The effect of anoxia on fowl spermatozoa: Recovery of fertilizing ability,motility, and cellular concentrations of potassium and ATP

Fowl semen when diluted in a glutamate-based medium without glucose, gradually lost its fertilizing ability during 4 hr of anaerobic incubation at 30°C. This incubation regime offered a system by which various in-vitro tests of spermatozoal viability could be assessed for their usefulness as monitors of fertilizing ability. Widely used tests such as spermatozoal enzyme leakage, dye exclusion, and morphology as assessed by light microscopy showed no change in spermatozoal status as the fertilizing ability declined. However the ability of sperm, during a short aerobic incubation to restore their motility and ATP and K⁺ concentrations, declined as did their fertilizing ability. When glucose was added to this re-aeration medium, spermatozoal motility, K⁺ and ATP concentrations, and fertilizing ability were restored to optimal levels. Thus the fertilizing ability of fowl sperm, following anaerobic storage at 30°C, appeared to be related to their ability to restore ATP and K⁺ concentrations and motility. An initial event in the loss of fertilizing ability was a loss in the ability of sperm to oxidise endogenous substrates. This could be restored by the addition of glucose.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.     

Management of pikeperch Sander lucioperca (Linnaeus, 1758) sperm quality after stripping

The aim of this study was to identify the potential for optimizing management of sperm quality during commercial reproduction of pikeperch Sander lucioperca. Sperm from different males is often pooled prior to fertilization or stored for short periods (hr) until ovulated eggs become available. A novel approach was applied to assess pooling effects by cross‐wise transfusion of sperm and seminal fluid (SF) of males with differing initial sperm quality. In addition, the effects of two different buffers (glucose and KCl) were tested, as well as a supplementation of melatonin and progesterone (1 mmol L^?1) to maintain or improve the quality of freshly stripped and incubated (0.5, 1, 2, 4 and 24 hr) sperm. Sperm motility and curvilinear velocity (VCL) were measured by computer assisted sperm analysis (CASA). The VCL proved to be a more sensitive, reliable parameter compared to motility, since significant differences occurred up to 3.5 hr earlier. Transfusion of SF between low and high quality sperm resulted in a significant decrease in sperm with high initial VCL (seven out of 22 transfusions), whereas VCL of low quality sperm could not be improved. In only one case did a transfusion result in an increased VCL. No treatment prevented a significant quality loss over 24 hr or even enhanced sperm performance. Conclusively, pooling sperm of different qualities as well as short‐term storage has a significant, negative impact on overall sperm quality. Pooling should only be considered when the sperm quality is known.     

Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility

An earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Trisbuffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer-directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb²⁺ and Cd²⁺, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb²⁺ was time- and concentration-dependent; the average percentage of hyperactivated sperm decreased from ～ 30% to<5% (n = 5) in 1 hr at a Pb²⁺ concentration of 25 μM. Cd²⁺ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 μM, the decrease in the percent of hyperactivated sperm was ～ 50% (n = 3). Hg²⁺, Zn²⁺, and Cr⁶⁺ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb²⁺ and Cd²⁺, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis. © 1995 Wiley-Liss, Inc.     

Concerted action between Ca2+ and hyperosmolality initiates sperm motility in amphioxus Branchiostoma belcheri tsingtauense

This study investigated the effects of different environmental conditions on the initiation and maintenance of sperm motility in amphioxus Branchiostoma belcheri tsingtauense. The findings were that: (1) hyperosmolality in the absence of Ca(2+) ions did not initiate amphioxus sperm motility; (2) addition of Ca(2+) into EGTA-containing Ca(2+)-free artificial sea water (ASW), in which no sperm were motile, restored sperm motility; (3) Ca(2+) failed to induce sperm motility under conditions of hypoosmolality; (4) K(+) channel blockers quinine and 4-aminopiridine did not suppress the initiation of sperm motility; and (5) changes in pH did not cause sperm motility in a solution isotonic to seawater without Ca(2+). In conclusion, we inferred that a concerted action between Ca(2+) and hyperosmolality was essential to initiate motility of amphioxus sperm, whereas K(+) and pH were indispensable to maintain motility.     

Influence of osmolality and ions on the activation and characteristics of zebrafish sperm motility

Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10 s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities (∼160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30 s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca²⁺. Therefore, whereas pH and concentrations of Ca²⁺ or K⁺ of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.     

