Protein tyrosine phosphorylation under capacitating conditions in porcine fresh spermatozoa and sperm cryopreserved with and without alpha tocopherol.

The aim of this study was to evaluate the capacitation behaviour of fresh and alpha-tocopherol frozen spermatozoa. Spermatozoa frozen with or without alpha-tocopherol and fresh semen were incubated under capacitating conditions. Aliquots were collected at 0, 15, 30, 45, 60, 90, 120 and 180 min of incubation time. Parameters of semen quality were evaluated by optical microscopy and capacitation was determined by the epifluorescence chlortetracycline technique. Protein tyrosine phosphorylation was examined by Western immunoblotting. Motility, viability and intact spermatozoa were higher (P < 0.05) in fresh semen compared with frozen samples. These parameters significantly decreased, in every treatment, throughout the incubation time. Fresh semen showed a progressive increase in capacitated spermatozoa, reaching 25 +/- 3% at 180 min. Cryopreserved semen had a fast increase at the beginning of incubation time (28 +/- 5% at 45 min and 28 +/- 3% at 30 min for samples with or without alpha-tocopherol, respectively). The amount of an MW 32 kDa tyrosine-phosphorilated protein, associated with capacitation, increased throughout incubation for fresh semen and spermatozoa cryopreserved with alpha-tocopherol. The supplementation with alpha-tocopherol preserved sperm plasma membrane, reflected not only in the acrosome integrity but also in a greater efficiency of energy production.

alpha-Tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm.

Sperm cryopreservation is associated with the production of reactive oxygen species (ROS) leading to membrane destabilization, which induces capacitation-like changes, increases protein tyrosine phosphorylation, and decreases their fertilizing ability. alpha-Tocopherol, a lipid peroxidation inhibitor, preserves the functionality of cryopreserved porcine sperm. Our aim was to evaluate the effect of alpha-tocopherol on sperm quality parameters as well as capacitation-like changes and modifications in protein tyrosine phosphorylation. Boar sperm frozen with or without 200 microg/mL of alpha-tocopherol were thawed and maintained at 37 degrees C for 10 min in BTS. Routine parameters of semen quality were evaluated by optical microscopy and membrane changes were determined by the epifluorescence chlortetracycline technique. Changes in protein tyrosine phosphorylation were examined using a specific anti-phosphotyrosine monoclonal antibody. Motility was higher (18%, P<0.05) in semen with alpha-tocopherol. Viability did not differ (P>0.05) between treatments. However, there was less (P<0.05) capacitation-like changes in semen with alpha-tocopherol compared to control samples. A MW 32 kDa tyrosine-phosphorylated protein was detected in extracts of cryopreserved sperm; the intensity of immunostaining was lower in semen containing alpha-tocopherol compared to the control (0.211+/-0.030 versus 0.441+/-0.034 arbitrary units). Additionally, this band was not detected in fresh sperm. The addition of alpha-tocopherol to the extender prior to cryopreservation of boar semen protected sperm membranes against oxidative damage and reduced both tyrosine phosphorylation and the capacitation-like state.

Nitric oxide induces acrosome reaction in cryopreserved bovine spermatozoa.

The aim of this work was to study the effect of nitric oxide on acrosome reaction (AR) and the participation of protein kinases and reactive oxygen species in the AR of cryopreserved bovine spermatozoa. Spermatozoa were capacitated in Tyrode's albumin lactate pyruvate medium with heparin (10 IU ml(-1)) and then incubated with different concentrations of sodium nitroprusside (SNP) (1-200 micromol l(-1)). Methylene blue and haemoglobin were used to confirm the role of nitric oxide as an inducer of the AR. The participation of protein kinase A (PKA) , protein kinase C (PKC) and protein tyrosine kinase was evaluated using specific inhibitors of these enzymes (H-89, 50 micromol l(-1); bisindolylmaleimide I, 0.1 micromol l(-1) and genistein, 3 micromol l(-1)). The role of hydrogen peroxide or superoxide anion was evaluated by incubation with catalase or superoxide dismutase respectively. AR percentages were determined by the fluorescence technique with chlortetracycline. The highest levels of AR were obtained in capacitated spermatozoa treated with 5-200 micromol l(-1) SNP (24.8 +/- 1.8%). The presence of PKA, PKC and protein tyrosine kinase inhibitors likewise decreased AR percentages. The addition of superoxide dismutase had no effect on the AR level but catalase completely blocked it. These results indicate that nitric oxide induces AR in capacitated spermatozoa involving hydrogen peroxide and the participation of PKA, PKC and protein tyrosine kinase as part of the signal transduction mechanism which lead to the AR in cryopreserved bovine spermatozoa.

