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.     

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.     

Use of a fluorescent dye to measure drug-induced changes in the membrane potential of boar spermatozoa.

The fluorescence emission intensity of the dye 3,3′ dipentyloxo-carbocyanine iodide equilibrated with washed boar spermatozoa and valinomycin or gramicidin varies with external potassium or sodium concentration in a manner indicating that dye fluorescence is related to the plasma membrane potential. The membrane potential in turn is shown to be dependent on energy metabolism. The drugs propranolol, lidocaine and diphendydramine, which possess local anesthetic-like properties, induce a rapid depolarization which can be reversed by valinomycin at low drug concentrations and a slower apparently energy dependent depolarization at higher drug concentrations that is not reversible. Low concentrations of these drugs decrease forward progression of sperm but have little effect on the percentage motile cells. Theophylline increases the frequency of contraction of drug-treated cells but not their forward progression, these findings are discussed in terms of the role of the sperm membrane in the control of motility.     

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.     

Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20°C or cooled down to 5°C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/PI. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V, and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5°C than at 20°C, which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature.     

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.     

Involvement of mitochondrial activity in mediating ELF‐EMF stimulatory effect on human sperm motility

It has recently been reported that the exposure of human spermatozoa to an extremely low frequency (ELF) electromagnetic field (EMF) with a square waveform of 5 mT amplitude and frequency of 50 Hz improves sperm motility. The functional relationship between the energy metabolism and the enhancement of human sperm motility induced by ELF‐EMF was investigated. Sperm exposure to ELF‐EMF resulted in a progressive and significant increase of mitochondrial membrane potential and levels of ATP, ADP and NAD⁺ that was associated with a progressive and significant increase in the sperm kinematic parameters. No significant effects were detected on other parameters such as ATP/ADP ratio and energy charge. When carbamoyl cyanide m‐chlorophenylhydrazone (CICCP) was applied to inhibit the oxidative phosphorylation in the mitochondria, the values of energy parameters and motility in the sperm incubated in the presence of glucose and exposed to ELF‐EMF did not change, thus indicating that the glycolysis was not involved in mediating ELF‐EMF stimulatory effect on motility. By contrast, when pyruvate and lactate were provided instead of glucose, the energy status and motility increased significantly in ELF‐EMF‐treated sperm. Under these culture conditions, the inhibition of glycolitic metabolism by 2‐deoxy‐D ‐glucose (DOG) again resulted in increased values of energy and kinematic parameters, indicating that gluconeogenesis was not involved in producing glucose for use in glycolysis. We concluded that the key role in mediating the stimulatory effects exerted by ELF‐EMF on human sperm motility is played by mitochondrial oxidative phosphorylation rather than glycolysis. Bioelectromagnetics 32:15–27, 2011. © 2010 Wiley‐Liss, Inc.     

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter–spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June–August plasma with respect to the value obtained in September–December plasma.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.     

α-Bungarotoxin binding by cell membranes : Blockage of sperm cell motility

Arbacia sperm cells in sea water suspension respond dramatically to the addition of bungarotoxin. Concentrations as low as 5 pM cause about half the cells to cease swimming, while total inhibition of all motile activity occurs abruptly at 0.5 μM. Nicotine acts cholinomimetically to increase sperm swimming speed at concentrations between 10 pM and 100 nM, while higher concentrations decreases motility. It is concluded that acetylcholine receptors, possibly nicotinic, located at the cell surface, may mediate the regulation of sperm motility.     

Sperm biology and control of reproduction in sturgeon: (II) sperm morphology, acrosome reaction, motility and cryopreservation

In the present review, sperm morphology, acrosome reaction, motility, short-term storage and cryopreservation are summarized and discussed in sturgeon (Chondrostei, Acipenseriformes). The elongated head of spermatozoon comprises an acrosome with 8?C12 posterolateral projections. Usually three endonuclear canals are observed in the nucleus. Proximal and distal centrioles and 3?C6 mitochondria are located in the midpiece region. The flagellum consists of an axoneme with a typical ??9?+?2?? structure of microtubules and presents a ribon-like structure due to two lateral membranous fins. Egg water, Ca²⁺ and Mg²⁺ can trigger acrosome reaction. Trypsin- and chymotrypsin-like activities are reported in sturgeon sperm. These physiological properties of sturgeon sperm are identified as serine activity with 33?kDa molecular mass and can be inhibited by their respective inhibitors. The K⁺ prevents sperm activation in seminal plasma, and hypo-osmolality or decrease of extracellular K⁺ triggers sperm activation. Extracellular Ca²⁺ is involved in flagellar beating pattern and sperm velocity. After activation, sperm motility, velocity, and flagellar beating frequency, wavelength and amplitude decrease, while number of waves and curvature increase. Sturgeon sperm can be stored for several days at 4?°C; however it is better to add K⁺ into the immobilizing medium because it prevents sperm activation during incubation. Regarding sperm cryopreservation, methanol is a better cryoprotectant than DMSO. Either short-term storage or cryopreservation of sperm generates damage to spermatozoa that lead to reduction of sperm motility performance. Some studies suggest using an activation medium containing Ca²⁺ for enhancing sperm motility performance of incubated or frozen-thawed sperm.     

