Participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa.

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrode's albumin lactate pyruvate medium in the presence of heparin (10 IU ml(-1) ) or forskolin (1-75 µm), a well-known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2',5'-dideoxyadenosine (6-25 µm). Spermatozoa capacitated with forskolin (25 µm) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25-µm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2',5'-dideoxyadenosine prevented forskolin-induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25-µm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.

Boar sperm functionality is related to α-tocopherol content after freezing-thawing.

Boar spermatozoa are sensitive to oxidative damage produced during cryopreservation. Our aim was to evaluate the participation of different antioxidants in the improvement of cryopreserved boar sperm functionality. Spermatozoa frozen with 200 µg ml(-1) α-tocopherol, 0.5 mm 17ß-oestradiol or seminal plasma were used to evaluate sperm parameters and capacitation-like changes. The 17ß-oestradiol and α-tocopherol concentrations were assessed by RIA and HPLC respectively. Motility was improved but lipid peroxidation and capacitation-like changes were diminished (P < 0.05) in antioxidant samples. A significant increase in 17ß-oestradiol concentration was detected in 17ß-oestradiol or seminal plasma samples. Alpha-tocopherol content increased in α-tocopherol, 17ß-oestradiol or seminal plasma samples, obtaining the lowest level in the α-tocopherol ones. The 17ß-oestradiol or seminal plasma components may be acting in the regeneration of the α-tocopherol antioxidant capacity. The α-tocopherol concentration may be conditioning the cryopreserved boar sperm functionality. The addition of antioxidants could be useful to reduce oxidative stress, thus improving the functionality of cryopreserved boar spermatozoa.

Nitric oxide and superoxide anion production during heparin-induced capacitation in cryopreserved bovine spermatozoa.

The aim of this work was to quantify NO,O(2)(-) and ONOO(-) production during heparin-induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin-dependent capacitation, O(2) uptake, and NO production. Conversely, O(2)(-) production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O(2) consumption (5.9 ± 0.6 nmol/min × 10(7) cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10(7) cells), and a five-fold increase in O(2)(-) production (1.3 ± 0.07 nmol/min × 10(7) cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O(2)(-) generation and the second-order rate constant of the reaction between these species. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O(2) uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O(2)(-) production by a membrane-bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.

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.

Variations in creatine kinase activity and reactive oxygen species levels are involved in capacitation of bovine spermatozoa.

The generation of reactive oxygen species (ROS) is associated with some factors such as oxidative substrate sources, mitochondrial function and NAD(P)H oxidase activity. In bovine spermatozoa, heparin capacitation produces a respiratory burst sensitive to diphenyleneiodonium (DPI). Creatine kinase (CK) is related to extramitochondrial ATP disponibility. Our purpose was to determine the variation in ROS level and its relation with NAD(P)H oxidase sensitive to DPI and CK participation, as factors involved in redox state and energy generation in capacitation. The chlortetracycline technique was used to evaluate capacitation. CK activity and ROS level were measured by spectrophotometry and spectrofluorometry respectively. The capacitation percentage was increased by heparin or quercetin treatment (P < 0.05) and no significant differences in sperm viability were observed. Samples treated with heparin or quercetin maintained the same ROS level as control (238.62 +/- 23.47 arbitrary units per 10(8) spermatozoa) (P > 0.05). CK activity decreased by 50% with heparin or quercetin (P < 0.05). In DPI presence, capacitation was inhibited and differential CK activities and ROS level variations were observed in heparin- or quercetin-treated samples (P < 0.05). In cryopreserved bovine spermatozoa, capacitation requires equilibrium between oxidative damage susceptibility and ROS levels. CK activity is associated with redox state variation and energy sources. In conclusion, capacitation induction depends on NADPH oxidase and the shuttle creatine-creatine phosphate, both sensitive to DPI.

Heparin and quercitin generate differential metabolic pathways that involve aminotransferases and LDH-X dehydrogenase in cryopreserved bovine spermatozoa.

