Copper: a biphasic regulator of caprine sperm forward progression

Abstract

Copper is essential for spermatogenesis and its presence has been demonstrated in male and female reproductive fluids in several mammalian species. However, little is known about the physiological significance of this trace element in the regulation of forward progression of mammalian sperm cells which is essential for sperm fertility potential in vivo. The purpose of this investigation was to determine the physiological role of the bivalent copper ion (Cu²⁺) on mammalian sperm forward motility using a chemically-defined medium and caprine cauda epididymal sperm model. Sperm forward motility was significantly enhanced by Cu²⁺ in a dose-dependent manner; maximal activation (approx 20%) was noted at the 5?µM level of the metal. Above 10?µM Cu²⁺ sperm motility decreased, showing that Cu²⁺ exerts a biphasic regulation on sperm motility. These findings have been confirmed using a spectrophotometric motility assay, an objective method of motility analysis. At lower concentrations (up to 5?µM), copper enhanced sperm membrane lipid peroxidation as well as the level of intra-sperm cyclic adenosine mono phosphate (c-AMP), but at a higher level it caused marked inhibition of both of the biochemical parameters. The observed correlation of Cu²⁺-dependent biphasic modulation of sperm membrane lipid peroxidation and intrasperm c-AMP with sperm forward motility is consistent with the view that Cu²⁺ regulation of sperm motility is mediated by membrane lipid peroxidation, which in turn modulates the level of intra-sperm c-AMP, a well-known activator of sperm motility.     

Toxicity of heavy metals using sperm cell and embryo toxicity bioassays with Paracentrotus lividus (Echinodermata: Echinoidea): comparisons with exposure concentrations in the Lagoon of Venice,Italy

Sperm cell and embryo toxicity tests using the Mediterranean sea urchin Paracentrotus lividus Lmk were performed to assess the toxicity of As3+, Cd2+, Cr3+, Ni2+, Pb2+, Cu2+, Zn2+, and Hg2+. The aim of this study was to improve information about the comparative sensitivity of sea urchin bioassays to the heavy metals, which are an important cause of contamination in the ecosystem of the Lagoon of Venice. Considering the data in mM/L, the order of toxicity is Hg2+ > Cu2+ > Zn2+ > As3+ > or = Cr3+ > or = Cd2+ > or = Pb2+ > or = Ni2+ for the sperm cell test and Hg2+ > or = Pb2+ > Cu2+ > Zn2+ > Cd2+ > Ni2+ > As3+ > or = Cr3+ for the embryo test. New toxicity data for metals expressed as median effective concentration (EC50) and no-observed-effect concentration (NOEC) are reported for the Mediterranean species. Accurate observations of embryotoxic effects at increasing metal concentrations were done, detecting some different behaviors in metal toxicity. Toxicity data compared with water column and pore-water concentrations recorded in the Lagoon of Venice (Italy) demonstrate the potential ability of bioassays using sea urchin to detect important contaminants in this ecosystem.     

HYALURONIC ACID INCREASES MOTILITY/INTRACELLULAR CA 2+ CONCENTRATION IN HUMAN SPERM IN VITRO

This study investigated the mechanisms of the stimulatory effect of hyaluronic acid on motility in human sperm in vitro. A method, involving the measurement of forward progression through an agarose gel, was used to measure sperm motility quantitatively. Changes in intracellular Ca 2+ concentrations in sperm were detected using the fluorescent dye Fluo-3. The effects of hyaluronic acid (6.5, 65, 650 ng/mL) and nifedipine (32 nM) on sperm motility were investigated. The effects of hyaluronic acid, nifedipine (32 nM), A23187 (32 µM), and a monoclonal antibody to human CD44 (1 µg/mL) on changes in intracellular CA 2+ concentrations were investigated. Hyaluronic acid significantly ( p <. 008) stimulated sperm motility and this was partially inhibited by nifedipine.A23187 significantly ( p <. 005) increased intracellular CA 2+ concentrations. Hyaluronic acid significantly ( p <. 04) increased intracellular Ca 2+ concentrations and this was inhibited by nifedipine and a monoclonal antibody to human CD44. Hyaluronic acid stimulated human sperm motility by increasing intracellular Ca 2+ concentration, partially via an influx of extracellular Ca 2+.     

