Levels of semenogelin in human spermatozoa decrease during capacitation: involvement of reactive oxygen species and zinc.

BACKGROUND: Semenogelin (Sg), the main protein of human semen coagulum, prevents sperm capacitation. The objective of this study was to examine the role of Sg and its mechanism of action. METHODS AND RESULTS: Sg blocked sperm capacitation triggered by various stimuli, via inhibition of superoxide anion (O(2)*-; luminescence assay) and nitric oxide (NO*; tested using diaminofluorescein) generation. Triton-soluble and -insoluble sperm fractions contained Sg and Sg peptides (immunoblotting), the level of which decreased with initiation of capacitation. This drop was prevented by superoxide dismutase and NO* synthase inhibitor and was reproduced by addition of O(2)*- and NO*. Zinc (Zn(2+)) blocked and a zinc chelator (TPEN) promoted the decline in Sg levels. There was a decreased labelling of Sg on the head in capacitating spermatozoa with the two fixation techniques tested (immunocytochemistry). Reactive oxygen species (ROS) (O(2)*- and NO*) caused, these changes, and zinc prevented them. Spermatozoa quickly internalized Sg upon incubation and Sg was then rapidly degraded in a zinc-inhibitable manner. CONCLUSIONS: Sg blocked capacitation mainly via inhibition of ROS generation. Spermatozoa appeared permeable to Sg and processed Sg in a zinc-inhibitable fashion. ROS themselves could promote sperm disposal of Sg which maybe one of the mechanisms that allows initiation of capacitation.

Effects of protease inhibitors and substrates on motility of mammalian spermatozoa.

A series of protease inhibitors were tested on the motility of human, rat, bull, and rabbit demembranated reactivated spermatozoa. Some inhibitors, including aprotinin, boc-gln-leu-lys-H, and D-phe-pro-arg-H, could inhibit motility as well as prevent initiation of motility. In general, with the exception of aprotinin, protease inhibitors were more potent in preventing the initiation of movement than in blocking motility of demembranated spermatozoa. Protease substrates could also block sperm motility. Of the substrates tested only those with arg or lys ester bonds were active. The inhibition of motility by protease substrates was reversible, as once spermatozoa hydrolyzed the added exogenous protease substrates, motility reappeared. The importance of ester bond in the inhibitory action of protease substrates was confirmed by experiments that showed the lack of effect of pre-hydrolyzed protease substrates. The results suggest that a serine protease with lys and arg ester bond specificity is involved in the control of sperm motility. The fact that protease substrates also block motility of intact spermatozoa further emphasizes the physiological relevance of this new regulatory system.

Capacitation-associated production of superoxide anion by human spermatozoa.

Previous results demonstrating the role of the superoxide anion in human sperm capacitation indirectly suggest that the superoxide anion is not only essential to this process, but is also generated by spermatozoa themselves. Our aim was to provide direct evidence for production of superoxide anion by capacitating spermatozoa. Three chemiluminescence probes were tested to detect the superoxide anion. Luminol- and lucigenin-amplified chemiluminescence of capacitating spermatozoa did not differ from that of background level. However, significant SOD-inhibitable chemiluminescence was observed when the Cypridina luciferin analog, MCLA, was used as probe. The chemiluminescence associated to capacitation of spermatozoa incubated in Ham's F-10 supplemented with fetal cord serum ultrafiltrate was 1270 +/- 77 mV/10 s (with 8 x 10(6) cells/ml; n = 37), and corresponded to levels of sperm hyperactivation (12 +/- 1%) and capacitation (17 +/- 2%) that were significantly different from those of control spermatozoa (4.9 +/- 0.8% and 6 +/- 1%, respectively). MCLA was cell impermeant and superoxide specific. The level of capacitation-associated chemiluminescence was directly related to sperm concentration up to 30 x 10(6) cells/ml. Sperm production of superoxide anion started at the beginning of incubation in capacitating conditions and was sustained over more than 4 h. This is the first direct evidence for the generation, by human spermatozoa, of a sustained level of superoxide anion that is associated with the progressive development of hyperactivation and capacitation.

Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production.

