Metabolism and motility of turkey (Meleagris gallopavo) spermatozoa in the presence or absence of oxygen, glucose and fructose

Sugar consumption by turkey spermatozoa for 1 hr at 37 degrees C was similar in the presence or absence of oxygen, and with glucose or fructose in the medium. Motility of the spermatozoa at the end of the above incubation period was lower under anaerobic than aerobic conditions. Fructose enhanced the oxygen uptake of spermatozoa in comparison with that in the glucose-containing medium. Omission of sugar from the medium depressed respiration but not motility of the spermatozoa. The rate of oxygen uptake by the spermatozoa during a 3-hr incubation period was higher in a 3.3-mM than a 15.0-mM glucose medium. Fructose was formed from glucose under aerobic but not under anaerobic conditions. Fructose originating from glucose was used for fructolysis, when the glucose reserve in the medium was almost exhausted.     

Alterations of oxygen uptake and the redox state of ubiquinone in rabbit sperm exposed to a variety of physiologic treatments

The rate of TEMPONE reduction by electrons originating from ubiquinone in intact rabbit spermatozoa was observed for control, high ionic strength (HIS) medium-treated, and HIS-seminal plasma-treated (HIS-SP) samples. The presence of TEMPONE in the incubation medium had no effect on oxygen consumption, demonstrating the utility of TEMPONE as a nonperturbing probe of the ubiquinol redox state. The rate of TEMPONE reduction was significantly increased over control levels for sperm incubated in hypertonic medium and was correlated to a decrease in oxygen consumption and a relative increase in ATP in the total adenine nucleotide pool. This increase in TEMPONE reduction in HIS sperm was reversed by treatment of sperm with seminal plasma, but seminal plasma had no effect on oxygen consumption or relative amounts of ATP in the adenine nucleotide pool. These observations are consistent with state 3 respiration in control sperm and state 4 respiration in HIS- and HIS-SP-treated sperm. Arrhenius data were obtained for ejaculated and epididymal sperm subjected to a variety of treatments. Lines fitted to plots of Arrhenius data revealed that each treatment affected the activation energy and intercept relative to controls. Evidence is presented for a phase transition occurring at 13 degrees C based on changes in the rate of TEMPONE reduction by ubiquinol. It was noted that, above the phase transition, rate constants for the reaction were dependent upon both treatment and temperature, but below the transition the differential effects of treatment were no longer apparent. The present study has demonstrated that events taking place in the respiratory chain can be closely monitored by measuring oxygen uptake and TEMPONE reduction, and that these events are affected by alterations in the sperm environment.     

Respiratory metabolism of the semen of the honey-bee, Apis mellifera

Mean respiratory quotients determined on samples from individual drones by Cartesian diver respirometry averaged 0·64, suggesting that phospholipids constitute the major source of energy. The average sperm density of semen was 7·76 millions/μl. Dilution of semen increased the rate of oxygen consumption by 68 per cent. Semen stored at 13 to 14°C for 1 month showed only 40 per cent of the oxygen uptake of freshly ejaculated semen. Streptomycin sulphate treated samples of semen showed significantly lower rates of oxygen consumption than untreated samples. These results suggest that the high density of sperm in the semen and low metabolic activity during storage may be at least partly responsible for the successful long-term storage of honey-bee spermatozoa.     

Respiration of bull spermatozoa in presence of exogenous glutathione (GSH)

In the investigations of the rate of oxygen uptake by sperm cells in frozen bull semen it was found that exogenous glutathione (GSH) in a 5 mM concentration failed to stimulate mitochondrial oxidative processes in the sperm cells. At the same time it was observed that the respiration of sperm cells changed in relation to the season of the year when ejaculates had been collected and preserved. The highest values were noted in winter and the lowest ones in summer. Moreover, in the observations on the motility of spermatozoa a difference was observed in the reaction of sperm cells to exogenous GSH, and this difference depended on the season. A significant (p less than or equal to 0.05) rise was disclosed in the number of sperm cells with maintained advancing motility in the presence of GSH in the medium as compared to controls without GSH, but this rise was found only in semen obtained and preserved in winter.     