A comparative evaluation of migration sedimentation method for sperm preparation

The effectiveness of semen preparation using the migration-sedimentation (MS) method was evaluated, and compared to density gradient centrifuge and swim-up combination (DGC+SU). Sperm selection using MS is based on motility, thus, deleterious effects for which centrifugation has been blamed, are believed to be avoided. Normozoospermic male patients who had more than 10% forward progressive motile sperm in their ejaculate were included in the study. Spermatozoa selected by two different methods were investigated and compared according to sperm motility, concentration, morphology, vitality, DNA fragmentation, and presence of persistent histones. The concentration and motility of sperm in the MS group was improved when compared to the DGC+SU group, but the difference between groups was not significant. The proportion of sperm with normal morphology was found to be 12.19 ± 6.45% vs. 10.67 ± 5.44%, vitality rate was 74.09 ± 16.65% vs. 70.45 ± 16.78%, DNA fragmentation rate was 3.91 ± 3.96%, vs. 2.95 ± 3.33%, presence of persistent histone proportion was 10.59 ± 13.40%, vs. 8.86 ± 7.89% in DGC+SU and MS groups respectively, without significance. The simple technique avoids centrifuge-based damage.     

硼作业工人精子质量的初步分析

目的研究硼暴露对男性精子质量的影响。方法于2002年在辽宁省某硼矿区及硼加工厂选择硼暴露组(60人)和对照组(9人)男性研究对象,进行体格检查和精液分析。精液分析使用综合光学可视精子分析仪和光学生物显微镜。结果对照组的精子密度、精子前向运动百分率、a级精子完全符合WHO标准,而暴露组各项指标均有不符合WHO标准的样本,高达44.8%的样本精子前向运动百分率低于WHO标准(≥50%)。暴露组和对照组的精子存活率、精子前向运动百分率、a级精子、精子的曲线运动速度和直线运动速度差异有统计学意义(P<0.01),并且暴露组精子各项指标的均值除精子密度外均低于对照组。结论硼暴露的工人精子质量有所下降。     

Curcumin: a potential vaginal contraceptive

The purpose of this investigation was to evaluate the sperm-immobilizing effects of curcumin, a plant-derived diferuloylmethane compound. Washed human healthy sperm were suspended in Ham's F10 and exposed to varying concentrations of curcumin. Sperm motility was evaluated and changes in sperm mitochondrial transmembrane potential (MTP) was quantified by flow cytometry. Incubation of normal human sperm with curcumin resulted in a dose- and time-dependent loss of sperm motility. At lower concentrations (30 g/mL), curcumin produced a significant (20%) decrease in sperm motility within 30 min without significant effects on sperm viability. An instantaneous (>50%) loss of sperm motility was observed with higher concentrations (300 g/mL) of curcumin and a total loss of sperm motility was achieved within 60 min. A significant reduction in sperm MTP was found with all doses of curcumin tested. Our results indicate that curcumin has a selective sperm-immobilizing effect, in addition to a previously studied anti-HIV property. This compound may have potential clinical applications as a novel intravaginal spermicidal agent for contraception and HIV prevention.     

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r2?=?0.236; P?=?0.002) and DNA fragmentation (r2?=?-0.318; P?40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.     