Spermatozoa must undergo capacitation prior to fertilization. In humans, this process appears regulated by oxidoreduction reactions. We investigated the possibility that these reactions involved the sulfhydryl-disulfide pair, which offers a reversible regulation of cellular processes. The effects of reagents targeted for sulfhydryl and disulfide groups on human sperm capacitation, superoxide (O2-.) generation and protein tyrosine phosphorylation were evaluated. The sulfhydryl targeted agents, phenylarsine oxide (PAO), diamide, dithiopyridine (DTP), N-ethylmaleimide (NEM), maleimidylpropionyl biocytin (MPB), p-chloromercuribenzoic acid (PCMB), and bromobimane analogs (mBBr and qBBr) triggered sperm capacitation to levels comparable to those observed with a biological inducer, fetal cord serum ultrafiltrate (FCSu). Capacitation induced by NEM, MPB, PCMB, and PAO was prevented by superoxide dismutase (SOD) and associated with an increased sperm production of O2-.. However, SOD did not affect the increase in protein tyrosine phosphorylation of spermatozoa treated with NEM, PAO, or MPB. Disulfide reductants, dithiothreitol (DTT), thioredoxin (TRX), glutathione (GSH), tris-(2-carboxyethyl) phosphine (TCEP), and tris-(2-cyanoethyl) phosphine (TCP) partially to totally inhibited FCSu-induced sperm capacitation and O2-. production. TCEP, DTT, and TRX decreased the capacitation-associated tyrosine phosphorylation of sperm proteins. The strong time-dependent increase of sperm membrane sulfhydryl groups exposed to the extracellular space occurring during the first hour of capacitation could indicate an important rearrangement of sulfhydryl carrying proteins during the initiation of capacitation. Therefore, protein sulfhydryl-disulfide status may be important for the regulation of human sperm capacitation and the mechanisms involved may be complex and multifactorial.

Human sperm hyperactivation and capacitation as parts of an oxidative process.

Capacitation of spermatozoa is essential for fertilization and is visually characterized by hyperactivated motility. Previous reports have shown that foetal cord serum (FCS) and superoxide anion, O2.-, can trigger human sperm hyperactivation (HA) and capacitation and that superoxide dismutase (SOD) could prevent these processes. We investigated further the role of O2.- and FCS components in human sperm HA and capacitation. Percoll-washed spermatozoa were incubated, at 37 degrees C, in Ham's F-10 medium with 7.5% of FCS, dialyzed FCS (> 12 kD), ultrafiltrate from FCS (FCSu; < 3 kD), or xanthine + xanthine oxidase + catalase (X +XO + cat). Spermatozoa incubated with FCSu were also supplemented with catalase to prevent the loss of motility often observed after 2-3 h of incubation. FCS and dialyzed FCS induced significant levels of HA (10 +/- 1% and 7.7 +/- 0.7%, respectively) that were, however, lower than those observed with FCSu (19 +/- 1%) or X + XO + cat (16 +/- 2%). Similar results were obtained when the lysophosphatidylcholine-induced acrosome reaction (LPC-AR, a measure of sperm capacitation) was evaluated. The presence of SOD in the incubation medium blocked the induction of HA and capacitation by FCS, FCSu, X + XO + cat, as well as the spontaneous HA and capacitation. The enzymatic activity of SOD was needed for the prevention of these processes. Desferrioxamine, up to 100 microM, had no effect on HA and LPC-AR induced by FCSu and X + XO + cat. Addition of SOD to already hyperactivated spermatozoa reversed the HA. These data suggest that spermatozoa need a sustained O2.- generation to maintain HA and proceed to capacitation. We hypothesize that FCSu or the O2.- generated by X + XO + cat activate enzymes, possibly a reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase at the level of sperm membrane.

Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives.

Spermatozoa undergoing capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2.-). Because, in neutrophils, the generation of O2.- is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human sperm capacitation. Human spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both sperm capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2.- and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and sperm capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and sperm capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the spermatozoa.

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa.

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.

Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma.

OBJECTIVES: To characterize the very vigorous type of motility observed in the semen of some infertile men and to compare the superoxide anion scavenging capacity of the seminal plasma from these men and that from normal men. SUBJECTS: Patients consulting for infertility related to sperm motility problems and men presenting as sperm donors. METHODS: Motility patterns and measurements of sperm motility parameters were evaluated by computer-assisted digital image analysis system. The superoxide anion scavenging capacity of seminal plasma was measured by inhibition of nitroblue tetrazolium reduction due to the superoxide anion generated by the combination xanthine plus xanthine oxidase. RESULTS: Spermatozoa from 9 of 68 semen samples with normal sperm concentration, morphology, and percentage of motility showed the typical motility patterns observed during hyperactivation (HA) and a significant level of HA (16% +/- 3%) as compared with those in semen (2.3% +/- 0.3%) from fertile volunteers. The superoxide anion scavenging capacity of the seminal plasma from men with sperm HA was 37% lower than that of control seminal plasma. CONCLUSIONS: Sperm HA can occur in whole semen and is associated with a low superoxide anion scavenging capacity in seminal plasma. It could be one of the causes for idiopathic infertility.