A thermodynamic study of sperm-egg interaction.

We have studied the binding of spermatozoa to the receptor sites on the vitelline coat (VC) of glycerol-treated eggs (ghost eggs) of the Ascidian, Ciona intestinalis (Protochordate). Glycerol treatment cytolyses the egg without affecting the ability of the VC to bind spermatozoa in a species-specific manner; however, in this system binding is not followed by the acrosome reaction. The ghost eggs are metabolically inert. As a base line for our analysis, we have studied the concentration-dependent heat evolved and oxygen consumption of spermatozoa when diluted in sea water. The process has been analyzed on the basis of equations derived by Liquori and Tripiciano to describe cell growth. Upon binding to the ghost eggs, the spermatozoa produce an explosive heat evolution (excess heat) which is not accompanied by oxygen consumption. The excess heat produced plotted against sperm concentration (at constant egg concentrations) gives an asymmetric bell-shaped curve. This is interpreted as being due to the competitive effect of sperm agglutination at a high sperm concentration. It is concluded that only spermatozoa that attach singly (monomeric spermatozoa) to the egg undergo metabolic activation.     

Sonication per se is not as deleterious to sperm chromosomes as previously inferred

Although sonication is a simple way to immobilize ("kill") spermatozoa prior to injection into oocytes, this has been thought to be destructive to sperm chromosomes. Mouse and human spermatozoa were immobilized by sonication and kept in various media for up to 2 h, then their nuclei were individually injected into mouse oocytes for the analysis of chromosomes at the first cleavage metaphase. In both the mouse and human, incidence of structural chromosome aberrations was much higher in the spermatozoa sonicated and stored in Biggers-Whitten-Whittingham medium for 2 h at 37.5 degrees C than in those stored for 5 min in the same medium. We concluded, therefore, that it is not sonication per se but a prolonged exposure of sperm nuclei to extracellular milieu that is detrimental to sperm chromosomes. The incidence of structural chromosome aberrations of mouse and human spermatozoa was significantly reduced when the spermatozoa were sonicated and stored in K(+)-rich nucleus isolation medium containing EDTA. This suggests that sperm chromosome degradation following sperm immobilization by sonication is partly due to detrimental effects of a Na(+)-rich medium and of DNase on sperm chromatin. Ideally, it should be possible to prepare artificial media that maintain the integrity of sperm chromosomes for many hours after immobilization.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Oxygen consumption of mouse sperm and its relationship to capacitation

The oxygen consumption of spermatozoa from the caudae epididymides of the mouse was measured in various media and in the presence or absence of substrates and inhibitory agents in order to investigate the relationship between oxidative metabolism and capacitation. When washed sperm were placed in a medium in which capacitation occurs, respiration increased abruptly, due to the availability of oxidizable substrates, and remained at a high, constant level throughout the period of measurement. There was no temporal change in rate during the time when capacitation was occurring, nor was the rate after capacitation had occurred higher than it was before. Capacitation does not occur in a medium in which polyvinyl alcohol is substituted for bovine serum albumin or to which ethyl alcohol is added. Prevention of capacitation by these means had no effect on the rate of oxygen consumption. The results support the conclusion that the increased respiration of sperm in a capacitating medium is due to the presence of oxidizable substrates and, as such, is an accompaniment of the process of capacitation rather than a factor in bringing it about.     

Mechanochemical Coupling in Flagella : II. Effects of viscosity and thiourea on metabolism and motility of Ciona spermatozoa

The relation between oxygen consumption and motility of Ciona spermatozoa has been measured by using pH stats to measure the acid production of spermatozoa swimming in dilute suspensions where their motility can be analyzed accurately, and calibrating the acid production by measuring it simultaneously with measurements of oxygen consumption, using more concentrated sperm suspensions. When the motility of the spermatozoa is inhibited by thiourea or by increased viscosity, their oxygen consumption decreases in proportion to the decrease in beat frequency. 80–85 % of their oxygen consumption appears to be tightly coupled to motility. The amount of movement-coupled oxidative metabolism per beat remains nearly constant, even when there are significant changes in the energy required per beat for movement against the viscous resistance of the medium. This implies that under these conditions, where the radius of curvature of flagellar bending remains constant, the amount of ATP used is determined by a stoichiometric relation to bending rather than by the energy requirement. The movement-coupled oxidative metabolism appears to be sufficient to generate approximately two molecules of ATP per beat for each molecule of the flagellar ATPase, dynein.     

