Sperm Energy Metabolism Research Articles

Structure and energy pathways of spermatozoa of the rove beetle Aleochara bilineata (Coleoptera, Staphylinidae)

The morphology of mature spermatozoa of the rove beetle Aleochara bilineata was examined by using scanning and transmission electron microscopy. They are about 1000 μm long and filiform. The acrosome and the nucleus are elongate and each about 20 μm long. A well-developed centriole adjunct region connects the nucleus with the sperm tail. The axoneme reveals the 9 + 9 + 2 pattern of the pterygote sperm flagellum. Two accessory bodies and two mitochondrial derivatives with paracrystalline inclusions are present. Cristae are reduced to the cortical zone of the derivatives. Cytochrome-c oxidase activity was detected within the cristae by DAB-reaction. The energy metabolism of the spermatozoa was investigated by using different inhibitors affecting the mitochondrial and cytoplasmic metabolic pathways. Sperm movement was used as an indicator for the utilization of ATP by the axoneme. In control experiments, the duration of motility was longer than 45 min. In the presence of atractyloside or potassium cyanide the motility duration was not affected. On the other hand, iodoacetic acid in the medium stopped sperm motility within 15 min. This indicates that sperm energy metabolism mainly depends on the glycolytic pathway.

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H 2O 2) and superoxide radicals (O 2 •−). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO ) were also generated. ROS generated by PMA-stimulated blood leukocytes (2–5 × 10 6/ ml) caused inhibition of sperm movement in 2 h ( p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 × 10 −10 moles/10 6 sperm cells) compared to control (0.84 × 10 −10 moles/10 6 sperm cells) ( p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 × 10 3 U/ml). Flow cytometric analyses of sperm treated with various doses of H 2O 2 (0.3 mM–20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H 2O 2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H 2O 2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O 2 •− and HO do not play a significant role in these processes. Low concentrations of H 2O 2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation. © 1999 Elsevier Science Inc.

Sperm structure and motility of the freshwater teleost Cottus gobio

When motility of spermatozoa of Cottos gobio was initiated with distilled water, the motility rate decreased to 0% within 1 min, and significant signs of osmotic alterations were observed at the end of the motility period. By contrast, in 50 mmol 1−1 NaCl solution, the motility rate persisted for 120–140 min. In both distilled water and in 50 mmol 1−1 NaCl solution, the main swimming type of spermatozoa was linear motion during the whole motility period. The initial swimming velocity (50.0 ± 2.1 μm s−1) measured 10 s after motility initiation was similar in both distilled water and in 50 mmol 1−1 NaCl solution. In distilled water, the velocity decreased to &lt;20 μm s−1 (locally motile) during the first minute of the motility phase. In 50 mmol 1−1 NaCl solutions, it remained at a constant level during the first 60 min of the motility period, but then started to decrease to &lt;20 μm s−1 after 120 min. When 5 mmol 1−1 potassium cyanide, antimycin or atractyloside was added to the 50 mmol 1−1 NaCl solution, the motility period was reduced to ≤2min. Ten millimoles per litre 2‐deoxy‐D‐glucose, malonate or a mixture of 5 mmol 1−1 atractyloside and 5 mmol 1−1 carnithine did not effect the duration of the motility period. This indicates that sperm energy metabolism depends mainly on respiration rate and fatty acid metabolism.

Endogenous substrate for energy metabolism and ultrastructural correlates in spermatozoa of the sea urchin Diadema setosum.

Energy metabolism in spermatozoa of the sea urchin Diadema setosum of the order Diadematoida was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol, but also triglyceride (TG). Glycogen and glucose were present at extremely low levels. Following dilution of dry sperm and incubation in seawater, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for a slight increase in the level of free fatty acid. High lipase activity was demonstrated in the spermatozoa. 14C-labeled fatty acid was oxidized to 14CO2. Ultrastructural study also showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and some vacuoles appeared. Thus, the results obtained strongly suggest that D. setosum spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules at the midpieces.

Relationship between intracellular calcium, energy metabolism, and motility of ram sperm.

