Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa

Mitochondrial membrane potential (ΔΨm) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC‐1 has been widely used to assess sperm ΔΨm; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with ΔΨm. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm ΔΨm. The results showed that TMRM accurately detects sperm populations displaying either high or low ΔΨm. Moreover, TMRM was able to measure sperm ΔΨm under the experimental conditions in which JC‐1 had previously presented difficulties. Differences in ΔΨm according to sperm and semen quality were properly detected and a positive correlation between ΔΨm and conventional semen parameters was observed. Finally, a positive correlation was found between the ΔΨm measurement by TMRM and by the widely used JC‐1. In conclusion, TMRM is a simple, time‐effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in ΔΨm with efficiency comparable to JC‐1 without its limitations.     

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.     

Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa

This study was designed to evaluate the effects of semen processing on the generation of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in spermatozoa, and to develop reliable indexes for the evaluation of sperm quality during sperm preparation. Swim-up and density gradient centrifugation methods were used to separate semen in oligoasthenoteratozoospermia (OAT), leucocytospermia (LC) and normozoospermia groups. Levels of ROS and MMP were measured by flow cytometry. Before preparation, the patients with abnormal semen parameters had a lower MMP and higher ROS, and there was a negative correlation between MMP and ROS. The levels of MMP and ROS increased significantly, especially ROS produced by swim-up. A significant difference was found between the correlation of MMP and total normal motile sperm count after preparation in the OAT group. The level of ROS was associated with the amount of white blood cells in the LC group. The MMP can be used as an objective index to evaluate the sperm quality of OAT patients, and the combination of MMP and ROS can be used to assess the efficiency of sperm preparation in LC patients. These findings can guide selection of the ideal sperm separation technique for different sperm samples.     

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.     

Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility

Aim: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. Methods: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. Results: The oxygen consumption averaged 0.24μmol/106 sperm×24 h, 0.28μmol/106 live sperm×24 h and 0.85μmol/106 live & motile sperm×24 h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/106 motile spermatozoa×24 h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/106 spermatozoa×24 h. The correlation of the oxygen/ energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, res     

Large volume cryoprotectant‐free vitrification: an alternative to conventional cryopreservation for human spermatozoa

Vitrification is a simple and cost‐effective method for the storage of human spermatozoa without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. As a result, solidification of living cells without the formation of ice crystals is achieved during cooling. This study aimed to compare cryoprotectant‐free vitrification to conventional cryopreservation protocols. Semen samples (n = 35) were collected from patients seeking diagnostic assistance at the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. Samples were processed using a discontinuous density‐gradient centrifugation method. Washed samples were split into two aliquots and cryopreserved either by means of cryoprotectant‐free vitrification (sucrose + 1% albumin) or conventional slow freezing (TEST‐yolk buffer). Post‐thawing, the sperm motion parameters, mitochondrial membrane potential (Δψm) and DNA fragmentation were compared between the two groups. No significant differences were observed in the sperm motility parameters (P > 0.05). Significantly higher percentages of Δψm (11.99% ± 4.326% versus 6.58% ± 1.026%; P < 0.001) and lower percentages of DNA fragmentation (2.79% ± 1.017% versus 3.86% ± 1.38%; P < 0.01) were observed when comparing cryoprotectant‐free vitrification to conventional cryopreservation. Cryoprotectant‐free vitrification is a rapid and promising alternative to conventional methods resulting in good‐quality spermatozoa post‐thaw.     

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。     

Overtime expression of plasma membrane and mitochondrial function markers associated with cell death in human spermatozoa exposed to nonphysiological levels of reactive oxygen species

In many cell types, the potential of reactive oxygen species to induce death processes has been largely demonstrated. Studies in spermatozoa have associated the imbalance of reactive oxygen species and phosphatidylserine externalisation as an apoptosis marker. However, the lack of consensus about time effect in the joint expression of these and other death markers has made it difficult to understand the set of mechanisms influenced beyond the concentration effect of reactive oxygen species to stimulate cell death. Here, the plasma membrane permeability and integrity, phosphatidylserine externalisation and mitochondrial membrane potential were jointly evaluated as death markers in human spermatozoa stimulated with H₂O₂. The results showed a profound and sustained effect of dissipation in the mitochondrial membrane potential and an increased phosphatidylserine externalisation in human spermatozoa exposed to 3 mmol⁻¹ of H₂O₂ at 30 min. This was followed by an increased membrane permeability after 45 min. The last observed event was the loss of cell membrane integrity at 60 min. In conclusion, mitochondria are rapidly affected in human spermatozoa exposed to reactive oxygen species, with the barely detectable mitochondrial membrane potential coexisting with the high phosphatidylserine externalisation in cells with normal membrane permeability.     

