Requirement of an extracellular energy substrate for the guinea pig sperm acrosome reaction induced by calcium ionophore

It is well established that calcium ionophore A 23187 induces acrosome reaction (AcR) of uncapacitated spermatozoa in the presence of extracellular Ca2+ ions. In the present study, we have investigated how extracellular energy substrates (glucose, pyruvate, and lactate) affect the ionophore-induced AcR of guinea pig spermatozoa. It was found that 0.3 microM concentration of A 23187 had the maximum effect to initiate AcR of guinea pig spermatozoa. Virtually no spermatozoa underwent their AcR when incubated in substrate-free modified Tyrode's medium containing 0.3 microM A 23187 and 2 mM Ca2+. At least one exogenous substrate is essential for the ionophore-induced AcR of spermatozoa. As for efficacy of the substrates, lactate was more effective than pyruvate and glucose. However, a better result was observed when lactate was added along with pyruvate. Malonate inhibited the ionophore-induced AcR but not the hyperactivated motility of spermatozoa. The mitochondrial electron transport chain blockers rotenone, antimycin, and oligomycin failed to inhibit AcR, although in the presence of these blockers spermatozoa were unable to show hyperactivated motility. These results suggest that the mitochondrial citric acid cycle, not the electron transport chain, is probably the energy source for ionophore-induced AcR of guinea pig spermatozoa.     

Biochemical and immunochemical characterization of boar sperm flagellar protein with role in hyperactivation/capacitation process

Investigations on specific and functionally active sperm antigens could bring about the elucidation of the mechanisms of gamete interaction and help the search for new approaches in prognosis and regulation of fertility. Previously, we reported that the monoclonal antibody (Mab) 3G4 against capacitated boar spermatozoa was capable of inhibiting boar sperm-porcine zona pellucida binding due to its inhibitory effect on sperm hyperactivation and capacitation. The cell and tissue specificity of Mab 3G4 was demonstrated in indirect immunofluorescence (IIF) and ELISA experiments against spermatozoa from different vertebrate species, as well as against extracts of boar reproductive and somatic organs. In the present IIF experiments, it was shown that Mab 3G4 recognized an antigen determinant on the flagellar midpiece region of ejaculated and capacitated boar spermatozoa. It was speculated that the Mab 3G4-corresponding antigen participates in pyruvate/lactate metabolism because of its specific localization in the sperm structure, which is responsible for producing forward motility and its involvement in processes that require the metabolism of pyruvate and lactate. As a possible approach toward investigating the participation of Ag 3G4 in pyruvate/lactate metabolism, Mab 3G4's effect on lactate dehydrogenase (LDH) was examined. Using an electrophoretic approach we provided evidence that Mab 3G4 stimulates LDH activity in the Triton X-100 and NP40 protein fractions of capacitated boar spermatozoa. In addition, we found that LDH isoenzymes stimulated by Mab 3G4 are of gametic C type. In Western blot, under nonreducing conditions, Mab 3G4 identified a single protein band with a molecular weight of 140 kDa. The biochemical and immunochemical experiments provided evidence supporting the involvement of 3G4 antigen in the sperm pyruvate/lactate metabolism.     

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.     

Selective inhibition of pyruvate and lactate metabolism in bovine epididymal spermatozoa by dinitrophenol and alpha-cyano-3-hydroxycinnamate

The disparity between the effects of the uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) on pyruvate metabolism in bovine spermatozoa has been characterized. In bovine epididymal spermatozoa metabolizing pyruvate, the uncouplers of oxidative phosphorylation, DNP (100 μm) and FCCP (0.4 or 5 μm), decreased the intracellular ATP concentration from 30 to ～10 nmol/10⁸ cells. Both uncouplers decreased, but did not abolish, sperm motility. DNP strongly inhibited pyruvate metabolism and stimulated the appearance of free carnitine from the acetylcarnitine pool. In contrast, FCCP enhanced the oxidation of pyruvate, diminished the reduction of pyruvate to lactate, and permitted the maintenance of the normal amount of acetylcarnitine. The effects of DNP and FCCP on mitochondrial pyruvate metabolism were examined in spermatozoa treated with filipin, which renders the plasma membrane permeable to small molecules. In these cells, DNP inhibited metabolism and respiration with pyruvate or lactate, but did not affect respiration supported by acetylcarnitine. Similarly, the pyruvate translocase inhibitor, α-cyano-3-hydroxycinnamate, markedly decreased the rate of metabolism of both pyruvate and lactate. With maximally inhibitory concentrations of DNP or α-cyano-3-hydroxycinnamate, the rates of pyruvate use and lactate use were the same. Metabolism of both lactate and pyruvate and production of ATP were inhibited by similar concentrations of DNP ($\text{I}{}_{50}\text{? 7}\text{μM}$). A common mitochondrial translocase for pyruvate and lactate in bovine spermatozoa is posited. This translocase is inhibited by minimally effective uncoupling concentrations of DNP.     