Collective sperm motility is characterized in terms of periodic aggregation or cooperation among cells, which are seen under a microscope as a continuous wave motion. In this study, sperm motility that was made dependent on mitochondrial activity (glycolysis inhibited) was significantly inhibited by mM exogenous calcium while glycolytic-dependent (mitochondrial respiration inhibited) motility was not inhibited under these conditions. At intracellular Ca2+ above 400 nM, sperm motility was inhibited independently of the source of ATP. At [Ca2+]i of approximately 110 nM, mitochondrial- or glycomitochondrial-dependent motility showed 75 or 0% inhibition, respectively, indicating higher sensitivity of mitochondrial-dependent motility to [Ca2+]i in comparison to glycolytic-dependent motility. Under the latter conditions, intracellular level of ATP was reduced by 16% only, indicating that the 75% inhibition of mitochondrial-dependent motility was not due to reduction in ATP. The inhibition of mitochondrial-dependent motility by mM extracellular Ca2+ can be prevented by incubating the cells at pH 6.5 instead of 7.6. At pH 7.6, calcium probably interacts with negative sites on the cell surface and interferes in cell-to-cell interactions, which are important to achieve collective motility. At more acidic pH (6.5) these negative sites are probably protonated; thus Ca2+ cannot interact with them and no inhibition of motility could be seen. Glycolytic-dependent motility was not inhibited by extracellular Ca2+, since the pH of the medium becomes acidic under these conditions. Like Ca2+, mersalyl, which interacts with Ca2+ binding sites on the cell surface, showed significant inhibition of mitochondrial-dependent motility without any effect on glycolytic-dependent motility. Collective motility was directly correlated with fertility. These data are significant for establishing better conditions for spermatozoa treatment when artificial insemination or in vitro fertilization is concerned.

Energy Metabolism of Spermatozoa of the Sand Dollar Clypeaster japonicus: The Endogenous Substrate and Ultrastructural Correlates1

Energy metabolism in spermatozoa of the sand dollar-sea urchin Clypeaster japonicus was examined. The spermatozoa contained triglyceride and cholesterol ester besides several kinds of phospholipids and cholesterol. Glycogen and glucose were present at extremely low levels. Following incubation of spermatozoa in seawater, the triglyceride content decreased rapidly. Other lipids, however, remained at constant levels. High lipase activity was demonstrated in the spermatozoa. Also, [1-14C]oleic acid was oxidized to 14CO2. Ultrastructural study showed that lipid globules were present at the bottom of the midpiece. After incubation in seawater, morphological changes in the lipid globules were observed and vacuoles of various sizes appeared near the lipid globules. Thus, it is concluded that C. japonicus spermatozoa obtain energy through oxidation of fatty acid from triglyceride stored in the lipid globules at the midpieces.

Endogenous Substrates for Energy Metabolism in Spermatozoa of the Sea Urchins Arbacia lixula and Paracentrotus lividus.

Energy metabolism was examined in the spermatozoa of the sea urchins Arbacia lixula and Paracentrotus lividus, which belong to the orders Arbacioida and Echinoida respectively. P. lividus spermatozoa contained various phospholipids and cholesterol, and their endogenous triglyceride (TG) content was quite low. After dilution of dry sperm in artificial seawater, the level of phosphatidylcholine (PC) decreased rapidly, but other phospholipids remained at constant levels. In contrast to those of P. lividus, the spermatozoa of A. lixula contained TG as well as phospholipids and cholesterol. Following incubation of A. lixula spermatozoa in artificial seawater, TG decreased, but there were no concomitant changes in the levels of phospholipids. Trace amounts of glycogen were present in both species. High lipase activity was demonstrated in A. lixula spermatozoa, but in P. lividus spermatozoa lipase activity was low and phospholipase A2 activity was high. It is thus concluded that A. lixula spermatozoa obtain energy for swimming through oxidation of endogenous TG, whereas P. lividus spermatozoa use PC as a substrate for energy metabolism. This suggests that the system of energy metabolism in spermatozoa is different in the orders Arbacioida and Echinoida.

Energy metabolism of spermatozoa during pronucleus formation induced in vitro by heparin-reduced glutathione. I. Glucose uptake.