Heparin and quercetin induce capacitation in spermatozoa through membrane receptor binding and inhibition of Ca-ATPase of the plasma membrane, respectively. Although capacitation is energy intensive, ammonia from amino acid metabolism can inhibit respiration and Krebs cycle activity. The objective was to determine activities of key enzymes in bull spermatozoa that contribute to the redox state and supply energy for capacitation. Malate dehydrogenase (MDH-NAD(+)), alanine and aspartate aminotransferases (ALT, AST), and lactate dehydrogenase-X (LDH-X) were measured spectrophotometrically (340 nm); mean (+/-S.D.) activities in control spermatozoa were 7.65+/-1.67, 0.45+/-0.05 and 0.74+/-0.14x10(-2)U/10(8) spermatozoa for MDH-NAD(+), ALT and AST, respectively, and were 2.83+/-0.66U/10(8) spermatozoa for LDH-X. Heparin decreased (P<0.05) activities of MDH-NAD(+), ALT, AST and LDH-X (78, 53, 66 and 66% of control levels, respectively); we inferred that amino acid catabolism was decreased. Quercetin decreased (P<0.05) activities of MDH-NAD(+) and ALT (60 and 49% of control levels), but activities of AST and LDH-X were not significantly different from controls; apparently maintenance of LDH-X activity supplied pyruvate for cellular metabolism. The proportion of capacitated spermatozoa in controls (8.5+/-1.73%) was substantially increased (P<0.05) by treatment with either heparin (36.2+/-4.5%) or quercetin (32.8+/-4.7%), there was no significant difference among groups for acrosomal integrity and sperm viability. In conclusion, heparin- or quercetin-induced capacitation affected different metabolic pathways that modulated the redox state and oxidative metabolism in cryopreserved bovine spermatozoa.

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.

Acrosome reaction in bovine spermatozoa: role of reactive oxygen species and lactate dehydrogenase C4.

After capacitation, mammalian spermatozoa accomplish the acrosome reaction (AR), a well-controlled exocytosis process crucial to fertilize mature oocytes that involves several protein kinases such as protein kinase A (PKA), C (PKC), and tyrosine kinase (PTK). Reactive oxygen species (ROS) are involved in both bovine sperm capacitation and AR. Lactate dehydrogenase C4 (LDH-C4) was associated with bovine and mouse sperm capacitation. Our aims were to study the participation of LDH-C4 to contribute with the status redox required for AR and the role of ROS in the regulation of PKA, PKC, and PTK involved in the exocytotic event. Sodium oxamate, an inhibitor of LDH-C4, prevented the AR induced by lysophosphatidylcholine (LPC) or NADH. Hydrogen peroxide promoted and superoxide dismutase (scavenger of superoxide), catalase (scavenger of hydrogen peroxide), diphenyleneiodinum, diphenyliodonium, cibacron blue, and lapachol (inhibitors of NADPH oxidase) prevented the AR, suggesting that ROS and a sperm oxidase are involved in the AR induced by these compounds. Inhibitors of PKA, PKC, and PTK also prevented the AR induced by LPC or NADH, suggesting the involvement of these kinases in the process. These results suggest that LDH-C4 may participate in the regulation of the redox status required to achieve the AR in bovine spermatozoa and that ROS are key elements in the regulation of protein kinases associated with the AR process.

Respiratory burst and NAD(P)H oxidase activity are involved in capacitation of cryopreserved bovine spermatozoa.

Heparin (a glycosaminoglycan) and quercetin (a calcium-ATPase plasma membrane specific inhibitor) induce bovine sperm capacitation. Mitochondria from frozen semen are capable of generating oxidative energy. The aim of the study was to determine oxygen uptake variation and the participation of diphenileneiodonium (DPI)-sensitive oxidases from spermatozoa capacitated with heparin or quercetin. Oxygen uptake was measured polarographically and 2 microM diphenileneiodonium (DPI) was used as a specific inhibitor of NAD(P)H-oxidases. Sperm capacitation was determined by the chlorotetracycline technique. Heparin produced a respiratory burst (17.0+/-3.2 microL O2/h/10(8) spermatozoa; mean+/-S.D.) versus control (11.3+/-0.9 microL O2/h/10(8) spermatozoa; P<0.05). Oxygen uptake and sperm hypermotility were inhibited by cyanide. Treatment with DPI blocked heparin capacitation and oxygen uptake (cyanide-sensitive) decreased to control levels. Respiration of quercetin-treated samples (cyanide-sensitive; 9.7+/-0.7 microL O2/h/10(8) spermatozoa) was not significantly different from the controls; oxygen uptake was not modified by DPI, but quercetin capacitation was inhibited (P<0.05). The effect of DPI with heparin confirmed that oxidases participate in capacitation induction. The addition of superoxide dismutase and/or catalase to heparin- or quercetin-treated samples, failed to modify oxygen uptake and blocked capacitation (P<0.05), suggesting that the superoxide anion (O2*-) participates in the capacitation induction. High mitochondrial activity from heparin-treated samples indicated that energy requirements, especially for hypermotility, were supported by the respiratory chain. Although a respiratory burst was not produced by quercetin, DPI-sensitive-oxidases (O2*- source) were necessary for capacitation. In cryopreserved bovine spermatozoa, heparin- or quercetin-induced capacitation required different levels of mitochondrial energy and DPI-sensitive oxidase activity.