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.     

Sperm metabolism of the telost fishes Chalcalburnus chalcoides and Oncorhynchus mykiss and its relation to motility and viability.

In the teleost fish Chalcalburnus chalcoides (Cyprinidae) the influence of metabolic inhibitors, substrates, coenzymes, and oxygen concentrations on spermatozoal parameters during motility and during immotile incubation was studied, the respiration rate was characterized, representative metabolite levels were measured, and the results were compared with Oncorhynchus mykiss (Salmonidae). In Chalcalburnus chalcoides the sperm motility rate, the average path swimming velocity, the motility duration, and the viability of immotile semen were significantly reduced in the presence of inhibitors of respiration (potassium cyanide, 2.4-dinitrophenol, atractyloside). Anaerobic conditions (<1 mg O(2)/liter) and inhibition of the tricarboxylic acid cycle by malonate and >7.5 mmol/liter succinate had similar effects on the sperm motility parameters and on the viability of immotile spermatozoa. Pyruvate and coenzyme A (an acyl-group carrier during oxidative carboxylation of pyruvate) prolonged the duration of sperm motility and the viability of immotile incubated spermatozoa, and also increased the spermatozoal respiration rate. Glucose levels significantly decreased during motility and during immotile storage and, under anaerobic conditions, the levels of lactate increased indicating that pyruvate derived from glycolysis. The respiration rate and the glycolytic rate significantly increased during motility. Therefore oxidative phosphorylation, tricarboxylic acid cycle, and aerobic glycolysis are central energy-supplying pathways for spermatozoa of Chalcalburnus chalcoides. The stimulatory effect of pyruvate and coenzyme A indicated that glycolysis is a rate-controlling pathway. Similar results were obtained for Oncorhynchus mykiss with the only exception that the stimulatory effect of coenzyme A was more significant than the stimulatory effect of pyruvate. When the sperm motility-activating saline solutions were optimized in aspects of energy supply, ionic composition, and osmolality, about 50% of the motile spermatozoa swam progressively (>20 mm/sec) for about 3 min in Chalcalburnus chalcoides and in Oncorhynchus mykiss. About 20% swam progressively for >2 hr in Chalcalburnus chalcoides and for >30 min in Oncorhynchus mykiss. J. Exp. Zool. 284:454-465, 1999.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Survival of ram testicular spermatozoa invitro: Effects of glucose,glucose metabolites,rete testis fluid-proteins,selected androgens and phospholipids

The effects of glucose, glucose metabolites, protein-enriched rete testis fluid (RTF), selected androgens and phospholipids on the survival of testicular spermatozoa $\text{in}$$\text{vitro}$ have been studied. The oxidative and glycolytic activity, motility and percentage of live cells were the criteria for assessing the viability of the spermatozoa washed free of the substances that had been added during storage. The addition of lithium lactate, sodium lactate and sodium pyruvate but not glucose was beneficial to the survival of testicular spermatozoa. Following 10 to 12 days storage at 4°C with added lactate or pyruvate testicular spermatozoa had a higher glycolytic activity than did control spermatozoa. The respiratory activity of stored testicular spermatozoa was maintained or depressed, depending on the density of spermatozoal suspension during storage. After 10 to 11 days storage in concentrated RTF or after exposure to selected androgens testicular spermatozoa utilized more glucose than after storage with lactate alone. However, this apparent response to androgens and RTF-proteins was the consequence of a higher survival rate of the spermatozoa rather than an increase in the metabolic activity of individual spermatozoa. These results indicate that metabolites of glucose may serve as substrates for spermatozoa in the epididymis while certain androgens and macromolecules occurring in reproductive tract fluids may play important roles in the survival of spermatozoa during their period of maturation in the epididymis.     

