Influence of fibronectin on the motility of human spermatozoa

The objective of the present study was to scrutinize the concentration of seminal fibronectin and the potential effects of exogenous fibronectin on human sperm motility. In addition, variability in the localization of fibronectin on human spermatozoa from andrological patients was studied, at both the light and electron microscopic levels. A total of 58 freshly ejaculated semen samples from patients attending for infertility treatment were submitted to sperm motility analysis and ELISA quantification of seminal plasma and cell-bound fibronectin. Immunofluorescence and immunoelectron microscopy revealed a relatively broad distribution pattern of fibronectin immunoreactivity on sperm heads and testicular spermatids. Addition of a fibronectin antiserum to vital spermatozoa in vitro at a moderate dilution (1:50) resulted in a significant increase in sperm motility. Purified plasma fibronectin, added at various concentrations to a preparation of live spermatozoa, was found to inhibit sperm motility in a dose-dependent manner. At concentrations from 0.18 to 0.5 mg fibronectin per ml ejaculate, no motile spermatozoa were recorded. Seminal plasma fibronectin ranged between 0.8 and 1000 μg/ml in infertility patients. There was a significant inverse correlation between sperm motility and seminal fibronectin in patients with oligo-astheno-teratozoospermia. In a preliminary study in patients with varicocele or hypogonadism, no such correlation was found.     

The influence of sperm density on the motility characteristics of washed human spermatozoa

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×10⁶ spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples ( P< 0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples ( P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 10⁶ spermatozoa/ml.
The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 10⁶ spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.     

Lipid peroxidation in human spermatozoa and maintenance of progressive sperm motility

Washed and deep frozen spermatozoa of 46 patients from an infertility clinic were separated into 3 different groups depending on their progressive motility (expressed as the sperm motile efficiency index according to Ishii et al ., 1977), determined 0 and 3  h after liquefaction, and were examined for their lipid peroxidation (LPO) potential by means of the thiobarbituric acid assay. Spontaneous and iron-catalysed generation (after 15, 30 and 60  min incubation) of thiobarbituric acid-reactive substances (TBARS) was measured spectrophotometrically. Spontaneous LPO revealed the highest generation of TBARS in the group of spermatozoa with initially normal progressive motility and decreased maintenance of progressive motility after 3  h of aerobic incubation. Iron-catalysed LPO generally revealed the highest amounts of TBARS after 60  min, especially in the aforementioned group with decreased motility maintenance. The differences between this group and the two other groups were highly significant. Consequently, spermatozoa with initially normal progressive motility but decreased maintenance of motility, generated higher amounts of stable LPO products than others, which suggests that loss of motility under aerobic incubation seems to be the consequence of enhanced LPO processes.     

Effect of zinc on human sperm motility and the acrosome reaction

This study has assessed the effect of zinc on human sperm motility and the acrosome reaction in vitro. Progressively motile human sperm were selected by swim-up and by glass bead columns and then incubated in a medium in which capacitation happened in an asynchronous way. Different doses of zinc (1, 10, 100 and 1000 microM) were added for periods of 2, 4 or 6 h. Other samples were incubated with zinc (1000 microM), and after 1 h incubation, the zinc was removed. Aliquots of each culture were used to evaluate progressive motility and the acrosome reaction using a triple-stain technique. Sperm motility was reduced when the amount of zinc added was greater than or equal to 100 microM, and these doses also caused a significant reduction in the % of sperm undergoing the acrosome reaction. After removal of zinc and further incubation in zinc-free medium for 1 h, an increase in the percentage of motile and acrosome-reacted sperm was observed. However, the increase in acrosome reaction did not reach the values observed in controls. Results suggest that extracellular zinc acts as an inhibitor of human sperm motility and the acrosome reaction (and/or capacitation and the acrosome reaction). This inhibitory effect is reversible and occurs in a dose-dependent fashion. The probable mechanisms involved are discussed.     