Glycolitic metabolism under basal conditions and its modifications by the combined action of heparin and GSH were studied in human sperm. Respirometric data indicated that the amount of U. L. [14C]-glucose converted to 14CO2 increased with the incubation time, being almost linear for up to 60 min and then leveling off at 120 and 150 min (594 and 620 nmol of [14C]-glucose/10(8) spermatozoa, respectively). When spermatozoa were incubated in the presence of heparin-GSH such behavior completely changed, showing a decrease (approximately 50%) in glucose metabolism with values of 254 and 366 nmol of [14C]-glucose/10(8) spermatozoa at the same incubation times as the basal consumption. When these results were compared with the kinetic of the swollen nuclei it was seen that at 30 min 44% of the spermatozoa have its nuclei swollen with a glucose uptake value of 91 nmol/10(8) spermatozoa, and at 150 min when nearly all the spermatozoa nuclei are swollen (95%) the glucose uptake increases fourfold more than the initial rate at 30 min. Therefore, it is possible to suggest the existence of an energy contribution by the sperm to the male pronuclei formation mechanism.

Energy metabolism of spermatozoa of the sea urchin Glyptocidaris crenularis

Energy metabolism in spermatozoa of the sea urchin Glyptocidaris crenularis was examined. The spermatozoa contained not only several kinds of phospholipids and cholesterol but also triacylglycerides (TG). Following dilution of the dry sperm in sea water, the TG content decreased rapidly. Other lipids, however, remained at constant levels, except for an increase in the level of free fatty acid. Oil red-O staining of spermatozoa showed that TG was principally located in part of the sperm midpiece. Also, high lipase activity was demonstrated in the spermatozoa. In both intact cells and a cell-free system, 14C-labeled fatty acids were oxidized to 14CO2. It is thus concluded that G. crenularis spermatozoa use TG as a substrate for energy metabolism.

Localization and Characterization of Phosphatidylcholine in Sea Urchin Spermatozoa&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.

The influence of an egg-associated peptide on energy metabolism in sea-urchin spermatozoa: the peptide stimulates preferential hydrolysis of phosphatidylcholine and oxidation of fatty acid

A study was made of the effects of a sperm-activating peptide (SAP-I: GlyPheAspLeuAsnGlyGlyGlyValGly) on energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. The swimming activity and respiratory rate in slightly acidic seawater (pH 6.6) have been shown to be somewhat less than in normal seawater (pH 8.2). Little change occurred in sperm lipid levels during incubation in seawater at pH 6.6. The addition of SAP-I to seawater at pH 6.6 enhanced the consumption of endogenous phosphatidylcholine (PC), with no change in the levels of other lipids. SAP-I also caused increase in 14CO 2 production from exogenous [1- 14C]oleic acid following incubation of spermatozoa at pH 6.6. However, the stimulated levels of PC consumption and fatty acid oxidation with SAP-I at pH 6.6 did not exceed those at pH 8.2. At pH 8.2, SAP-I had no effect on PC metabolism. Activities of phospholipase A 2 and fatty acid oxidation were markedly influenced by pH and increased at pH exceeding 7. SAP-I is thus concluded to stimulate sea-urchin sperm energy metabolism which depends on the oxidation of endogenous PC. It follows from these results that PC metabolism is activated following increase in the intracellular pH of spermatozoa.

Effects of deep freezing on the energy metabolism of bovine spermatozoa during in vitro capacitation: A cytochemical approach

In order to study the effects of deep freezing on the energy metabolism of bovine spermatozoa, a cytochemical quantitative study was carried out by a microdensitometric method on cytochrome oxidase and lactate dehydrogenase (LDH) activities. These were evaluated in situ on individual frozen-thawed bull spermatozoa collected at different times during in vitro capacitation. The results showed that in bull spermatozoa both the initiation of motility and capacity to fertilize eggs were associated with the anaerobic rather than aerobic glycolysis. The freezing-thawing processes and storage in liquid nitrogen induced a general enhancement of both the enzymatic activities examined. The high ionic strength treatment gave rise to a significant but reversible decrease in both the cytochrome oxidase and LDH activities in the fresh as well as in the frozen-stored sperm. The findings, based on cytochemical observations of energy metabolism of spermatozoa and evaluated during in vitro capacitation, suggest that the respiration and the anaerobic glycolysis of spermatozoa seem to be slightly impaired by the freezing-thawing and storage processes.

The relationship between glycolysis, mitochondrial respiration, protein-carboxyl methylation and motility in hamster epididymal spermatozoa.