In vitro capacitation of goat spermatozoa

Goat epididymal and ejaculated spermatozoa were incubated in Krebs-Ringer bicarbonate buffer containing pyruvate and lactate as energy source. A 3 hr incubation for epididymal and 4 hr for ejaculated spermatozoa was required for the capacitation and acrosome reaction to take place. Calcium is an essential requirement which was needed for motility maintenance/activation and for the initiation of acrosome reaction. A 2-fold increase in cAMP content was measured over 3 hr period of incubation of goat epididymal spermatozoa which was not seen when calcium ions were either omitted or chelated with EGTA. There is thus a definite involvement of Ca2+ ions and cAMP in capacitation and acrosome reaction of goat spermatozoa.     

Sulfhydryl groups in relation to the metabolism and motility of human spermatozoa

1. The motility and metabolism of human spermatozoa are inhibited by substances which have an affinity for sulfhydryl groups. 2. These inhibitions can be prevented, and in part, reversed, by the addition to the cell + inhibitor system of sulfhydryl compounds such as cysteine or glutathione. 3. Cysteine and glutathione, under aerobic conditions or in a system in which these substances can be oxidized, show widely different effects on the motility of the spermatozoa. Cysteine destroys the motility of the spermatozoa, whereas glutathione has no effect upon it. 4. Possible mechanisms of these effects are discussed.     

Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility

Loss of forward motility of rabbit epididymal spermatozoa in high K+ phosphate buffer is inhibited by taurine, hypotaurine, epinephrine and bovine serum albumin. Pyruvate and lactate also show this effect. The rate of lipid peroxidation in these spermatozoa, as measured by rate of formation of malondialdehyde, is also inhibited by these agents. A close linear correlation between percent inert spermatozoa and malondialdehyde was found, which was independent of the rate of peroxidation. Complete cessation of motility was observed at 0.5 nmol malondialdehyde/10(8) cells in the absence or presence of these agents, which is the same value found in other suspending media in a previous study [Alvarez and Storey (1982) Biol. Reprod. 27:1102-1108]. Albumin was the most effective agent in preventing loss of motility and inhibiting lipid peroxidation. Hypotaurine was the next most effective, followed by taurine, epinephrine, pyruvate and lactate. Hypotaurine reduces the amount of rate of superoxide production, as measured by the rate of reduction of acetylated ferricytochrome c by O(2), from rabbit sperm under these conditions and concomitantly reduces inactivation of the superoxide dismutase in these cells. Since superoxide seems to be the major inducer of lipid peroxidation in rabbit sperm, the protective effect of hypotaurine, which should be readily permeant to the plasma membrane, may be ascribed to scavenging of intracellular superoxide. The mechanism of the protective action of albumin is not known. Rabbit epididymal spermatozoa lose motility over time if Ca2+ or Mg2+ are omitted from the suspending medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa

Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.     

Mechanism for procaine-mediated hyperactivated motility in guinea pig spermatozoa

Hyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine-treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca²⁺ did not inhibit procaine-induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca²⁺ for sperm to express hyperactivated motility. © 1994 Wiley-Liss, Inc.     

Cloacal gland foam enhances motility and disaggregation of spermatozoa in Japanese quail (Coturnix japonica)

The adult male Japanese quail produces white foam from the cloacal gland, which is transferred to the female proctodeum during natural mating. The physiological role of foam on quail spermatozoa is still unclear. Therefore, attempts have been made to understand the effect of cloacal foam on motility and metabolism of quail spermatozoa. The profile of various biochemical constitutes in the foam extract was investigated. The addition of foam extract to neat semen completely disaggregated the clumps of spermatozoa leading to vigorous motility. The metabolic rate (MBRT) of the spermatozoa was significantly increased with the addition of foam extract. The foam extract was sub fractionated into seven different fractions by using the molecular cut off devices. Among all the seven sub-fractions from the foam extract, the addition of < 1 KDa sub-fraction contained lactate and has enhanced sperm motility and metabolism. Another fraction (3-10 KDa) has non-protein and non-heparin components which completely disaggregated the clumped quail spermatozoa. However, the remaining fractions did not show any effect on quail spermatozoa. It can be concluded from the present investigation that the lactate present in foam might be a fuel for sperm metabolism and motility. Furthermore, low molecular weight (3-10 KDa) components in the foam may responsible for sperm disaggregation.     