Positive correlation between the level of protein-carboxyl methylase in spermatozoa and sperm motility.

Levels of protein-carboxyl methylase (PCM) activity were measured in spermatozoa from infertile patients with less than 50% sperm motility and compared with those of normal fertile controls. When spermatozoa were washed by a standard centrifugation procedure, the level of PCM activity in a subgroup of patients with sperm motility ranging from 0% and 20% (24.0 +/- 5.2 pmol/mg protein, mean +/- standard error of the mean) was significantly different from that of controls (35.9 +/- 2.3 pmol/mg). However, when the entire population of patients with sperm motility ranging from 0% to 50% (32.6 +/- 6.2 pmol/mg) was compared with controls, no significant difference was observed in sperm PCM levels. With this standard washing procedure no significant relationship (r = 0.28; P greater than 0.05) between sperm PCM activity and motility was observed. By contrast, when spermatozoa were washed on a Percoll gradient, to eliminate other cellular elements, both groups of patients with 0% to 20% (14.6 +/- 2.5 pmol/mg) and with 0% to 50% (21.5 +/- 2.4 pmol/mg) sperm motility had sperm PCM levels significantly lower than that of controls (34.7 +/- 3.6). A highly significant relationship (r = 0.78; P less than 0.001) was observed between the levels of sperm PCM activity and motility.

Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers.

OBJECTIVE: To investigate whether activated poly-morphonuclear neutrophils (PMN) can affect sperm motility and to assess the capacity of seminal plasma and of scavengers of reactive oxygen species to prevent any loss of motility. DESIGN: Blood PMN were isolated, and their capacity to generate reactive oxygen species was investigated. The effect of activated PMN was tested on the motility of Percoll-washed spermatozoa in the presence and absence of reactive oxygen species scavengers or seminal plasma (whole or fractionated). PARTICIPANTS: Fertile volunteers and patients attending the Infertility Clinic at the Royal Victoria Hospital. RESULTS: The production of reactive oxygen species by activated PMN occurred in the 1st hour after stimulation and was linearly correlated with the concentration of PMN. Percoll-washed spermatozoa had reduced motility in the presence of PMN, and the drop in motility was more severe as the concentration of PMN was increased. Catalase and dimethylsulfoxide reduced the toxic effect of PMN on sperm motility, whereas superoxide dismutase was without effect. Seminal plasma (50%, vol/vol) allowed a highly variable protective effect against the loss of sperm motility that appeared to originate predominantly from the low molecular weight (< 10 kd) fraction but also from the high molecular weight (> 12 kd) fraction of seminal plasma. CONCLUSION: These data suggest that [1] the presence of PMN, even at 0.6 x 10(6) cells/mL, in semen can be hazardous for spermatozoa, [2] H2O2 and .OH are responsible for the loss of motility, and [3H] seminal plasma confers a variable protection due to high and low molecular weight factors.

Determination of neutrophil concentration in semen by measurement of superoxide radical formation.

OBJECTIVE: To develop an assay that measures the concentration of functional neutrophils in human semen. DESIGN: Human blood neutrophils were first isolated to establish linearity and proportionality for the determination of functional neutrophil concentration. Thereafter blood neutrophils in seminal plasma and neutrophils in semen of patients were measured. SETTING: Blood and human samples were obtained from clinics of the Royal Victoria Hospital. PATIENTS, PARTICIPANTS: Blood from normal men or from patients presenting with chest pain or trauma were used. Semen samples were also obtained from healthy fertile donors or from unselected patients attending the infertility clinic. MAIN OUTCOME MEASURES: Active neutrophil concentration in semen by a colorimetric assay. RESULTS: The assay developed is based on the reduction of nitroblue tetrazolium of pale yellow color to blue formazan by the superoxide anions produced by stimulated neutrophils. The intensity of the derived blue color is proportional to the concentration of active neutrophils. This assay is simple, requires only 10 minutes of preparation time, and detects neutrophil concentrations greater than 0.5 x 10(6) neutrophils/mL of semen, independent of sperm concentration.

Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility.

