N. Beorlegui

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

Summary Bovine spermatozoa from frozenthawed semen are sensitive to lipid peroxidation. Vitamin E protects sperm membrane against oxidative damage. Sperm capacitation produces structural changes on the plasma membrane. Reactive oxygen species could be involved in the capacitation process. The aim of this work was to study the influence of natural antioxidants on the plasma membrane and the influence of reactive oxygen species during bovine sperm capacitation. Sperm samples were frozen in a standard diluent, with and without vitamin E (1 mg ml‐1). Heparin (60 μg ml‐1) was used as a sperm capacitation inductor. Sperm capacitation was evaluated by chlorotetracycline assay. Lipid peroxidation was determined by the 2‐thiobarbituric acid assay. A diminution of thiobarbituric acid reactive substances was observed in sperm samples frozen with vitamin E (P < 0.05). The addition of vitamin E to the freezing diluent had no effect on the capacitated pattern (P > 0.05).

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.

Comparative study of functional and biochemical parameters in frozen bovine sperm

Summary. In order to correlate functional and biochemical parameters, frozen bovine sperm samples presenting diverse motility were studied. Specimens collected from four bulls (A, B, C and D) were graded according to motile spermatozoa percentage and vigour, regarding the sample from bull A as highest motility, those from bulls B and C of intermediate motility and that from bull D of low motility. In order to evaluate lipid peroxidation enhanced by the addition of sodium ascorb‐ate and ferrous sulphate, thiobarbituric acid reactive substances (TEARS) were measured. Values were 0.34 ± 0.18 and 4.95 ± 0.31 nmol TEARS/108 spermatozoa for samples A and D, respectively. Superoxide dismutase (SOD) activity was 144±16.48 and 44±4.0 IU/1010 spermatozoa for A and D, respectively, while intermediate values were recorded for samples B and C. The capability to undergo acrosome reaction induced by calcium ionophore A23187 was significantly lower for sample D, but differences were negligible for the remaining three samples inter se. Motile spermatozoa percentage correlated closely both with SOD activity (r = 0.92) and with TEARS production (r=−0.80), but not with acrosome reacted percentage (r=0.65). From correlation data, it would be inferred that motility is not entirely indicative of sperm quality.

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. Atter 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 (R2 = 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.

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

Summary. 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.

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.

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 Tyrodes albumin lactate pyruvate medium with heparin (10 IU ml−1) and then incubated with different concentrations of sodium nitroprusside (SNP) (1–200 μmol 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 μmol l−1; bisindolylmaleimide I, 0.1 μmol l−1 and genistein, 3 μmol 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 μmol 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.

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.

Heparin- and superoxide anion-dependent capacitation of cryopreserved bovine spermatozoa: Requirement of dehydrogenases and protein kinases

Capacitation is part of an oxidative process necessary for bovine spermatozoa to acquire fertilizing capacity. This process includes the generation of reactive oxygen species (ROS) and the participation of protein kinases such as A (PKA), C (PKC) and tyrosine kinase (PTK). A redox status is required to support both sperm motility and capacitation. Our aim was to determine the requirement of lactate dehydrogenase C4 (LDH-C4) and isocitrate dehydrogenase (NADP-ICDH) and of protein kinases in cryopreserved bovine sperm capacitation. The presence of inhibitors of both LDH-C4 and NADP-ICDH prevented the heparin-induced capacitation. H89, GF109203X or genistein blocked capacitation triggered by heparin or the superoxide generator system xanthine–xanthine oxidase–catalase (XXOC) suggesting the requirement of PKA, PKC and PTK in this process. Taken together these results suggest that LDH-C4 and NADP-ICDH contribute with the redox status to support bovine sperm capacitation and that PKA, PKC and PTK are involved in different mechanisms induced by different inducers that lead bovine spermatozoa to be capacitated.

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 α‐tocopherol frozen spermatozoa. Spermatozoa frozen with or without α‐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 α‐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 α‐tocopherol. The supplementation with α‐tocopherol preserved sperm plasma membrane, reflected not only in the acrosome integrity but also in a greater efficiency of energy production.