Endogenous protein-carboxyl methylase activity can be measured in intact motile spermatozoa by using [3H]methionine as a precursor of the methyl donor, S-adenosylmethionine (SAM). Since the conversion of methionine to SAM requires ATP, the relationship between the energy metabolism of spermatozoa and methylation was investigated using inhibitors of glycolysis and mitochondrial respiration. When hamster spermatozoa from cauda epididymides were incubated in 12.2 mM glucose, glycolysis was progressively inhibited as concentrations of 2-deoxyglucose (2-DOG) increased. On the other hand, endogenous protein-carboxyl methylation showed a biphasic response being stimulated at low concentrations of 2-DOG and inhibited at higher concentrations. Sperm movement was also altered by 2-Dog. Increasing concentrations of 2-DOG in the incubation medium resulted in an increase in beat amplitude and a corresponding decrease in beat frequency. When the glucose concentration of the medium was reduced to 5 mM, protein methylation was inhibited at all concentrations of 2-DOG. The biphasic effect of 2-DOG on methylation at 12.2 mM and its monophasic effect at 5 mM suggested that this reaction was related to the effective glucose concentration. To investigate this possibility, endogenous protein-carboxyl methylation was measured after incubation of sperm in glucose concentrations ranging from 0-48.8 mM. Low glucose concentrations stimulated protein methylation (up to 6.1 mM for maximal effect), but further increases in the glucose concentration (10-48.8 mM) suppressed the rate of protein methylation to that observed in the absence of glucose. 2-Deoxyglucose decreased conversion of pyruvate to CO2 consistent with diminished mitochondrial respiration. The inhibition of mitochondrial respiration by antimycin A also inhibited endogenous methylation and motility. The results of these studies suggest that maximal endogenous protein-carboxyl methylation in intact spermatozoa from hamster epididymides depends upon active glycolysis as well as mitochondrial respiration.

Effect of gossypol on the motility and metabolism of human spermatozoa.

Gossypol, a polycyclic compound isolated from cotton seeds, had a dose-dependent inhibitory effect on human sperm motility. The drug also inhibited powerfully fructolysis and glycolysis by human spermatozoa. Both lactate and CO2 formation from the 14C-labelled sugars was inhibited, and the prevention of CO2 formation from [1-14C]pyruvate and [2-14C]pyruvate by gossypol indicated a direct effect on the tricarboxylic acid cycle. Repeated washing of the sperm cells after gossypol pretreatment failed to abolish the inhibitory effect on CO2 production. The profound disturbances of the sperm energy metabolism induced by gossypol were also reflected by a striking fall of the sperm ATP content. Gossypol had little effect on glucose utilization by minces of human vaginal mucosa, indicating the specificity of gossypol.

Modification of the dehydrogenase activities of human sperm after freezing in liquid nitrogen

Freezing in liquid nitrogen does not result in appreciable modification of the activities of hydrolases or peptidases in human sperm. In contrast the activities of 6-P-gluconate dehydrogenase and, particularly, of glucose 6-phosphate dehydrogenase are severely diminished or even abolished after freezing and thawing. This is not the result of a denaturation of the enzymes, but rather a leak into the surrounding medium. These results are discussed with reference to the role of the pentose pathway in the energy metabolism of human sperm; this enzyme leakage from the spermatozoon could constitute a reliable test assay for evaluation of cell damage due to deep freezing.

Energy Metabolism of Spermatozoa. VII. Interactions Between Lactate, Pyruvate and Malate as Oxidative Substrates for Rabbit Sperm Mitochondria

Rabbit spermatozoa, like other mammalian spermatozoa, have part of their lactate dehydrogenase in the mitochondrial matrix. This enables lactate to be utilized directly as a NAD-linked oxidizable substrate by the mitochondria, but it also enables pyruvate to act as an oxidant of intramitochondrial NADH which should inhibit 02 uptake. These reactions have been investigated in hypotonically treated rabbit epididymal sperm (HTRES) by means of 0, uptake rate determinations and fluorimetric measurements of the reduction level of intramitochondrial NAD. Pyruvate is a potent inhibitor of lactate oxidation; the inhibition is overcome by malate. Pyruvate alone is oxidized slowly but is oxidized rapidly when malate is present. Lactate alone is oxidized rapidly but malate is required for maximal 02 uptake. Fluorimetric measurements show a high reduction level for intramitochondrial NAD with lactate as substrate; this level is increased by malate. Pyruvate reoxidizes intramitochondrial NAD reduced by lactate and inhibits 0, uptake; reversal of this inhibition by malate partially restores the NAD reduction level. Malate plus pyruvate maintain a NAD reduction level sufficient for rapid 02 uptake. We conclude that malate is essential to pyruvate and lactate oxidation by rabbit sperm mitochondria and functions by 2 mechanisms: a) The NADH generated from malate dehydrogenase does not have access to LDH and is made directly available to the respiratory chain. b) The oxaloacetate from malate oxidation stimulates pyruvate dehydrogenase by reaction with the product acetyl C0A. These mechanisms appear to act in concert to prevent inhibition of respiration by pyruvate oxidation of intramitochondrial NADH catalyzed by LDH.