Flow and image cytometry for quality assessment of fresh and frozen human sperm samples

A previous study reported that sperm mitochondrial activity and sperm motility can be evaluated by combined flow and image cytometry, suggesting their potential interest in fertility clinical applications and for studying the effect of physical and chemical agents that modify sperm motility and/or metabolism. This paper focuses on the effect of freezing sperm in liquid nitrogen, extensively used in artificial insemination (AI), combined with different sperm manipulations (washing, swim-up in capacitating medium, CM) using flow and image cytometry on sperm samples from three fertile donors. Rhodamine 123 (Rh123) uptake profiles were bimodal both for fresh and frozen/thawed samples. The mean value of fluorescence of active cells (m+) remained nearly similar after freezing while the percentage of active cells (%C+) was significantly decreased and the percentage of dead cells (%dc, revealed by propidium iodide uptake) significantly increased. In all experiments, the decrease of MOT (percentage of motile sperm) due to freezing was concomitant, to a smaller extent, with a drop in %C+ and an increase in %dc; there was a good relationship between velocity (VCL, VSL) or trajectory characteristics (ALH) and the mean fluorescence values of active cells (m+). Sperm immobilization was neither found related to a major decrease of Rh123 fluorescence nor to an increase in dead cells.     

A new role of anandamide in human sperm: Focus on metabolism

The endocannabinoid system and the presence of CB1 receptor (CB1‐R) target of the anandamide were identified in human sperm, however the anandamide action in this context needs to be further elucidated. At this purpose we analyzed the effects of anandamide on human sperm capacitation and motility. Afterwards, we focused on lipid and glucose sperm metabolism and also investigated the interrelationship between anandamide and insulin secretion by sperm. By intracellular free Ca²⁺ content assay and proteins tyrosine phosphorylation, we evidenced that anandamide did not induce capacitation process and a negative effect was obtained on sperm motility. The blockage of CB1‐R by the specific antagonist SR141716 increased both capacitation and sperm motility suggesting an involvement of the CB1‐R in the acquisition of sperm fertilizing activity. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that anandamide exerts a lipogenetic effect on human sperm lipid metabolism. Concerning the glucose metabolism, anandamide increases GSK3 phosphorylation indicating that it is involved in the accumulation of energy substrates. G6PDH activity was not affected by anandamide. Interestingly, AEA is involved in insulin secretion by sperm. As insulin had been demonstrated to be an autocrine factor that triggers capacitation, the endocannabinoid might be inserted in the signaling cascade that induces this process. Altogether these findings highlight a pivotal involvement of the CB1‐R in the control of sperm energy homeostasis and propose a new site of action for endocannabinoids in the control of energy metabolism. J. Cell. Physiol. 221: 147–153, 2009. © 2009 Wiley‐Liss, Inc     

Metabolism of intratesticular spermatozoa of a tropical teleost fish (Clarias gariepinus)

Sperm metabolism of a tropical fish species, the African catfish, Clarias gariepinus, was studied by measurements of sperm enzyme activity and metabolite levels. We also analysed the effect of metabolites, co-enzymes and enzymatic blockers on sperm motility behaviour and viability. Similar to other teleostean species, African catfish spermatozoa have the capacity for glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, lipid catabolism, beta-oxidation and osmoregulation. In immotile spermatozoa, lipid catabolism, beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation were important primary energy-delivering pathways; sperm oxygen consumption was 0.39-0.85 microg O(2)/min/ ml of testicular semen. During motility, glycolysis, lipid catabolism and beta-oxidation of fatty acids occurred simultaneously, which is atypical for teleosts, and the spermatozoal respiration rate increased drastically by 15-25-fold. Also in contrast to other teleostean sperm cells, ATP levels remained stable during motility and immotile storage. The sperm cell status was unstable in the African catfish. Although the spermatozoa have osmoregulation ability, and even though balanced physiological saline solutions were used for sperm motility activation and sperm incubation, the motility and viability of spermatozoa quickly decreased at 28 degrees C, the spawning temperature of the African catfish. Cyclic AMP and inhibition of phosphodiesterase activity could not prolong sperm motility and viability. In contrast, at 6-10 degrees C motility was prolonged from approximately 30 s to >5 min, probably due to decreased metabolic rates.     