AGGREGATION OF HUMAN SPERM AT HIGHER TEMPERATURE IS DUE TO HYPERACTIVATION

Chemotaxis of sperm cells to chemicals and hormones, such as progesterone, helps us to understand the concept of sperm transport. Here, the hypothesis was that heat increased sperm hyperactive motility, which caused the sperm to aggregate at the higher temperature. The objectives were (1) to determine the concentration of sperm at both halves of an artificial female reproductive tract made from a hermetically sealed cryopreservation straw filled with culture medium and placed with each end at different temperatures, and (2) to analyze the motility or kinematic parameters and hyperactivation of sperm found at the different temperatures. Cryopreserved-thawed human donor sperm (N = 6) were pooled and processed through 2-layer colloid solution. Analyses of the motile sperm were carried out and the washed sperm were homogeneously mixed and pipetted into several 0.5-mL French cryopreservation straws and heat-sealed. The control substance, consisting of acid-treated sperm, was also placed in several straws. The plastic straws of sperm were placed half at 23oC and half was at either 37 or 40oC. After 4 h, sperm at different sections of the straws were analyzed using the Hamilton Thorn motility analyzer (HTM-C). After 4 h of incubation, the concentration of sperm was doubled at the 40oC heated half of the straw when compared with the other half of the straw at 23oC. There were no differences in sperm concentration in the straw kept half at 37oC and half at 23oC. There were significantly higher percent motility, mean average path velocity, straight line velocity, lateral head displacement, and percent hyperactivation in sperm at the 40oC tempera ture. The aggregation of sperm at the higher temperature of 40oC may be due to enhanced motility, increased sperm velocities, and a 10-fold increase in hyperactivation at that temperature. The 37oC temperature was not sufficient to attract sperm. Sperm cells migrating into the higher temperature site of ovulation begin nonprogressive hyperactivation movement, which is the physiological ''brake" to detain the sperm at the site of ovulation.     

Effect of sperm concentration, medium osmolality and oocyte storage on artificial fertilisation success in a myobatrachid frog (Limnodynastes tasmaniensis)

The present study optimised artificial fertilisation and oocyte storage conditions in Limnodynastes tasmaniensis (Myobatrachidae). Data on general reproductive biology, the effect of sperm motility and concentration, medium osmolality and oocyte storage on artificial fertilisation success are presented. Egg number was most strongly correlated with bodyweight (r = 0.819). Sperm yield was correlated with testes weight (r = 0.827), which was strongly correlated with snout-vent length (r = 0.772). Optimal artificial fertilisation occurred in 0-7 mOsm kg(-1) amphibian Ringer, similar to ranid, bufonid and hylid species. High fertilisation rates were achieved using spermatozoa with little forwards progressive motility at comparatively low concentrations (3 x10(4) sperm cells mL(-1)) and with no relationship between percentage sperm motility and fertilisation success (correlation of fertilisation rate with sperm motility after activation: r = -0.145). Oocytes stored in 5 mOsm kg(-1) solutions showed no significant decline in fertilisability after 2 h, showing that swelling of the jelly surrounding the eggs does not prevent sperm from fusing with the oocyte in this species. Fertilisability of oocytes was extended to > 4 h in medium to high osmolality solutions (124-271 mOsm kg(-1)). These data allow for the future use of L. tasmaniensis in developing assisted reproductive technology protocols for foam-nesting myobatrachid species, many of which are now threatened with extinction in the wild.     

EFFECTS OF EXPOSURE TO NEW INHALATIONAL ANESTHETICS ON SPERMATOGENESIS AND SPERM MORPHOLOGY IN RABBITS

The effects of chronic exposure to new inhalation anesthetics (sevoflurane and isoflurane) on spermatogenesis and sperm morphology were examined in 23 rabbits, randomly divided in 3 groups. Rabbits received 20 exposure hours (four hours/day × 5 days), as follows: group I: 2.3% (1.2 MAC) sevoflurane + 2L/min oxygen, group II: 1.3% (1.2 MAC) isoflurane + 2L/min oxygen, and group III (control): 2L/min oxygen. Semen was collected on the 12th, 19th, 26th, 33rd, and 41st days of exposure. Sperm concentration, motility and morphological changes were evaluated. On the 41st day, testicular biopsies were taken and observed with light microscopy. Sperm concentration and motility significantly decreased in the sevoflurane and the isoflurane groups, compared to control. There were no significant changes in the control group. It is concluded that chronic exposure to the new inhalational anesthetics had negative effects on spermatogenesis and sperm morphology.     