速冻和缓慢冷冻法对精子运动特征的影响

目的了解冷冻方法对人精子运动特征的影响。方法精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子运动特征分析。结果冷冻复温后精子运动能力与冷冻前精子运动能力比较明显下降(P<0.001,P<0.05);速冻与缓慢冷冻方法保存的精子运动参数相比较差异均无显著性(P>0.05)。结论冷冻保存易导致精子运动能力下降,速冻与缓慢冷冻方法对精子运动参数影响无明显差异。     

Factors affecting sperm motility VIII. Velocity and survival of human spermatozoa as related to temperatures above zero

The effect of temperature on motility and survival of ejaculated human spermatozoa was investigated with the aid of the multiple exposure photography (MEP) method for objective sperm motility determination. Fresh specimens from healthy donors were analyzed while being heated or cooled gradually, or during their storage at various temperatures from 0 to 48°C. Sperm velocity increased steadily from zero to 50.4 nm/sec between freezing point and body temperature. Thereafter, their activity dropped dramatically and total immobilization occurred at 45°C. The induced immobilization was reversible providing exposure to those extreme temperatures was short enough to prevent permanent damage. Sperm survived up to 24–48 h when stored at 23°C, while at body temperature, their survival in vitro was much shorter and rarely extended beyond 12 h. Their longevity was still shorter at higher or lower temperatures, especially when approaching 48°C. With the aid of the combined supravital staining and MEP methods it was found that temperatures of extreme levels induced mainly immobilization rather than a spermicidic effect. The possible mechanism of thermal effect on sperm motility and some of its practical implications are discussed.     

The effect of serum on motility of human spermatozoa in culture

The motility of spermatozoa was higher at 6-22 h in Ham's F10 culture medium supplemented with 20-30% human serum than with lower proportions of serum or with 1% human serum albumin. Heat treatment (56 degrees C, 1 h), charcoal extraction and dialysis (18,000 molecular weight cut-off) of the serum did not reduce sperm motility suggesting that high molecular weight components are responsible for maintenance of motility. Renewing the medium (Ham's F10 with 30% serum) at 7 h resulted in better sperm motility and velocity at 22 h. At 22 h the pO2, pCO2, pH and sodium concentrations were not different in replenished and control cultures, but the concentration of glucose was higher and that of potassium lower if the medium was changed. These results suggest that addition of 20-30% human serum and renewal of medium at intervals is beneficial for sperm culture and may be of use in in vitro fertilization.     

Reactive oxygen species induce reversible capacitation in human spermatozoa

Leucocytospermia has been associated with reduced sperm motility and decreased capacity for sperm-egg interaction. This effect could be mediated through reactive oxygen species (ROS), which, at high concentrations, induce lipid peroxidation and cellular death. The high impact on sperm capacitation reported in other mammalians should be more accurately assessed in the human because premature activation could affect sperm fertilizing capacity. The aim of this study was to evaluate both the effect of ROS on sperm capacitation and the protective role of seminal plasma. Spermatozoa selected by Percoll gradient were incubated with polymorphonuclear (PMN) granulocytes isolated from blood and activated by phorbol-12-myristate 13-acetate (PMA). Different seminal plasma concentrations were added immediately or after 3-h incubation. Afterwards, ROS production was evaluated by luminescence and sperm capacitation by chlortetracycline stain. In PMN granulocytes and sperm suspensions, the basal ROS production was < 32 x 103 relative luminescence units (RLU). After stimulation with PMA, the rate of ROS production by PMN increased to 1,287 x 103 RLU. Incubation of sperm with activated PMN resulted in an increase of sperm capacitation (37% versus 19% in the control). Immediate addition of seminal plasma caused a significant reduction in ROS (P < 0.01) and prevented sperm from capacitating. A higher effect in inhibition of sperm capacitation was observed when seminal plasma had been added after 3-h incubation. The results suggest that human sperm capacitation can prematurely be induced by exogenous ROS and this effect can be reversed by seminal plasma. Thus, human sperm capacitation is another functional parameter that may be affected by nonphysiological ROS production.     

Fertilization of hamster ova by human spermatozoa in relation to other semen parameters

Different indices of the zona-free hamster ovum test system were interrelated. High correlations were found between the fertilization percentage and the average number of penetrated spermatozoa/ovum (r = 0.99) and between the fertilization percentage and the number of spermatozoa attached to the ova surfaces (r = 0.72). Fertilization percentages from 63 subfertile patients were correlated with different semen factors assessed by multiple exposure photography (MEP). Low correlations were found between sperm concentration and fertilization percentage (r = 0.29) and between fertilization percentage and motile sperm count (r = 0.33). Spermatozoa progressing with high average velocity had low fertilization rates compared to specimen with moderate average velocity. Effects of sperm washing on the in vitro fertilizing capacity were studied in a group of 40 subfertile men. The fertilization percentages of 8 specimens -with visual seminal plasma abnormalities- increased significantly when immediate dilution of semen was applied.     