Maturation and regulation of the motility of spermatozoa in the epididymis of the tammar wallaby (Macropus eugenii).

Demembranated spermatozoa from the rete testis developed vigorous flagellation when reactivated with ATP, but showed no forward progression such as that seen in samples from the cauda epididymidis. The proportion of spermatozoa that were reactivated was smaller for samples from the rete testis than from the cauda epididymidis. Studies in vitro of undiluted micropuncture samples from the epididymis indicated that the activity of spermatozoa is suppressed as they develop the capacity for motility. However, as spermatozoa spontaneously became activated during the collection or subsequent incubation of undiluted samples, it was concluded that the suppressive action is labile. The activity of spermatozoa in vitro was examined in diluted samples from the cauda epididymidis. A concentration of 2.5 mmol extracellular calcium/l was better than lower concentrations. Diluents at pH 5.5 completely inhibited sperm motility when they contained 20 mmol lactate/l (but not glutamate) and the effect was reversed by readjusting the diluent to pH 7.4. However, lactate was not considered to suppress sperm motility in situ, as the plasma from the cauda epididymidis contained only 2.7 +/- 0.5 mmol lactate/l. There was no effect of sodium concentration (1 and 115 mmol/l), pH (5.5 and 7.4) or amiloride (0 and 1 mmol/l) on sperm motility, indicating that motility is not dependent on the concentration of sodium above 1 mmol/l or on a sodium-proton exchange system. The relative viscosity of plasma from the cauda epididymidis did not affect the motility of spermatozoa.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica)

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.     

Cryogenic preservation of fish and mammalian spermatozoa

Various combinations of sucrose, reduced glutathione and potassium bicarbonate were tested for the cryogenic preservation of salmon spermatozoa. When a fast freezing procedure was followed, the extender that gave the best results was composed of 1 part of dimethyl sulphoxide (DMSO), as a protective agent, and 7 parts of a medium containing 125 mM-sucrose, 6.50 mM-reduced glutathione and 100 mM-potassium bicarbonate. Salmon and cod spermatozoa were kept frozen in this extender for 1 year. Freezing resulted in a reduction in the number of motile spermatozoa but had no significant effect on the degree of progression of motile spermatozoa or on their fertilizing capacity. When glycerol replaced DMSO in the extender and a slow freezing procedure was followed, similar results were obtained for the spermatozoa of bull or man; although the number of motile spermatozoa was reduced, there was no effect on the progressive motility score.     

Ammonia effects on pyruvate/lactate production in astrocytes--interaction with glutamate

Ammonia exerts a multitude of metabolic and non-metabolic effects on brain tissue. In the present communication we have investigated its effect on lactate production rates, pyruvate production rates and pyruvate/lactate ratios in mouse cerebrocortical astrocytes and neurons in primary cultures. No effects were found in neurons. All three parameters were affected by ammonia in astrocytes, but less potently and to a smaller degree in cells that had been treated with dibutyryl cyclic AMP (morphologically differentiated cells) than in untreated cells (morphologically undifferentiated cells). In the differentiated cells ammonia had virtually no effect up to a concentration of 1.0 mM, but at 3.0 mM it increased lactate production and decreased pyruvate/lactate ratio significantly. In the undifferentiated cells ammonia greatly increased lactate accumulation (by 80% at 3.0 mM) and it inhibited pyruvate accumulation (by 40% at 3.0 mM). It thereby reduced the pyruvate/lactate ratio progressively within the entire range 0.1-3.0 mM ammonia. In support of the hypothesis that the ammonia-induced reduction of pyruvate/lactate ratio is secondary to depletion of cellular glutamate by formation of glutamine (and glutathione) and a resulting interruption of the malate-aspartate shuttle (MAS), the addition of glutamate to the incubation medium significantly diminished the ammonia-induced reduction of pyruvate/lactate ratio, whereas it had no effect on the increased lactate production. It is discussed that MAS interruption may have additional consequences in astrocytes.