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 microg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (P<0.01) affected by 27 microg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.     

Neurochemical control of Arbacia sperm motility

Spermatozoa appear to function as multi-subunit, single cell, excitable systems at least partially under acetylcholinesterase control. The motility of Arbacia sperm systematically exposed to a variety of agents which influence acetylcholine metabolism or cell membrane stability changed in a dose-dependent manner. Acetylcholine by itself had only minimal effects, but evoked a dramatic response when administered in the presence of dimethyl sulfoxide. Twenty mM DMSO, alone, did not alter the sperm swimming speed but at this concentration it potentiates acetylcholine action which now nearly doubles the initial rate of motility. Subsequent return to control rate follows hydrolysis of the acetylcholine or desensitization of the acetylcholine receptors. Hemicholinium depresses sperm motility at all concentrations above 100 μM. Exposure to procaine at first excites the cells to more than double the control swimming speed at 1 mM; 10 mM procaine decelerates the sperm to 50% of the control rate. Subsequently complete stoppage occurs at these concentrations, presumably by stabilization of the cell membrane. Strychnine speeds the cells by 50% at 10μM and slows them by 50% at 5 mM; curare also increases and decreases motility, but only by about 20%.     

Metabolism of motile zebrafish sperm

As metabolism of motile fish sperm is not well understood, the current study examined the metabolism of saline-activated zebrafish (Danio rerio) sperm. Activation of sperm with inhibitors of oxidative phosphorylation (potassium cyanide, 2,4 dinitrophenol or carbonyl cyanide 3-cholorophenylhydrazone) negatively impacted sperm motility by 60-90 s postactivation. Incubation of quiescent sperm with 2,4 dinitrophenol prior to activation resulted in a 67% decrease in the percent motile sperm assessed 15s postactivation. Thus, production of ATP in quiescent sperm is important for motility upon activation and nascent ATP production via oxidative phosphorylation by motile sperm appears important at 60-90 s postactivation. Exposure of sperm to iodoacetamide, an inhibitor of creatine kinase, at activation was without effect. However, incubation of quiescent sperm with iodoacetamide prior to activation resulted in a 77% reduction in percent motile sperm and decreased velocity and wobble at 15s postactivation. These results suggest that creatine kinase and phosphocreatine shuttle are physiologically important at, or shortly after the initiation of motility. Finally, sperm were exposed to lactate, pyruvate, or acetate as well as to several monosaccharides upon activation. The results provided no evidence supporting any metabolic role of exogenous organics (potentially from the female via ovarian fluid) in sperm once motility has begun.     

Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro

In previous reports from this laboratory, we identified the presence of a novel alpha-D-mannosidase on the surface of rat, mouse, hamster, and human spermatozoa [J Cell Biol 1989; 109:1257-1267 and Biol Reprod 1990; 42:843-858]. Since it has been suggested that mannosyl residues on the egg zona pellucida may be important for sperm-egg binding, studies were undertaken to examine the potential role of the sperm alpha-D-mannosidase during fertilization. Incubation of mouse spermatozoa in the presence of increasing concentrations of the inhibitory sugars, alpha-methyl mannoside, alpha-methyl glucoside, D-mannose, or D-mannitol, resulted in a dose-dependent decrease in the number of spermatozoa bound per egg without a deleterious effect on sperm motility or on the sperm acrosome, and a dose-dependent inhibition of the sperm mannosidase activity. Galactose, however had no effect on sperm-egg binding or on sperm mannosidase activity. Two nucleotide sugars (UDP-GlcNAc and UDP-gal) were also tested and shown to reduce sperm-egg binding but with only a minimal effect on sperm mannosidase activity. In additional studies, spermatozoa incubated in the presence of a mannose-containing oligosaccharide exhibited a dramatic reduction in sperm-egg binding that correlated with a similar inhibition of sperm mannosidase activity. The oligosaccharide substrate did not affect sperm motility or the sperm acrosome. These studies suggest that the sperm alpha-D-mannosidase may play an important role during fertilization.     

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.