage in stallion spermatozoa.

The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.     

Effect of cooling and seminal plasma on the capacitation status of fresh boar sperm as determined using chlortetracycline assay

Insemination of sows with frozen-thawed spermatozoa results in lower fertility, in part due to spermatozoa having undergone a capacitation-like reaction. The present study employed chlortetracycline (CTC) staining analysis to investigate the effect of adding 20% (v/v) boar seminal plasma (SP) to boar spermatozoa on the temporal progress of capacitation and the acrosome reaction in spermatozoa cooled to 5 degrees C or incubated at 39 degrees C. Based on CTC staining patterns, seminal plasma appeared to reverse capacitation in spermatozoa that had undergone capacitation while incubated at 39 degrees C in a capacitation-supporting medium from 59.7 to 36.6% capacitated (P<0.001). Similarly, the addition of SP to boar spermatozoa cooled to 5 degrees C resulted in both the prevention of the capacitation-like reaction, and the reversal of an established capacitation-like reaction from 63.3 to 34.2% capacitated (P<0.001). These observations indicated that some constituent(s) of boar SP both prevent spermatozoa from undergoing capacitation as well as reverse capacitation in spermatozoa that have already undergone the process.     

Endogenous digitalis-like factor as a stimulator of endothelin secretion from endothelial cells

Effects of human urine-derived endogenous digitalis-like factor (EDLF) and ouabain on endothelin (ET) secretion were examined in cultured endothelial cells. ET was secreted in a linear fashion over 5 hours from bovine pulmonary artery endothelium into serum-free medium. EDLF stimulated ET secretion in a dose-dependent manner. In contrast, ouabain did not affect ET secretion at the concentration of 10(-9)-10(-5) M. These results indicate that human urine-derived EDLF is distinct from plant-derived ouabain and act as a stimulator of ET secretion by endothelial cells.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

On the presence of 3H-GABA uptake mechanism in bovine spermatozoa

In the present study, existence of (3)H-GABA uptake mechanism in bovine spermatozoa and the modulation of (3)H-GABA transport by GABA itself were evaluated. The hypothesis was tyrosine phosphorylation affects transporter (GAT) function. (3)H-GABA uptake assays were performed on bovine spermatozoa and it resulted to be temperature- and time-dependent and K(m) was 1.48muM. Uptake was inhibited by the metabolic inhibitor ouabain and different blockers of GAT-1 (beta-alanine, l-DABA, nipecotic acid, tiagabine). Extracellular GABA up-regulated GABA transport, while the addition of SKF89976A, a high affinity inhibitor of the rat brain GABA transporter, reduced GABA uptake. Tyrosine phosphorylation affects transporter function since genistein, a broad-spectrum tyrosine kinase inhibitor, decreased (3)H-GABA uptake. Reduction in uptake did not occur in the presence of daidzein, an inactive genistein analogue. Furthermore, the genistein-mediated reduction in transport could be prevented by the tyrosine phosphatase inhibitor pervanadate. The action of these drugs on GABA transport is likely mediated through the GABA transporter GAT-1 since SKF89976A blocked a majority of GABA uptake. Wash-out experiments indicated that the genistein effect was reversible. When the experiments were conducted using "in vitro" capacitated spermatozoa there was no detectable uptake. Present results demonstrate that the carrier-mediated GABA uptake system in bovine spermatozoa modulates its function in response to extracellular GABA, that changes in lipid distribution and membrane composition which occur during capacitation eliminates GABA uptake and suggest the involvement of tyrosine phosphorylation in GABA transport.     

Decreased ouabain binding in cystic fibrosis fibroblasts in potassium-free medium

We have previously demonstrated that cystic fibrosis (CF) cells show increased survival compared to normal cells when exposed to ouabain in medium lacking potassium. In this report, we show that the CF cells bind significantly less ouabain than the normal cells in potassium-deficient medium. Using age-matched normal and CF skin fibroblast strains, we show that (1) at ouabain concentrations <20 × 10⁻⁹ M binding for both normal and CF cells is linear with time for 1 h and reaches equilibrium after 4 h; (2) at ouabain concentrations between 2 and 20 × 10^-9 M, the initial rate of binding for CF cells is approx. 70% of the normal cells; (3) under equilibrium-binding conditions, Scatchard analysis reveals that three different CF strains have 12, 16, and 44% fewer ouabain-binding sites than their matched normal controls. In addition, studies in potassium-free medium of the inhibition of ⁸⁶Rb flux (a K⁺ analogue) into the cell by ouabain show no differences between CF and normal cells. We have also previously shown that in a low glucose, potassium-deficient medium the CF cells survived ouabain exposure no better than normal cells. In this report, equilibrium-binding studies with [³H]ouabain clearly show that CF cells bind less ouabain under these conditions than normal cells. These results indicate that ouabain resistance in CF cells is not solely a function of differences in ouabain binding. Furthermore, the differential ouabain killing may not be due to ion transport differences, but rather to as yet unknown mechanisms. CF cells thus appear to be unlike previously characterized ouabain-resistant mutants.