OBJECTIVES: To determine whether reactive oxygen species (ROS) produced by abnormal or deficient spermatozoa and polymorphonuclear leukocytes (PMN, activated or not) can affect normal sperm motility. To determine what level of ROS is detrimental to spermatozoa. SUBJECTS: Patients consulting the Infertility Clinic at the Royal Victoria Hospital and healthy volunteers. METHODS: Normal spermatozoa (not producing ROS) were incubated with ROS-producing spermatozoa or PMN (activated or not), and motility was analyzed using a computer-aided sperm analysis system. The proportion of ROS produced by spermatozoa and released extracellularly was estimated by the decrease in the chemiluminescence observed in the presence of catalase and superoxide dismutase. RESULTS: There was no consistent effect on motility when ROS-producing and non-ROS-producing spermatozoa were mixed. This lack of effect could be due to the relatively low level of ROS produced by spermatozoa as well as the fact that only a third of these ROS are released outside spermatozoa. To cause a low but significant decrease (15 +/- 4%) in sperm motility after a 5-hour incubation, the level of ROS imposed on spermatozoa must be equivalent to ROS released by 1 x 10(6) activated PMN/mL. CONCLUSIONS: The production of ROS by deficient spermatozoa is low and of no consequence to the motility of normal spermatozoa present in the same sperm preparation. However, 1 x 10(6) activated PMN/mL, with a 1,000-fold higher ROS production, have detrimental effects on the motility of normal washed spermatozoa.

Increased reactive oxygen species formation in semen of patients with spinal cord injury.

OBJECTIVES: To evaluate reactive oxygen species production of semen samples and Percoll-washed spermatozoa from men with spinal cord injuries and to determine if there is a relationship between this reactive oxygen species production and sperm motility. PARTICIPANTS: Semen samples from healthy volunteers and infertile patients were collected by masturbation. INTERVENTIONS: Semen samples from men with a spinal cord injury were obtained by electroejaculation or by masturbation after treatment with physostigmine. MAIN OUTCOME MEASUREMENTS: Motility was measured using the CellSoft computer-assisted analysis system (Cryo Resources Ltd., Montgomery, NY). Luminol-amplified chemiluminescence was used to measure reactive oxygen species production. RESULTS: Semen samples and Percoll-washed spermatozoa from men with a spinal cord injury produced reactive oxygen species at much higher frequency and levels than equivalent preparations from infertile men or healthy volunteers. There was an inverse relationship between the percentage of motility and reactive oxygen species production in Percoll-washed spermatozoa from men with a spinal cord injury. CONCLUSION: Semen samples and Percoll-washed spermatozoa from men with spinal cord injury produce high levels of reactive oxygen species that may be related to the low sperm motility and infertility observed in these men.

Origin of a motility inhibitor within the male reproductive tract.

Seminal plasma contains a motility inhibitor of demembranated reactivated spermatozoa. We investigated its origin within the reproductive tract. The highest level of inhibitor was detected in seminal vesicle fluids from the three species investigated (bull, rat, rabbit). Significant levels of inhibitor were also observed in prostatic fluids. Testes and epididymal fluids, as well as bulbo-urethral and coagulating gland homogenates were essentially devoid of inhibitor. On a mg protein basis, the inhibitor in seminal vesicle fluid was about four times less active than the inhibitor of seminal plasma. The high level of inhibitor in seminal plasma can not be explained by the synergistic effect of the combination of seminal vesicle, prostatic and epididymal fluids. Dialysis experiments suggested that the high level of inhibitor in seminal plasma was mainly due to the presence of a dialysable activator. This activator is capable of potentiating up to four-fold the inhibitor present in seminal vesicle fluid.

Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes.

Mammalian spermatozoa are sensitive to oxygen-induced damages mediated by lipid peroxidation of the cell membrane. The aim of this study was to evaluate whether reactive oxygen species (ROS) could also induce axonemal damage. When Percoll-separated spermatozoa were treated with hydrogen peroxide, or the combination xanthine and xanthine oxidase (X + XO), there was a progressive decrease, leading to a complete arrest, in sperm flagellar beat frequency. Once demembranated in a medium containing magnesium adenosine triphosphate (Mg.ATP), ROS-immobilized spermatozoa still reactivated motility; however, the percentage and duration of motility obtained in these tests gradually decreased to zero in the next hour. In 50% of the cases, motility of intact spermatozoa spontaneously reinitiated after 6 to 24 hours of immobilization due to ROS treatment, although with percentages and beat frequencies lower than those of untreated spermatozoa. Studies using ROS scavengers (such as catalase, superoxide dismutase, and dimethylsulfoxide) indicated that hydrogen peroxide was the most toxic of the ROS involved, but that .O2- and .OH probably also played a role in immobilization of spermatozoa by ROS. The data suggest that ROS induce a chain of events leading to sperm immobilization, that axonemes are affected, and that limited endogenous repair mechanisms exist to reverse these damages.

Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility.

Under moderate conditions, reactive oxygen species (ROS) have been shown to inhibit sperm motility after several hours of incubation. The rapid decrease in flagellar beat frequency observed within the first hour of contact between ROS and spermatozoa was associated with a rapid loss of intracellular adenosine triphosphate (ATP). Motility of intact spermatozoa ceased when their ATP concentration was reduced by 85 +/- 5%. Axonemal damage was confirmed when ROS-treated spermatozoa could not reactivate motility after demembranation in a medium containing magnesium adenosine triphosphate (Mg.ATP). However, in conditions allowing rephosphorylation of the axonemes (addition of cyclic adenosine monophosphate, or cAMP, and protein kinase or sperm extracts to the demembranation medium), the motility could reactivate. Three lines of evidence suggested that ATP depletion induced by ROS treatment was responsible for the effects observed in spermatozoa. First, the rapid decrease in intracellular ATP observed after ROS treatment was closely followed by a decrease in beat frequency, loss of intact sperm motility, and axonemal damage due to insufficient phosphorylation. Second, incubation of spermatozoa with the combination pyruvate-lactate allowed maintenance of sperm ATP at a normal level and prevented the effects of ROS; furthermore, spermatozoa immobilized after ROS treatment, then supplemented with pyruvate-lactate, were able to reinitiate motility in parallel with an increase in their ATP level. Third, treatment of spermatozoa with rotenone, an ATP depleting agent, produced effects similar to ROS treatment and could also be reversed by the addition of pyruvate-lactate. These data are consistent with the conclusion that ROS treatment produced axonemal damage mostly as a result of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of a motility inhibitor within spermatozoa may explain the poor sperm motility of some infertile men.

The presence of motility inhibitors in seminal plasma and within spermatozoa from control and infertile men with poor sperm motility was investigated using demembranated reactivated human spermatozoa. No difference was found in the inhibitory capacities in seminal plasma of patients with poor sperm motility (less than 50%) when compared with that of fertile controls with motility above 50%. No correlation was observed between inhibitory capacity and sperm motility. However, when extracts of spermatozoa from these patients were tested for the presence of inhibitor, it was observed that three of nine patients had an inhibitor in their sperm extract. By contrast, all sperm extracts from fertile control subjects were devoid of inhibitor. It was concluded that the presence of a motility inhibitor in seminal plasma does not explain the poor sperm motility observed in patients. The presence of a motility inhibitor within spermatozoa, however, may represent an important factor in the etiology of the poor sperm motility observed in some patients.

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction.

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Low levels of nitric oxide promote human sperm capacitation in vitro.

The influence of nitric oxide on human sperm hyperactivation and capacitation, as well as its mechanism of action and its possible origin from spermatozoa were studied. Percoll-washed spermatozoa from healthy volunteers were incubated in Ham's F-10 medium supplemented or not with the nitric oxide-releasing agents, diethylamine-NONOate or spermine-NONOate, in combination or not with superoxide dismutase or catalase (scavengers for the superoxide anion and for hydrogen peroxide, respectively), or with sodium nitrate, sodium nitrite, or preincubated NONOates. Sperm hyperactivation, capacitation, and nitric oxide synthase activity were determined. High concentrations (0.3 to 1 mM) of NONOates reduced sperm motility. However, a lower concentration (0.1 mM) of the two NONOates had no effect on the percentage of sperm motility or of hyperactivation but resulted in a significant increase in sperm capacitation (24% +/- 4%) when compared to that of control spermatozoa (Ham's F-10 alone, 12% +/- 2%). Nitric oxide released by the NONOates appeared responsible for this effect because sodium nitrate or nitrite or preincubated NONOates (to exhaust the formation of nitric oxide) had no influence on sperm capacitation. Catalase, but not superoxide dismutase, abolished the capacitating action of the NONOates. No nitric oxide synthase activity was detected in spermatozoa, whether they were in their basal state or already capacitated. Furthermore, the nitric oxide synthetase inhibitor L-NG nitroarginine methyl ester did not block sperm capacitation induced by fetal cord serum ultrafiltrate. It is therefore concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of nitric oxide induce human sperm capacitation, and this action likely involves hydrogen peroxide.