Maintenance of motility of fowl spermatozoa in vitro is prolonged by a low molecular weight factor derived from cultured chick embryo cells.

Gel filtration of a conditioned medium composed of the supernatant fluid removed from a 5-day culture of skeletal muscle cells from 9-day-old chick embryos with Bio-Gel P-2 revealed one peak of motility-prolonging activity (about 0.3 kDa), which was not present in fresh medium. Spermatozoa incubated in this fraction of the conditioned medium maintained their motility for at least 36 h at 37 degrees C. Both the formation of lipid peroxide and the leakage of lactic dehydrogenase of spermatozoa incubated in the conditioned medium fraction were lower than those incubated in the corresponding fresh medium. Initial rate of oxygen consumption of the spermatozoa incubated in the conditioned medium fraction increased compared with that of the fresh medium fraction. These results suggest that a low molecular weight factor(s) supplied by cultured cells effectively prolongs the motility of fowl spermatozoa, and that the effect could result from inhibition of the structural damage to the sperm membrane.     

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.     

Interactive effects of organic and inorganic selenium with cadmium and mercury on spermatozoal oxygen consumption and motility in vitro

The effects of cadmium (Cd), mercury (Hg), and three different chemical forms of selenium (Se) (selenite, selenocystine, and selenomethionine) on ram spermatozoal motility and oxygen consumption in vitro were studied over a 4-mo period. Concentrations of 10(-6) to 10(-2) M Cd and Hg were injurious to spermatozoa as indicated by depressed motility and reduced oxygen uptake. Equimolar concentrations of Se as selenite, selenocystine, or selenomethionine counteracted the toxicity of Cd and Hg at low concentrations (10(-5) and 10(-6) M) but not at higher concentrations (10(-4) to 10(-2) M). Gel filtration (Sephadex G-75) of seminal plasma and solubilized sperm prepared from semen incubated with Cd or Hg with or without the Se compounds revealed that Cd or Hg eluted with the void volume proteins in all treatments. Incubation of ram spermatozoa with any of the three chemical forms of Se ranging from 10(-6) to 2.5 X 10(-5) M significantly improved sperm motility and oxygen consumption.     

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation.Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24 h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24 h post activation.A forward progressive movement was maintained for at least a 20 h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6 h post activation and severe alterations were observed in sperm morphology after 24 h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase.The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.     

Calcium alters capacitation and progressive motility of uterine spermatozoa from +/+ and congenic tw32/+ mice.

The importance of calcium-dependent sperm processes for fertilization in vitro is well known, but their interaction with sperm transport in vivo is not yet clear. To determine whether exposure to calcium alters sperm physiology after incubation in the uterus, spermatozoa from +/+ mice were incubated in medium with 1.7 mM calcium prior to artificial insemination (AI). Spermatozoa from congenic tw32/+ mice were also tested because their flagella are hypersensitive to calcium. As a control, spermatozoa were incubated in calcium-deficient medium before AI. When recovered from the uterus 60 min post-AI, neither prior exposure to calcium nor genotype affected numbers of spermatozoa, or percentage of motile or acrosome-reacted spermatozoa. However, significantly more calcium-treated spermatozoa were capacitated and significantly fewer were progressively motile than spermatozoa preincubated without calcium. In addition, significantly fewer spermatozoa from tw32/+ mice than from +/+ mice were progressively motile. These results suggest that uterine sperm physiology is changed by prior exposure of sperm to calcium. Since the level of progressive motility of spermatozoa recovered from the uterus was correlated with their ability to reach the oviduct (as determined in a previous study), these data support the hypothesis that progressive motility of uterine spermatozoa is important for passage to the oviduct and fertility.     