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.     

Wave parameters of the sperm flagellum as predictors of human spermatozoa motility

Summary. We characterized the undulatory movement of the sperm's flagellum as a sigmoid wave by measuring the absolute and linear speed of the sperm, period, amplitude and length of the flagellum's wave, and the segment comprised between the head and the origin of the movement in the flagellum. These parameters were correlated with traditional ones used to determine the pattern of movement of the sperm. Our results show that wave parameters are useful predictors of sperm motility. They correlate among themselves, and thus, a few wave parameters may characterize the sperm motility. The advantage of wave parameters is that they can be easily obtained and can be eventually associated to the sperms' internal morphology.     

Assessment of mitochondrial potential: implications for the correct monitoring of human sperm function

Mitochondrial membrane potential (MMP) is an indicator of sperm functionality that can be assessed using specific fluorescent markers. However, the ability of distinct probes to dynamically evaluate sperm MMP has not been determined. In the present study, human sperm samples were independently labelled with MitoTracker Green, MitoTracker Red and JC-1. The ability of each probe to correctly monitor MMP was determined by incubating sperm with MMP disruptors (KCN, FCCP and valinomycin). Similarly, the effect of distinct fixatives (formaldehyde and methanol) was also tested. The three mitochondrial probes provided similar results, and were able to monitor changes in MMP when sperm had been previously incubated with MMP-disrupting agents. However, only JC-1 could, to a small extent, mirror MMP alterations after sperm labelling. Unexpectedly, the three probes were able to stain some pre-fixed sperm, even though this behaviour was very variable, especially for MitoTrackers. On the other hand, none of the probes was shown to be reliably fixable. Of the three probes tested JC-1 seems to be the most adequate, nevertheless, the choice of an MMP-specific probe may depend on the aim of each experimental setting and appropriate controls must always be performed.     

The motility of demembranated human spermatozoa is inhibited by free calcium ion activities of 500 nmol/L or more

A number of studies have demonstrated that high calcium ion activities inhibit sperm motility, but little is known about the effect of different calcium activities close to the physiological range. Therefore, we investigated whether raising calcium activities within the submicromolar range would inhibit the motility of demembranated human spermatozoa. Spermatozoa were demembranated with Triton X-100 and motility was measured objectively by computer assisted semen analysis. Motility, reactivated by 1 mol adenosine 5'-triphosphate (AlphaTauP)/L, was short lived, with maximum activity only sustained for about 1 min. Reactivated motility was not affected by 50 micromol cAMP/L. The amplitude of lateral head displacement was significantly greater at room temperature than at 37 degrees C, but there were no significant differences between the percentage of sperm motile or their velocity at the two temperatures. The calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at 1 mmol/L was included in the demembranation-reactivation medium, and free calcium ion activities were calibrated using the fluorescent calcium probe Fura-2. Calcium ion activities of > or =500 nmol/L significantly inhibited the percentage of demembranated-reactivated spermatozoa that were motile, and the velocity and lateral head displacement of these cells. The range of intracellular calcium activities in spermatozoa from 24 cryopreserved ejaculates was 110-534 nmol/L; roughly twice the value in fresh spermatozoa. Therefore, calcium ion activities in the range observed in cryopreserved spermatozoa can inhibit the activity of demembranated human spermatozoa.     

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.     

The relationship between morphology, motility and zona pellucida binding potential of human spermatozoa

