Relation between respiratory activity and sperm parameters in boar spermatozoa cryopreserved with alpha-tocopherol and selected by Sephadex.

Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo-osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.

Participation of phosphofructokinase, malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa.

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.

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.

Phosphofructokinase and malate dehydrogenase participate in the in vitro maturation of porcine oocytes.

Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2-oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10(-5) and (2.54 ± 0.32) 10(-5) , and for MDH, the U were (4.72 ± 0.42) 10(-5) and (4.38 ± 0.25) 10(-5) for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10(-3) and (0.94 ± 0.12) 10(-3) , and for MDH (9.08 ± 0.93) 10(-3) and (1.89 ± 0.10) 10(-3) for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2-oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm.

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.

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.

Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm.

The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

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.

In vitro evaluation of the quality and fertilizing capacity of boar semen frozen in 0.25 ml straws.

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.

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.

Interaction of proteins with spherical polyelectrolyte brushes in solution as studied by small-angle x-ray scattering.

We use small-angle x-ray scattering (SAXS) as a tool to study the binding of proteins to spherical polyelectrolyte brushes (SPB) in situ. The SPB consists of a solid core of approximately 100 nm diam onto which long polyelectrolyte chains [poly(styrene sulfonic acid, PSS) and poly(acrylic acid, PAA)] have been densely grafted. The proteins used in this investigation, Bovine Serum Albumine (BSA) and Bovine Pancreatic Ribonuclease A (RNase A), adsorb strongly to these SPB if the ionic strength is low despite their negative charge. Virtually no adsorption takes place at high ionic strength. SAXS demonstrates that both proteins are distributed within the brush. The findings reported here give further evidence that the strong adsorption of proteins to SPB is due to the "counterions release forces": The patches of positive charge on the surface of the proteins become multivalent counterions of the polyelectrolyte chains. Thus, a concomitant number of co- and counterions is thereby released and the entropy of the entire system is increased. The repulsive Coulombic interaction as well as the steric repulsion between the proteins and the brush layer are counterbalanced by this effect. The data discussed here demonstrate that the adsorption of proteins in SPB presents a new principle for the immobilization of proteins.