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca²⁺. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca²⁺ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca²⁺ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca²⁺ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca²⁺ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.     

Effect of anti-sperm gamma globulins on metabolism of human spermatozoa.

Rabbits were immunized with saline extracts of human spermatozoa Presence of antibodies to spermatozoa was confirmed by Ouchterlony gel diffusion, microscopic sperm agglutination, and immunofluorescent techniques. Anti-sperm gamma globulin significantly decreased the average oxygen consumption of 10⁸ washed human spermatozoa. Sperm incubated in the presence of anti-sperm gamma globulin had a significantly lower rate of lactic acid accumulation than sperm incubated in normal rabbit gamma globulin. It appears that anti-sperm antibodies can influence both aerobic respiration and glycolysis of washed human spermatozoa.     

Male infertility and mitochondrial DNA

The mitochondrial machinery plays a key role in the energy production and maintenance of spermatozoa motility. In this paper 200 idiopathic oligo-asthenozoospermic patients were classified on the basis of rapid progressive motility ("a") and sperm concentration. Mitochondrial enzymatic activity was studied and correlated to the viability of sperm cells. Mitochondrial DNA purified from both motile and non-motile sperm of the same individuals was amplificated using PCR. Results suggested that only motile sperm have organelles functional in oxygen consumption, unequivocally demonstrating that motility depends on the mitochondrial activity. Mitochondrial DNA of oligo-asthenozoospermic patients seemed to present some defects that made DNA unavailable for amplification.     

Ionic factors affecting motility,respiration and fertilization rate of the sperm of the bivalvePecten maximus (L.)

Fertilization of the scallopPecten maximus occurs after gametes were naturally released in sea water by the bivalve which has undergone stimulation. The motility of the spermatozoa requires their dilution in sea water (1/40). Dilution triggers an immediate increase of oxygen consumption by sperm, reflecting an activation of a cyanide-sensitive respiration of a cellular origin. When scallops were stimulated by thermal shocks or by serotonin injection, sperm sampled at the urogenital pore output duct shows a respiration-motility activation after sea water dilution which is not seen in sperm scarified from the gonad. Dilution of kidney-sampled sperm into acidic (pH 5) or Na⁺-free artificial sea water reversibly inhibits both respiration and motility. In all cases fertilization rate of sperm is correlated to the increase of respiratory rate and motility measured after dilution in different media. Whether the scallop was stimulated or not, the pH of haemolymph and pericardic fluids were one pH unit below the value of sea water, the pH of the gonad and of the kidney tissues being more acidic (6.5 in average). Our results suggest that the acidic pH of the genital tract maintains the spermatozoa in a quiescent state and that capacitation occurs when male gametes move from the gonad to the kidney from where it is naturally released.Abbreviations ASW artificial sea water - SW sea water - TRIS trishydroxymethyl-aminomethane     

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

The purpose of the present study was to investigate the optimal concentration of osmolarity, calcium and bicarbonate for sperm penetration and formation of pronuclei (PN), and to investigate the time required for capacitation, penetration across the zona pellucida and formation of PN in bovine cumulus-free oocytes matured in vitro. Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro. Bovine sperm penetrated the zona pellucida in medium containing 240 to 440 mOsm, whereas PN formation was observed in a narrow range of osmolarities, from 280 to 360 mOsm. Maximal penetration by spermatozoa and PN formation was obtained in the medium with 2.5 mM calcium. High rates of spermatozoa penetration were observed in the medium with 37 to 49 mM NaHCO3. However, PN were formed regardless of the concentration of NaHCO3. The times required for sperm capacitation and penetration through the zona pellucida were 260 and 50 min, respectively. The first development of PN was recorded at 120 min after sperm penetration. Therefore, our study suggests that fertilization ability of spermatozoa in vitro appears to be more stable in high concentrations of NaCI. Oocytes are more sensitive to osmotic stress than spermatozoa. Calcium is required for both sperm penetration and PN formation in cumulus-free oocytes, but bicarbonate may be needed mainly for the penetration of spermatozoa.     

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.