Activation of bovine epididymal sperm respiration by caffeine: Its transient nature and relationship to the utilization of acetyl carnitine

Caffeine, which stimulates the motility of freshly extruded bovine epididymal spermatozoa, caused a large but transient increase in the respiratory activity of these cells incubated in a modified Ringer buffer without exogenously added substrate. In spermatozoa that were incubated without added substrate for 2 h at 30 °C or for 15 min at 37 °C, caffeine addition failed to increase respiratory activity even transiently. However, subsequent addition of pyruvate to these aged and caffeine-treated cells resulted in a rapid increase in the respiratory rate, nearly equal to that observed after caffeine addition to fresh cells or to cells stored at 4 °C. These observations indicate that the loss in metabolic response to caffeine is a result of the active metabolism of the spermatozoa.In freshly prepared sperm that were incubated without added substrate, the acetyl carnitine content declined and the free carnitine content of the sperm increased in amounts sufficient to account for the entire respiratory increment produced by caffeine addition. Respiratory stimulation by caffeine was sustained in the presence of those exogenously added substrates that are capable of entering the acetyl carnitine pool, such as acetate, pyruvate, l(+)-lactate, glucose, fructose or β-hydroxybutyrate. Tricarboxylic acid cycle intermediates were not effective.These observations clarify the relationship between the stimulatory effects of caffeine and the metabolic state of the spermatozoan and suggest the importance of the acetyl carnitine pool to the activation of sperm motility and oxidative metabolism.     

Accumulation of carnitine esters of beta-oxidation intermediates during palmitate oxidation by rat-liver mitochondria

Rat-liver mitochondria were incubated with [14C]palmitate in the presence of L-malate, fluorocitrate, and L-carnitine. The specific activities of acetyl groups incorporated into citrate, ketone bodies and acetyl-L-carnitine were measured. During state-4 oxidation of [1--14C]palmitate the specific activity of the acetyl-CoA pool was 1.3-times higher than that of the average acetyl group of palmitate, indicating an incomplete breakdown of the palmitate molecule. Accumulation of carnitine esters was observed in this condition. The acyl moieties of carnitine esters formed during the state-4 oxidation of [U-14C]palmitate or [16(-14)C]palmitate were analysed by radioactive gas-chromatography. Substantial amounts of beta-oxidation intermediates were found. The accumulation of carnitine esters of C6-C14 intermediates can quantitatively explain the high specific activity of the acetyl-CoA pool during the state-4 oxidation of [1(-14)C] palmitate. The localization and control of beta-oxidation are discussed.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.     

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.     

Calcium transport in bovine sperm mitochondria: effect of substrates and phosphate.

Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH.     

Cryopreservation of epididymal stallion sperm

Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters.     

Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.

The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.     

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.     

Clearance of exogenous carnitine in bovine semen: Potential diagnosis of epididymal function and patency of the ductus deferens

Carnitine content in the ejaculate depends mainly on the capability of the epididymis wall to transfer carnitine from the blood and on the patency of ejaculatory ductus systems. An elevation of carnitine in semen subsequent to an intravenous injection of carnitine is expected. Intravenous injections of carnitine (L-isomer and DL-isomers) caused a significant (P <0.05) elevation (more than 10-fold) in blood carnitine. However, carnitine injection failed to increase net secretion of carnitine into the ejaculate and blood elimination half-life was 2.3 hours. Mean concentrations of carnitine in the electroejaculate (3.0 nmoles/ml) were significantly lower than in the ejaculate following natural mating (180 nmoles/ml). Vasectomy decreased net carnitine per ejaculate to about 1/5 the prevasectomy value, when ejaculate was collected following natural mating. However, vasectomy did not affect carnitine concentrations in semen collected by electroejaculation. Twenty-one percent of the carnitine in semen originated in the accessory glands and 79% in the epididymides. Carnitine in the electroejaculate was originated almost exclusively in the accessory glands. It was concluded that the diagnostic value of carnitine in semen is limited. Some considerations are: secretion of carnitine is not organ specific, there are large individual variations, there is a negative effect of electroejaculation, and a carnitine loading dose technique is not feasible. However, there is a diagnostic potential in using carnitine assay to detect epididymides occlusion, but only when ejaculate is collected by an artificial vagina.     

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.     

Role of carnitine esters in brain neuropathology

L-Carnitine (L-C) is a naturally occurring quaternary ammonium compound endogenous in all mammalian species and is a vital cofactor for the mitochondrial oxidation of fatty acids. Fatty acids are utilized as an energy substrate in all tissues, and although glucose is the main energetic substrate in adult brain, fatty acids have also been shown to be utilized by brain as an energy substrate. L-C also participates in the control of the mitochondrial acyl-CoA/CoA ratio, peroxisomal oxidation of fatty acids, and the production of ketone bodies. Due to their intrinsic interaction with the bioenergetic processes, they play an important role in diseases associated with metabolic compromise, especially mitochondrial-related disorders. A deficiency of carnitine is known to have major deleterious effects on the CNS. Several syndromes of secondary carnitine deficiency have been described that may result from defects in intermediary metabolism and alterations principally involving mitochondrial oxidative pathways. Mitochondrial superoxide formation resulting from disturbed electron transfer within the respiratory chain may affect the activities of respiratory chain complexes I, II, III, IV, and V and underlie some CNS pathologies. This mitochondrial dysfunction may be ameliorated by L-C and its esters. In addition to its metabolic role, L-C and its esters such as acetyl-L-carnitine (ALC) poses unique neuroprotective, neuromodulatory, and neurotrophic properties which may play an important role in counteracting various disease processes. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach, which may limit further developmental damage, cognitive loss, and improve long-term therapeutic outcomes. The neurophysiological and neuroprotective actions of L-C and ALC on cellular processes in the central and peripheral nervous system show such effects. Indeed, many studies have shown improvement in processes, such as memory and learning, and are discussed in this review.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Metabolism of pyruvate and citrate in lactobacilli

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.