Motility activation, respiratory stimulation, and alteration of Ca2+ transport in bovine sperm treated with amine local anesthetics and calcium transport antagonists

Optimal concentrations of dibucaine and other structurally related tertiary amines, variously classified as local anesthetics, Ca²⁺ transport antagonists or calmodulin-directed agents greatly stimulate respiration and the motility of bovine spermatozoa in a reversible manner. Because dibucaine also increases lactate production by sperm made dependent upon glycolysis, the induced metabolic stimulation is probably a secondary response to the greater energy demands resulting from increased motility. Microscopic and time lapse photomicrographic examinations indicate that dibucaine increases the proportion of motile cells and alters the predominant linear swimming path to a peculiar figure eight pattern of movement. Frame by frame analysis of video recordings indicate that this pattern of movement closely resembles, or is identical to the characteristic “motility activation” that occurs during the capacitation sequence which obligatorily precedes fertilization of many, if not all, mammalian species. Dibucaine and the Ca²⁺ transport antagonists, D600 and TMB-8, inhibit the net uptake of Ca²⁺ by sperm suspensions. The dose-response relationships indicate that inhibition of Ca²⁺ uptake does not bear a causal relationship to the activation of motility and metabolism and further suggest a common action of these agents rather than selective effects of D600 and TMB-8 upon Ca²⁺ channels in the sperm plasma membrane. In addition, dibucaine and D600 each induce release of that Ca²⁺ which was accumulated by intact sperm in a preliminary incubation in the absence of the drugs and also inhibit uptake of Ca²⁺ by digitonin-treated sperm. Apparently, therefore, local anesthetics have a direct deleterious action on sperm mitochondrial function. Treatment with the high concentrations of local anesthetics that are required to inhibit uptake of Ca²⁺ completely results in a rapid and irreversible immobilization of the sperm. This loss of motility is either not mediated, or mediated indirectly, through an action of the drug on mitochondrial function because sperm similarly become immotile when a glycolytic substrate is supplied simultaneously.     

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.)

In the present study, we investigated the possibility of spontaneous carp spermatozoa activation by freeze-thawing. To evaluate this, the parameters of spermatozoa motility percentage, velocity, ATP content level and fertility rate of sperm were used. The motility and velocity of spermatozoa activated by freeze-thawing were characterized by motile spermatozoa with a median value of 16% and a velocity of 98 μm/s. In addition, the motility and velocity of sperm from the thawed samples were significantly lower than in the control (median value of 100% for sperm motility and 175 μm/s for sperm velocity). Furthermore, a spontaneously activated spermatozoa motility terminated within five minutes post-thaw time. After freeze-thawing the ATP level significantly decreased with post-thaw time (46 nmol ATP/10⁹ and 10 nmol ATP/10⁹ at 25 s and 10 min after thawing, respectively). Fertility of spermatozoa was not significantly affected within 10 min post-thaw. On the other hand, the fertility of frozen-thawed sperm was significantly lower if compared to fresh sperm. We conclude that the freeze-thawing procedure spontaneously activated spermatozoa motility in common carp. However, this activation did not negatively affect the fertility of frozen-thawed sperm.     

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     

Roles of Na(+)/K(+)-dependent ATPase, Na(+)/H(+) antiporter and GLUT hexose transporters in the cryosurvival of dog spermatozoa: effects on viability, acrosome state and motile sperm subpopulation structure

To assess the roles of Na⁺/K⁺-dependent ATPase, Na⁺/H⁺ antiporter and GLUT hexose transporters in the cryosurvival of dog sperm, semen samples were frozen in a standard freezing medium supplemented with the specific inhibitors of these factors ouabain, amiloride or phloretin, respectively. The presence of ouabain did not counteract the effects of freeze-thawing on the percentages of motile sperm and altered acrosomes, although a small recovery effect was observed in motility parameter means. Amiloride had a similar effect, although motility was more intensely recovered. Phloretin significantly (P < 0.05) impaired viability when added at a maximal concentration of 10⁻4 M (57.3 ± 5.1% vs 76.5 ± 5.7% in cells frozen without inhibitors), although partial recovery of motility parameters was also observed. These effects were accompanied with specific changes in both motility parameters and the percentages of motile sperm in each of the 4 subpopulations comprising the motile sperm population of the ejaculate. Our findings indicate a role for Na⁺/K⁺-dependent ATPase and Na⁺/H⁺ antiporter in the mechanisms involved in determining specific sperm motility patterns in response to freeze-thawing, although neither pump seems to be important for the resistance of cell membrane structures to freezing-thawing. In addition, a role for GLUTs in regulating water exchange in dog sperm during freeze-thawing seems unlikely. In contrast, the precise structure of dog sperm in terms of its motile subpopulations was found to condition both cryosurvival and sperm cell sensitivity to the inhibitors used.     

The retention of 125I-labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg-yolk citrate extender

Cryopreservation of bovine sperm in egg-yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with ¹²⁵I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES)-buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol-free EYC and held for 3 hr, or c) frozen-thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze-thawing dislodged most egg-yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11-18 bands of ¹²⁵I polypeptides. There was no difference (P > 0.05) in the amount of ¹²⁵I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES-buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of ¹²⁵I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of ¹²⁵I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.     