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.

Reactive oxygen species in bovine embryo in vitro production.

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7'-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.

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.

Effect of alpha-tocopherol and ascorbic acid on bovine in vitro fertilization.

The aim of this work was to study the effect of alpha-tocopherol (vitamin E) and ascorbic acid on the in vitro fertilization process. Frozen bovine semen was prepared using extenders with and without addition of vitamin E. Samples were capacitated with heparin in the fertilization medium. In vitro matured oocytes were inseminated with spermatozoa frozen with and without vitamin E and, after thawing, fertilized in TALP medium (control) and in TALP medium with vitamin E (1 mg/ml), with ascorbic acid (5 mM) and with vitamin E plus ascorbic acid. Gametes were incubated in the respective fertilization medium for 48 h; those frozen without vitamin E yielded 75, 76, 69 and 49% of fertilized oocytes in the control, vitamin E, ascorbic acid and vitamin E plus ascorbic acid media, respectively. The last value was significantly different (P < 0.01). In bovine sperm frozen with vitamin E, fertilization rates were 74, 50, 47 and 34%, respectively for the 4 groups. Values observed for the different supplements were significantly different inter se (P < 0.01), except between the media with vitamin E and with ascorbic acid. These results indicate that preserved antioxidant capacity of vitamin E impairs the success of the in vitro fertilization process.

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.

Effect of lactate dehydrogenase activity and isoenzyme localization in bovine oocytes and utilization of oxidative substrates on in vitro maturation.

Oocyte nutritional metabolism changes during maturation in order to increase the energy available to support metabolic requirements. The aim of this work was to study pyruvate and lactate utilization as oxidative substrates on IVM and lactate dehydrogenase (LDH) activity and localization of their isoenzymes in bovine oocytes. Immature cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles in ovaries obtained from slaughtered cows. The COCs and denuded oocytes were separately cultured in TCM-199 with steer serum (controls) and were supplemented with pyruvate, lactate or lactate plus NAD for 24 h at 39 degrees C in 5% CO2:95% humidified air. No significant differences were found in IVM rates of COCs matured according to the various treatments (P>0.05). The IVM rate in denuded oocytes without supplementation was 47.8%. The presence of pyruvate in the culture medium resulted in an increased number of matured denuded oocytes (59.4%; P<0.05), but the addition of lactate failed to improve the IVM rate of matured denuded oocytes (47.6%, P>0.05). When the medium was supplemented with lactate plus NAD, the IVM rate of denuded oocytes likewise failed to differ from that obtained with the addition of pyruvate (59.9%, P>0.05). The LDH activity in immature and matured COCs and denuded oocytes was (3.1+/-1.6) 10(-3), (3.3+/-1.6) 10(-3) U/COC, (5.2+/-2.0) 10(-5), (5.4+/-3.5) 10(-5) U/oocyte with pyruvate as substrate, and (1.2+/-0.5) 10(-3), (1.0+/-0.5) 10(-3) U/COC, (2.2+/-0.1) 10(-5), (2.5+/-1.4) 10(-5) U/oocyte respectively, with lactate; no significant differences due to maturation status were observed (P>0.05; n = 9 for each LDH activity). Electrophoresis disclosed that the principal band corresponded to the LDH-1 isoenzyme in oocytes, while there was no predominance of any isoenzyme in cumulus cells. Due to the fact that LDH-1 is the main oocyte isoenzyme, the pyruvate used during oocyte maturation could be partly produced from lactate when the NAD supply is adequate. Cumulus cells would be responsible for providing pyruvate and/or lactate as oxidative substrates to be used by the bovine oocyte and this supply would be regulated by the LDH activity in these cells.

Energy requirement of bovine spermatozoa for in vitro capacitation.

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.

Effect of natural antioxidants on frozen bovine semen preservation.