Effects of Timing of Ovum Recovery,Cumulus Cells,Sperm Preincubation Time,and Ph on in Vitro Fertilization in C57BL/6 Mice

The effects of the time of ova recovery following hCG injection, the presence of cumulus cells, duration of sperm preincubation time, and pH on in vitro fertilization in C57BL/6 mice were investigated. Significantly more ova were recovered at 14 h than at 12 h post-hCG injection. Although the number of ova recovered at 16 h was similar to that at 14 h, the percentage of ova showing degeneration increased. The presence or absence of cumulus cells had no effect on ovum fertilization rates, although sperm incubated with cumulus-intact ova underwent the acrosome reaction sooner than those incubated with ova lacking cumulus cells. Sperm motility was sustained slightly longer in the presence of cumulus-free ova than in the presence of cumulus-intact ova. The average percent fertilization of eggs combined with sperm preincubated 1 h was higher than that of sperm preincubated 0 and 0.5 h. Longer preincubation times resulted in a linear decrease in the percent motility and an increase in the percent acrosome reactions. A plot of the number of sperm attaching to the egg vs. coincubation time produced a bell-shaped curve in each case. The greatest number of sperm attaching to the egg occurred between 45 min and 1 h. When the medium was at pH 7.4, fertilization rates were higher than at pH 7.0, 7.2, or 7.6, as were the percent sperm motility and the number of sperm attached to ova. A pH of 7.6 induced 60% of the sperm to undergo the acrosome reaction immediately, and within 1 h all motility was lost.     

汽车尾气对人精子运动能力影响的探讨

西文分析了交警和男性师生精液的精子运动能力参数和血铅水平。结果显示：与对照组比较，精子活动能力降低、精子琥珀酸脱氢酶活性明显抑制。而且ＳＤＨ活性与血铅水平呈铡相关。交警精子活动能力减弱可能和铅干扰精子线粒体能量代谢有关。     

Effects of mechanical stresses on sperm function and fertilization rate in mice

In this study, we investigated whether any of the observed changes in mouse sperm function tests secondary to mechanical stresses (centrifugation and pipetting) correlate with sperm fertilization ability. Chinese Kunming mice were used as sperm and oocyte donors. Sperm samples were allocated evenly into centrifugation, pipette, and control groups. Sperm plasma membrane integrity (PMI), mitochondrial membrane permeability (MMP), baseline and stimulated intracellular ROS, and sperm fertilization ability were measured by hypo-osmotic swelling, flow cytometry, and fertilization tests. Parallel studies were conducted and all tests were repeated six times. Our results showed that after centrifugation, the progressive motility, average path velocity, and overall sperm motility and PMI decreased significantly (p < 0.05). In addition, the MMP level decreased significantly in viable sperm when the centrifugation condition reached 1,400 g × 15 minutes (p < 0.05). When pipetting was performed two or more times, progressive motility, average path velocity, and overall sperm motility decreased significantly (p < 0.05); when it was performed four or more times, sperm membrane integrity and intracellular basal ROS level of viable sperm was also significantly decreased (p < 0.05). In conclusion, various mechanical stresses seem to affect sperm function, however this does not appear to alter fertilization rate. Laboratory handling steps should be minimized to avoid unnecessary mechanical stresses being applied to sperm samples.     