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.     

Original endomorphin-1 analogues exhibit good analgesic effects with minimal implications for human sperm motility

To search a novel analgesic characterizes the effects on human sperm motility as minimal as possible. A new class of endomorphin-1 (EM-1) analogues was synthesized by combining successful chemical modifications including N-terminal guanidino modification, Phe⁴ was chlorinated, replaced of l-Pro²-Trp³ by d-Ala²-Gly³ or d-Pro²-Gly³ at position 2 and 3. Their bioactivities were measured by radioligand binding assay, metabolic stability, antinociception activity and sperm motility effects. In radioligand binding assays, analogue GAGP shown a μ-opioid receptor affinity about 17.7-fold higher and a 57.3-fold higher δ-opioid receptor affinity than EM-1. In the metabolic stability assays, GAGP had the longest half-lives and 16.6-fold higher than EM-1. In the tail-flick test in mice, GAGP showed the best analgesia. In sperm motility assays, the group of GAGP (10^?5, 10^?7 mol/L) decreased of the percentage of a + b grade, and no significant when compared with initial value. In GAGP (10^?6 mol/L) group, sperm motility was progressively increased, although it was not statistically significant. But at the groups of morphine (10^?7 mol/L) and GAGD (10^?7 mol/L), these caused significant reduction between 0 and 90 min. We found that analogues GAGP, activating μ-opioid receptor and partial δ-opioid receptor, exhibit good analgesic effects with minimal implications for human sperm motility. It might be important in potential application as drug candidates of analgesic without implications for human sperm motility.     

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.)

In the present study, we investigated the possibility of spontaneous carp spermatozoa activation by freeze-thawing. To evaluate this, the parameters of spermatozoa motility percentage, velocity, ATP content level and fertility rate of sperm were used. The motility and velocity of spermatozoa activated by freeze-thawing were characterized by motile spermatozoa with a median value of 16% and a velocity of 98 μm/s. In addition, the motility and velocity of sperm from the thawed samples were significantly lower than in the control (median value of 100% for sperm motility and 175 μm/s for sperm velocity). Furthermore, a spontaneously activated spermatozoa motility terminated within five minutes post-thaw time. After freeze-thawing the ATP level significantly decreased with post-thaw time (46 nmol ATP/10⁹ and 10 nmol ATP/10⁹ at 25 s and 10 min after thawing, respectively). Fertility of spermatozoa was not significantly affected within 10 min post-thaw. On the other hand, the fertility of frozen-thawed sperm was significantly lower if compared to fresh sperm. We conclude that the freeze-thawing procedure spontaneously activated spermatozoa motility in common carp. However, this activation did not negatively affect the fertility of frozen-thawed sperm.     

The Mitochondrial Toxin Produced by Streptomyces griseus Strains Isolated from an Indoor Environment Is Valinomycin

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC₅₀) were 10 to 63 ng (dry weight) ml of extended boar semen⁻¹. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml⁻¹. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC₅₀ of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen⁻¹, and the EC₅₀ of commercial valinomycin was 2 ng ml of extended boar semen⁻¹. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

Sodium as a mediator of non-phorbol tumor promoter action. Down-modulation of the epidermal growth factor receptor by palytoxin

Previous studies have shown that palytoxin, a non-(12-O-tetradecanoylphorbol-13-acetate)-type tumor promoter, is able to down-modulate the epidermal growth factor (EGF) receptor through a sodium-dependent pathway in Swiss 3T3 cells. A role for sodium is supported by the observation that the sodium proton exchanger monensin and the sodium-conducting ionophore gramicidin mimic palytoxin action by causing a decrease in both high and low affinity EGF binding. However, in addition to causing sodium influx, these agents can induce other cellular effects including changes in membrane polarization, intracellular pH, and macromolecular synthesis. To determine whether any of these factors might be responsible for palytoxin action in our system, we examined the role of each of them in palytoxin-induced inhibition of EGF binding. Although palytoxin depolarizes the membrane, the observation that potassium-induced depolarization of the membrane does not cause a decrease in EGF binding, in conjunction with the fact that monensin hyperpolarizes the membrane, indicates that depolarization of the membrane is not responsible for palytoxin-induced changes in the EGF receptor. An investigation of intra-cellular pH suggests that the palytoxin effects are not mediated by proton flux. In addition, nigericin-mediated changes in intracellular pH do not cause an inhibition of EGF binding. Finally, studies conducted in the presence of cycloheximide indicate that protein synthesis is not required for palytoxin action and that inhibition of EGF receptor biosynthesis does not account for palytoxin-induced loss of EGF-binding sites. These results suggest that sodium may act as a second messenger in the signal transduction mechanism by which palytoxin modulates the EGF receptor.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.