Toxicity of ornidazole and its analogues to rat spermatozoa as reflected in motility parameters

Ornidazole, a 5-nitro-imidazole derivative, has contraceptive properties in rats. As some ornidazole passes through the body unmetabolized after administration, the aim of this study was to investigate if ornidazole itself has a direct effect on sperm motility and whether these effects are limited or potentiated by the epididymal epithelium or structural changes to the molecule. Cauda epididymal spermatozoa or cauda epididymal tubules were incubated with ornidazole or ornidazole analogues, and motility parameters were subsequently measured by means of a computer-assisted sperm analysis (CASA) system. Incubation of spermatozoa in 2.5 mmol/L ornidazole for 4 h reduced their motility significantly, whereas incubation of epididymal tubules for 8 h in 10 mmol/L ornidazole was required to alter the velocity parameters of the enclosed spermatozoa upon release, suggesting that extratubular non-metabolized ornidazole can participate in inhibiting the motility in vivo. The in vitro toxicity of ornidazole derivatives depends on the halogen present and on the position of the nitro-group. The putatively inactive (R)- and the active (S)-ornidazole exhibited equivalent depression of sperm motility by direct incubation. This observation, and the differences between the in vitro and the in vivo efficacies of various ornidazole analogues, indicates distinct mechanisms of motility inhibition in the two experimental systems.     

Pentoxifylline increases the level of nitric oxide produced by human spermatozoa

Pentoxifylline (PF) is a xanthine derivative drug primarily used to treat peripheral vascular disorders. It is currently used in assisted reproductive technologies to enhance human sperm motility. However, the mechanism by which this enhancement occurs is not fully understood. Given that nitric oxide has been identified as a trigger to sperm motion, we asked whether nitric oxide modulates the stimulatory effect of PF on sperm motility. A total of 41 semen samples from infertile males were studied. Nitric oxide production in the presence of 5 mm PF was tested using different bio‐analytical methods (spectrophotometry, fluorometry and fluorescence microscopy). The spectrophotometric determination showed higher levels of nitrite, an indirect measure for nitric oxide, in sperm samples supplemented with PF compared to controls. The fluorometric experiment showed higher 4, 5‐diaminofluorescein triazole, a product from the reaction between nitric oxide and 4, 5‐diaminofluorescein diacetate, after adding PF to spermatozoa. The fluorescence microscopy images of the spermatozoa supplemented with PF showed higher green fluorescence, indicating higher 4, 5‐diaminofluorescein triazole levels, compared to controls. It is concluded that PF enhances nitric oxide production in human spermatozoa, which explains, at least in part, the mechanism by which PF stimulates human sperm motility.     

Oxygen uptake by mitochondria in demembranated human spermatozoa: a reliable tool for the evaluation of sperm respiratory efficiency

In this work we report a relatively simple and fast method for analysing oxygen consumption and therefore mitochondrial functionality, in individual human ejaculates. This oxygraphic method requires a low number of cells, is highly reproducible and linearly correlates with sperm concentration. Our results have shown that oxygen uptake by mitochondria of demembranated sperm cells from normozoospermic subjects is significantly stimulated by a large set of respiratory substrates and ADP. The respiratory control ratio (RCR) values indicate a good coupling between respiration and phosphorylation by sperm mitochondria and thus a well preserved integrity of the mitochondria themselves. Interestingly, whereas the rates of oxygen uptake, as expected, changed with different sperm concentrations, the RCR values remained constant, thus demonstrating a linear response of the assay. In asthenozoospermic subjects, however, a significant decrease in the sperm respiratory efficiency was found. The results obtained suggest that this method, besides its potential clinical application, could be useful for a deeper understanding of the biochemical properties of sperm mitochondria and their role in ATP production in human spermatozoa.     

A new CentriSwim procedure to increase the recovery of motile spermatozoa

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.     

The effects of pentoxifylline on the generation of reactive oxygen species and lipid peroxidation in human spermatozoa