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways.

The effect of nitric oxide (NO*) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05-100 microM), a NO* donor. The participation of NO* was confirmed by the use of scavengers, i.e. methylene blue (50,100 microM) and hemoglobin (20-40 microg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NA) in concentrations ranging from 1 to 500 microM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO*-induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 microM; bisindolylmaleimide I, 0.1 microM and genistein, 3 microM). The role of hydrogen peroxide or superoxide anion in NO*-induced capacitation was evaluated by incubation with catalase (20-100 microg/ml) or superoxide dismutase (SOD, 0.05-0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 microM SNP treatment (31 +/- 5.15%) were similar to those of heparin treated samples (33 +/- 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO*- scavengers (P <0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 +/- 0.71, 12.75 +/- 1.41, 9.00 +/- 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO* may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO* acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.

Sperm evaluation in cryopreserved bovine semen recovered by two selection methods.

Previous experiments have established that various semen manipulation techniques are able to increase the qualitative features of the spermatozoa used in different techniques of assisted reproduction, but practically no comparative data on frozen-thawed bovine semen have been found. The aim of this study was to compare the efficacy of two sperm selection methods: centrifugation on Percoll gradient and filtration through a Sephadex ion-exchange column, to improve the recovery of motile and morphologically normal spermatozoa, without inducing sperm damage, from cryopreserved bovine semen samples. Semen samples were thawed and centrifuged on a discontinuous Percoll gradient, or were filtered through a Sephadex G-15-120 column with the addition of ion exchangers. Sperm concentration, percentages of motile spermatozoa, acrosome integrity, superoxide dismutase activity and lipid peroxidation were evaluated in recovered samples and controls. The motility of spermatozoa obtained by Sephadex ion-exchange filtration (88.87 +/- 6.37%) and by Percoll gradient centrifugation (83.00 +/- 6.21%) were significantly greater than that of control samples (60.14 +/- 8.44%). Other results disclosed that both sperm selection methods significantly increased the percentage of intact acrosome and superoxide dismutase activity. In both cases, the number of recovered spermatozoa diminished significantly versus untreated samples. Although the number of recovered spermatozoa was low, these methods were effective to select viable sperm from cryopreserved bovine semen.

Lactate dehydrogenase-C4 is involved in heparin- and NADH-dependent bovine sperm capacitation.

Lactate dehydrogenase C4 isoenzyme (LDH-C4) is involved in the energy metabolism of spermatozoa. Sperm capacitation is considered part of an oxidative process; an NADH oxidase of plasma membrane could be responsible for superoxide anion generation which is required for capacitation. The role of LDH-C4 and the requirements of NADH in cryopreserved bovine sperm capacitation were studied. LDH-C4 activity was 5.52 +/- 3.41, 15.72 +/- 6.04 and 15.22 +/- 1.92 Units 1010 spermatozoa-1 in plasma membrane, sperm suspension and cytosol fraction, respectively; these activities were inhibited by sodium oxamate. To study the influence of oxidative substrates in capacitation, three different TALP (T) media were used: TP (pyruvate); TL (lactate) and TC (citrate); heparin or NADH was then added. There were no significant differences in the percentage of capacitation induced by heparin or NADH in TALP medium; similar levels of capacitation were achieved with TL alone or TL +heparin and TP +NADH; capacitation was inhibited with sodium oxamate in all treatments used. Cytosolic NADH may be required as a substrate for sperm oxidase. Lactate influx through plasma membrane may be utilized by cytosolic LDH-C4, increasing reduced coenzymes required for capacitation. Plasma membrane LDH-C4 may participate in the production of lactate to obtain intracellular reducing equivalents to be used by sperm oxidase for in vitro sperm capacitation.

Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction.

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.

Intracellular calcium variation in heparin-capacitated bovine sperm.

This report investigates the mechanisms by which heparin induces capacitation of sperm. Capacitation was determined with chlortetracycline and intracellular calcium concentration, [Ca2+]i, with FURA 2-AM. After 15 minutes incubation with heparin [Ca2+]i was increased 60% over basal, reaching a plateau thereafter. Sixty percent of calcium entry was inhibited by methoxy-verapamil, suggesting that activation of voltage dependent calcium channels (VDCC) may be involved in the process. A significant correlation (R1 = 0.88, p < 0.006) was found between the percentage of capacitated spermatozoa and the [Ca2+]i increase. The effects of heparin on both processes were blocked by the protein kinase C (PKC) inhibitors staurosporine (100%) and GF-109203X (90%). It is concluded that heparin may induce sperm capacitation and calcium influx mainly through VDCC (approx. 60%), and also through other membrane systems (40%). Both systems of calcium entry as well as the capacitation process appear to involve on PKC activity.

Mitochondrial calcium uptake in bovine frozen sperm.

Mitochondria from bovine frozen semen are capable of preserving oxidative energy. The purpose of this work was to study calcium uptake by the inner mitochondrial membrane which, together with the plasmatic membrane, is involved in spermatic motility and viability regulation. Mitochondrial coupling was confirmed. In the active state, there was a drop in oxygen uptake rate using calcium concentrations ranging from 20 to 100 microM. In the resting state, 75 microM calcium concentration mimicked the active respiration state. In both states, concentrations greater than 100 microM induced mitochondrial uncoupling. Maximal mitochondrial uptake was recorded in the active state with 10 microM lactate. At calcium concentrations greater than 100 microM, there was a drop in mitochondrial calcium uptake for both respiration states. The ability to preserve oxidative energy allows the spermatozoon to take up mitochondrial calcium, thus regulating its functional activity.

Influence of antioxidants on SOD activity in bovine sperm.

SOD activity and susceptibility to peroxidation on spermatozoa from frozen and fresh bovine semen were determined either in presence or not of synthetic (BHA) or natural (vitamin E) antioxidants. In sperm suspensions incubated with vitamin E, SOD activity was higher than in the control samples and the ones treated with BHA. It was found a highly significative correlation between malondialdehyde production and SOD activity. SOD activity could be used in bovine spermatozoa as a metabolic indicator of membrane integrity.

Phosphorylant capacity study and lactate mitochondrial oxidation in frozen bovine sperm.

Frozen-stored bovine sperm-pellets of proven fertility were used, and the response to respiratory chain effectors was studied, thus demonstrating the energy conservation capacity. It was further observed that the assayed suspensions used lactate oxidatively, which proves the LDH-X mitochondrial activity (the presence of oxidative substrates is fundamental in capacitation and acrosome reaction processes). The suspensions were treated with 10mM phosphate buffer hypotonic medium to eliminate plasmalema and cytoplasmic content. Lactate respiration was sensitive to respiratory chain effectors, such as oligomycin and antimycin. To evaluate the LDH-X contribution to mitochondrial respiration, lipoate dehydrogenase was inhibited through 5-methoxyindole-2-carboxylic acid (MICA) in the presence of pyruvate-malate and citrate-malate, obtaining with the addition of lactate, oxygen uptakes of 18% and 51% with respect to respiration with the mentioned substrates. In the MICA dose-effect curve, a major sensitivity to inhibitor in active state mitochondrial respiration is obtained when pyruvate-malate is used. Lactate competence with pyruvate by mitochondrial LDH-X was observed. The results obtained would allow the thorough study of the necessity of oxidative energy in the capacitation and fertilization processes, and of the LDH-X role in frozen-stored bovine sperm.