Effects of physiological levels of exogenous steroids on metabolism of testicular, cauda epididymal and ejaculated bovine sperm.

Testicular, cauda epididymal and/or ejaculated sperm were incubated in vitro with exogenous steroids at essentially physiological concentrations (<160 nM). A charcoal stripping technique was used to reduce the amount of endogenous steroid bound to sperm. Effects of 6 or 12 individual steroids on energy metabolism of charcoal stripped and nonstripped sperm were- evaluated. Metabolic differences among nonstripped testicular, cauda epididymal and ejaculated sperm were confirmed. Charcoal-stripped sperm had metabolic characteristics differing from those of non-stripped sperm, but these differences were not abolished by adding steroid. No consistent alteration of metabolic characteristics, e.g., glucose composition, adenine nucleotide levels, oxygen consumption, or carbon dioxide production was associated with inclusion of any steroid in the incubation mixture. We concluded that no single androgen at physiological concentration alters energy metabolism of sperm.

Reversible inhibition of the motility of human spermatozoa by tetraphenylboron

The motility of washed suspensions of human spermatozoa was completely inhibited by tetraphenylboron at concentrations that had little effect on sperm energy metabolism. The inhibition of motility was reversed by quaternary ammonium salts, albumin, caffeine, dibutyryl cyclic AMP and potassium ions. The addition of ouabain to cells redndered immotile by tetraphenylboron prevented reinitiation of motility by potassium but not by the other compounds. These observations, together with the effect of tetraphenylboron on the fluorescence of sperm suspensions treated with 1-anilinonaphthalene 8-sulphonic acid, suggest that the binding of tetraphenylboron to sites on the sperm plasma membrane is involved in the inhibition of sperm motility and the cyclic AMP may be involved in the regulation of ion transport across the plasma membrane.

Energy Metabolism of Spermatozoa. II. Comparison of Pyruvate and Fatty Acid Oxidation by Mitochondria of Rabbit Epididymal Spermatozoa

Energy Metabolism of Spermatozoa. II. Comparison of Pyruvate and Fatty Acid Oxidation by Mitochondria of Rabbit Epididymal Spermatozoa

ATP levels in hamster spermatozoa during capacitation in vitro.

Capacitated sperm have been reported to exhibit elevated rates of motility, oxygen uptake, and glucose consumption. In order to investigate the biochemical basis for this elevation of energy metabolism, we have developed a serum-based test-tube system that capacitates sufficient quantities of hamster sperm to permit biochemical analysis. The rate of motility of sperm incubated in this system is greatly elevated over that of controls. Measurement of sperm ATP content revealed that the capacitating sperm maintained an ATP level one-third to one-fifth that of the control sperm whose content was about 50 nmoles ATP/108 sperm. This reduction in ATP content was found to be caused by the low molecular weight fraction of human serum. It was accompanied by large reductions in sperm ATP/ADP ratios. Data are presented that show the reduction in ATP content of capacitating sperm was not due to external ATPase, differential cell lysis, or nucleotide extraction artifacts. The average concentration of ATP within uncapacitated sperm was approximated to be 5 mM and that of capacitated sperm about 1 mM. This is 100 times the amount known to be required for hamster sperm flagellation and indicates that under these conditions ATP level does not control motility. Evidence from the literature is presented indicating that sperm motility is instead controlled via cAMP and possibly cAMP-dependent protein kinase. In capacitating hamster sperm an increase in cAMP synthesis has been reported. This observation is consistent with the elevated motility described here and would account for the reduction of sperm ATP levels and ATP/ADP ratios to new values. These lower values are known to activate sperm oxidative phosphorylation and glycolysis, thus accounting for the reported elevation in energy metabolism of capacitated sperm. Stimulation of sperm motility, with the consequent increase in energy metabolism, may be required for the penetration of the zona in fertilization.