The influence exerted by natural antioxidants (Vitamin E or sodium ascorbate) was studied in various thermal treatments of semen and their effect on respiratory activity and membrane integrity during cryopreservation. Frozen bovine semen samples of diverse quality were employed in the presence and absence of antioxidants. Both in good-quality samples subjected to cold shock and in those of poor-quality standard-cooled, low superoxide dismutase activity was observed concomitantly with high malondialdehyde production; as regards oxygen uptake there was no evidence of mitochondrial coupling. In good-quality samples standard-cooled in the presence of antioxidants, greater superoxide dismutase activity, intact acrosome percentage and mitochondrial coupling were recorded as well as lower malondialdehyde production than in the controls. Natural antioxidants would seem to exert a protective effect on the membrane of the cryopreserved spermatozoon in samples from good-quality semen.

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen.

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome reacted cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96% of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.

Study of evaluation criteria used for in vitro bovine oocyte selection and maturation.

Production of bovine preimplantation embryos in vitro requires beneficial maturation conditions and high quality oocytes at the germinal vesicle stage. The current classification of oocytes is based on character of the cumulus cell investment around the oocyte. We wished to study the nuclear stage of immature oocytes selected for in vitro maturation according to cumulus cell character and, in the other hand, to compare the relationship among 3 parameters utilized to evaluate in vitro maturation of bovine oocytes (degree of cumulus expansion, meiotic maturation rate and in vitro fertilization rate) when fetal calf serum, steer serum and bovine follicular fluid supplementation were used. Ovaries were collected at an abattoir and the oocytes harvested. As regards selection criteria, immature oocytes were classified as Class A, B, C and D according to the character of the cumulus cells. A high percentage of Class A oocytes (87.7%) were in the germinal vesicle stage with respect to the other classes (p < 0.05). Significant differences were found in the meiotic maturation rate in Class A oocytes (76.5%) versus those of the other classes (p < 0.05). The meiotic maturation rate diminished to 47.5% when Class A oocytes were denuded and then matured in vitro (p < 0.05). As regards maturation criteria, there was no cumulus expansion when oocytes were matured in TCM-199 without supplementation, partial expansion with the addition of fetal calf serum and full expansion when supplemented with steer serum or bovine follicular fluid. No significant differences were found in the meiotic maturation rate for the various treatments. In vitro fertilization rate was significantly lower in media without supplementation versus supplemented media (p < 0.05), but no significant differences were found between the supplemented media inter se. There is no direct relationship between the three studied parameters to evaluate in vitro maturation. Class A oocytes are the most likely to mature in vitro as they not only have a close association with their surrounding cumulus cells, but are also very numerous in the germinal vesicle stage. The degree of cumulus expansion and the meiotic maturation rate have a relative importance in evaluating in vitro maturation, as oocyte maturation implies not only nuclear events but also at other cellular levels, as evaluated by in vitro fertilization.

Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved bovine spermatozoa.

Spermatozoa require a preparatory process called capacitation to fertilize mature oocytes. Two events related to capacitation of mammalian spermatozoa are an increase in intracellular Ca(2+) and protein tyrosine phosphorylation. The sites that regulate intracellular Ca(2+) concentration are plasma membrane and mitochondria. There are different systems for mitochondrial Ca(2+) influx and efflux. Our aim was to study the involvement of mitochondrial Ca(2+) cycle during heparin-induced capacitation in cryopreserved bovine spermatozoa. Samples were incubated at 38°C for 45 min, in TALP medium, in the presence of: (a) heparin (H), a well known capacitation inducer; (b) H+CGP 37157, a specific inhibitor of mitochondrial Ca(2+) efflux; (c) H+RU 360, a specific inhibitor of Ca(2+) influx to the mitochondria and (d) H+CGP 37157+RU 360. In every treatment, capacitation (by CTC), progressive motility (by optical microscopy), viability (by the eosin/nigrosin technique) and protein tyrosine phosphorylation (by Western Immuno-blotting), were evaluated. The addition of CGP 37157 (20 µM) decreased progressive motility (p<0.05), without affecting capacitation or protein tyrosine phosphorylation, indicating the importance of calcium efflux for maintaining progressive motility. RU 360 (5 µM) significantly reduced capacitation without affecting progressive motility, sperm viability or protein tyrosine phosphorylation, showing that inhibition of the mitochondrial calcium uptake, negatively affect the capacitation process. The addition of both inhibitors showed the effect of RU 360. According with these results, there would exist a differential participation of the income and outcome mitochondrial calcium carriers, in the capacitation process. In conclusion, this research demonstrates the importance of normal mitochondrial calcium cycle in the achievement of sperm capacitation and the maintenance of progressive motility in cryopreserved bovine spermatozoa.