Summary. Prediction of the fertilizing potential of human gametes under in vitro conditions has been a major field of interest of assisted reproductive programmes. However, sperm morphology has been regarded as a predictor of human in vitro fertilization rate. This paper prospectively evaluates the relationships among normal sperm morphology and (1) motion characteristics viz. curvilinear velocity (VCL), straight line velocity (VSL), and linearity (LIN) (n = 37) and (2) spermzona pellucida binding capacity under HZA conditions (n = 144) of two separate groups of infertile couples. Semen was evaluated for sperm concentration, percentage motility, forward progression, and percentage normal morphology (strict criteria). The motility characteristics were measured using a computerized Sperm Motility Quantifier (SMQ). The zona binding potential of sperm was evaluated using the hemizona assay. Firstly, the VCL significantly differred between the P-pattern and both the G (72.9 ± 7 vs. 86.3±16 μm s^?1; P = 0.04) and N patterns (72.9 ± 7 vs. 91.0 ± 15 μm s^?1; P = 0.002). The VSL differed only between the P and N patterns, being 19.7 ± 7 vs. 32.6±15 μm s^?1 (P = 0.02), respectively. No significant differences in LIN were noted between any of the three patterns. The sperm concentration differed significantly between the P and both the G (37.9±35 vs. 80.8 ± 9 × 10⁶ ml^?1; P = 0.03) and the N patterns (37.9 ± 35 vs. 89.7 ± 72 × 10⁶ ml^?1; P = 0.05). Significant differences were observed in the percentage motility between the P and both the G (38.0 ± 21% vs. 43.7 ±9%; P = 0.03) and the N patterns (38.0 ± 21% vs. 52.1±8%; P = 0.04). In the second study, the hemizona indices (HZI) differed significantly between the P and both the G (29.3 ± 26% vs. 57.6 ± 62%; P = 0.01) and the N patterns (29.3 ± 26% vs. 102.4 ± 80%; P < 0.001). The G and N patterns also differed significantly in their HZI (57.6 ± 62% vs. 102.4 ± 80%; P = 0.005). Sperm concentration differed between the P and both the G (32.8 ± 29 vs. 76.1±54 × 10⁶ ml^?1; P < 0.001) and the N patterns (32.8 ± 29 vs. 95.44 ± 61 × 10⁶ ml^?1; P < 0.001). The percentage motility differs significantly between the P pattern and both the G (41.2± 17% vs. 50.9±11%; P = 0.002) and the N patterns (41.2±17% vs. 53.4±11%; P = 0.001). Sperm morphology seems to be indicative of important functional characteristics of spermatozoa, for example motility and zona pellucida binding.     

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 μg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.     

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

目的:研究精子特异性钙离子通道(CATSPER1)蛋白和 mRNA 在人类睾丸和射出精子中的表达,检测抗人 CATSPER1 抗体体外对人类精子前向运动的影响以初步探讨人 CATSPER1的功能及其作为免疫避孕靶点的可能性。方法:用逆转录聚合酶链反应(RT-PCR)和间接荧光免疫组化法分别检测液氮冻存人睾丸组织和12例正常精液标本中 CATSPER mRNA 和蛋白的表达。12例正常精液标本经 Percoll 不连续密度梯度离心法上游,上游后精子分别与终浓度为20 mg/mL、4 mg/mL、0.8 mg/mL 的抗人 CATSPER1抗体孵育,以抗体的储存液(0.01mol/L PBS,pH7.4)为对照,1 h、2 h、6 h 后用计算机辅助精液分析仪(CASA)分析前向运动精子百分率(WHO 际准,a 级 b 级)和快速前向运动精子百分率(WHO 标准,a 级)。结果:CATSPER1的 mRNA 存在于人类睾丸组织和射出精子中。CATSPER1蛋在人类睾丸主要表达于精子细胞,并定位于射出成熟精子尾部的主段。实验中用所有浓度的抗人 CATSPER1机体体外在1 h、2 h、6 h 时段均使前向运动精子百分率和快速前向运动精子百分率下降,都与对照组差异显著。结论:CATSPER1在人类睾九中呈减数分裂及减数分裂后式表达,存在于射出精子中的 mRNA 将是比睾丸穿刺方便、更易接受的用于研究 CATSPER1和不育检查的靶点。这些结果也提示人 CATSPER1是免疫避孕的有效靶点。     

High HbA1c levels affect motility parameters and overexpress oxidative stress of human mature spermatozoa

The aim of this study is to investigate, by a validated in vitro model, the effect of diabetic plasma on ejaculated human spermatozoa. Plasma of 51 male diabetic patients (mean age 62.28 ± 9.28 years) was selected according to their HbA_1c level: low HBA_1c ≤ 5% (31 mmol/mol), moderate HBA_1c 6%–8% (42–64 mmol/mol) and high HBA_1c ≥ 10% (86 mmol/mol). The plasma was tested on eighteen normal semen samples by analysing gametes motility using a computer Sperm Class Analyzer® and their corresponding oxidative stress (OS) status using thiobarbituric acid-reactive substances assay. The results indicated that diabetic plasma affected all sperm motility parameters with high HbA_1c showing the most important deleterious effects. Low gametes' straight-line velocity was observed in high HbA_1c level, mainly after 20 min of co-incubation (8.78 ± 0.47 µm/s). Also, the highest lipid peroxidation (nmoles MDA/10⁸ SPZ) was observed in high HbA_1c values (0.92 ± 0.09), higher than those in spermatozoa treated with H₂O₂ (0.85 ± 0.04). Conclusively, a direct impact of diabetic plasma on spermatozoa is revealed with overexpression of OS as the underlying mechanism. These findings suggested that it is strongly recommended to control clinically the glycaemic level and OS in diabetic patients for the maintenance of male fertility.     

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.     

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.