Summary. The aims of this study were to compare the in vitro effects of 3.6 mM and 7.2 mM pentoxifylline on the ability of spermatozoa to generate reactive oxygen species (ROS) and on lipid peroxidation (LPO). Semen samples were obtained from 10 asthenozoospermic men who had been previously identified as producing ROS after addition of Phorbol 12-myristate 13-acetate (PMA) during the screening of patients attending with male factor infertility. Spermatozoa were prepared by a swim-up technique from unprocessed semen and divided into 3 aliquots. To the control aliquot [A] an equal volume of BWW medium was added. To aliquots B and C an equal volume of BWW medium containing pentoxifylline was added to obtain final concentrations of 3.6 and 7.2 mM, respectively. ROS production was measured from peak luminescence (mV 10⁻⁷ sperm) using a lucigenin chemiluminescent probe. LPO was also measured in the medium surrounding the spermatozoa after 30 min exposure to pentoxifylline using the thiobarbituric acid (TBA) assay for malondialdehyde (MDA). The reduction in ROS production was significantly greater in the samples exposed to 7.2 mM pentoxifylline as compared with the control and 3.6 mM pentoxifylline samples. There was no significant difference in peak luminescence between control and 3.6 mM pentoxifylline specimens. Both concentrations of pentoxifylline caused comparable reductions in MDA concentration in the medium ( P <0.05) surrounding the spermatozoa compared with control after 30 min exposure. Extracellular ROS generation may damage surrounding healthy spermatozoa. These findings suggest that higher concentrations of pentoxifylline are protective against ROS release in susceptible spermatozoa and may also reduce collateral LPO.     

Changes from puberty to adulthood in the concentration, motility and morphology of mouse epididymal spermatozoa

A systematic study of the concentration, motility and morphology of epididymal spermatozoa was undertaken in mice of the OF1 strain, in order to characterize the changes observed during puberty. The comparative development of these 3 parameters was followed from days 30 to 90. A detailed morphological system of classification was established covering both individual and multiple abnormalities. At puberty, the first spermatozoa to appear were few in number, showed poor motility and were extremely atypical. Subsequently, the number of atypically-shaped spermatozoa diminished, and their concentration and motility exhibited parallel increases. At 60 days, the values for these 3 parameters and the proportions of normal and abnormal spermatozoa became stable. During puberty, various forms of disruption of the midpiece as well as the presence of extremely atypical detached flagella resulted in a special pattern of sperm morphology, although all of these abnormal features disappeared at 40 days.     

Serum factors stimulate the motility of human spermatozoa

Motile human sperm were collected from a Percoll gradient and the effects on sperm motility of human serum, various serum fractions, follicular fluid and seminal plasma were assessed. In culture medium alone (RPMI-1640) sperm motility was lost after about 5 h. The addition of male blood serum both enhanced sperm motility and prolonged viability very significantly. Albumin, seminal plasma and follicular fluid all stimulated sperm motility but to a much lesser extent than did blood serum. No difference was noted between male serum or female serum which had been collected during the follicular or luteal phases of hormone-stimulated cycles and which contained high levels of oestradiol. Serum fractions obtained by separation on Sephacryl S-300 column were tested for their ability to enhance sperm motility. The most pronounced effect, much superior to that achieved by the albumin fraction, was obtained by a fraction with a molecular weight of around 200 kD. In conclusion, certain factors in human serum, which are different from albumin, strongly support sperm motility. The high serum concentrations of oestradiol resulting from hormone stimulation for in-vitro fertilization do not invalidate the use of serum from the same patient during sperm preparation, or in the medium used for ovum insemination and culture.     

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.     

Aquaporin3 expression and the potential role of aquaporins in motility and mitochondrial membrane potential in human spermatozoa

Aquaporins are water selective channels which play important roles in cell volume regulation during the transmission of spermatozoa to female tract. This study investigated the expression of aquaporin3 and determined the role of aquaporins in human sperm motility and mitochondrial membrane potential (MMP). RT-PCR and flow cytometry analysis were done to investigate aquaporin3 expression levels, and immunolocalisation of aquaporin3 in the spermatozoa was detected using immunocytochemical analysis. The sperm suspension was divided into four groups of spermatozoa: (a) Spermatozoa at 0 hr, (b) spermatozoa in control group, (c) spermatozoa treated with HgCl₂ (as an aquaporin inhibitor) and (d) spermatozoa treated with HgCl₂+ and 2-mercaptoethanol. The sperm samples were examined in terms of sperm motility and mitochondrial membrane potential. Results confirmed aquaporin3 expression in human spermatozoa and immunocytochemistry results showed an intense immunoreactivity in whole sperm tail. After 60 min, HgCl₂ showed a significant decrease in motility and MMP compared to the control group. At this time point, 2-mercaptoethanol in the HgCl₂+ 2-mercaptoethanol group reversed the effects of HgCl₂ as compared to the HgCl₂ group. Present study showed the expression and immunolocalisation of AQP3 in human spermatozoa and the potential role of AQPs in the sperm motility and MMP.