吸烟对精子DNA完整性、精子参数的影响

目的:探讨吸烟对精子DNA完整性、精子参数的影响。方法:调查191例男性不育患者的吸烟情况,采用吖啶橙荧光染色后检测精子DNA完整性,计算机自动分析精子密度与活力,精子形态检测系统下人工修正方法进行精子形态分析。结果:吸烟组精子活力显著低于不吸烟组(P<0.05),精子密度和形态低于不吸烟组,但差异无统计学意义(P>0.05)。吸烟组精子DNA完整率异常例数显著高于不吸烟组(χ2=5.393,P<0.05),精子DNA完整率异常组精子密度显著低于精子DNA完整率正常组(P<0.05),精子DNA完整率异常组的精子活力、形态与正常组相比差异无统计学意义(P>0.05)。结论:吸烟影响精子DNA完整率;精子DNA完整率异常与精子参数异常相关;吸烟可能通过影响精子DNA完整性影响精子参数。     

Spermicidal activity of extract from Cestrum parqui

OBJECTIVE: The leaf extract of Cestrum parqui was examined for its effects on sperm motility in vitro. METHODS: Washed sperm were prepared by discontinuous buoyant density gradient centrifugation and incubated with varying concentrations of extract from C. parqui (40-250 microg/mL) at 37 degrees C and 5% CO(2). Sperm motility and viability were assessed at different intervals ranging from 5 to 240 min. Morphological changes in human ejaculated spermatozoa after exposure to the extract were evaluated under transmission electron microscope. RESULTS: A dose- and time-dependent effect of this extract on sperm motility and viability was observed. The maximal spermicidal effect was observed with a 250-microg/mL dose of the extract. Transmission electron microscope revealed a significant damage to sperm membrane in both head and acrosomal membranes, notable swelling and disruption. CONCLUSION: The present study indicates that this natural extract has potential spermicidal effect in vitro.     

流产患者丈夫精子因素初步分析

目的:探讨流产的精子因素。方法:采用精液常规分析、精子形态分析、吖啶橙荧光染色方法对156例自然流产1～4次患者的丈夫及80例正常生育男性的精子进行分析。结果:对照组与流产组a级精子百分率、a+b级精子百分率、精子密度、精子活率、正常形态精子百分率、异常形态精子百分率、尾部异常精子百分率和精子DNA完整率方面差异均有统计学意义(P<0.05)。结论:精子运动参数、形态参数、精子DNA损伤可能与流产有关。     

Semen collection from mice: electroejaculation

The effects of device type (electrostimulator, function generator or computer-generated waveforms), waveform (square, triangle or sine wave), probe type (ring or strip) and anaesthetic compound (ketamine/xylazine combination or pentobarbitone sodium) were investigated on electroejaculation (EEJ) responses of C57B1 x CBA and C57Bl/6J mice. Ejaculates were analysed for total sperm count and motility variables using computer-assisted sperm analyses. Automated computer-generated waveforms delivered through a sound card were more effective and reproducible compared with waveforms generated by function generator and electrostimulator. Sine waves and triangle waves were found to be more effective in producing ejaculate than square waves. As an anaesthetic, pentobarbitone sodium tended to outperform ketamine/xylazine across waveforms and strains. Strip probes failed to produce any ejaculate regardless of the device or waveform employed. Sperm obtained by EEJ exhibited poor motility and C5B1/6J mice had lower motility variables than C57BI x CBA mice.     

A simple glycerol-based freezing protocol for the semen of a marsupial Trichosurus vulpecula, the common brushtail possum

Frozen storage of semen and embryos is now a well established part of the breeding of many eutherian mammals but it has not been applied to marsupials. This paper reports the first successful technique for the frozen preservation of marsupial spermatozoa. Semen was collected by electroejaculation under anaesthesia from a pool of five brushtail possums. The ejaculated semen was diluted 1:1 with Krebs Henseleit Ringer, centrifuged at 800 g for 5 min, resuspended in the test cryoprotectant media at 1, 2 and 5 x 10(6) spermatozoa mL-1 and 7, 10.5, 14 and 17.5% glycerol and then drawn up into 0.25 mL plastic straws. The spermatozoa were rapidly frozen in the vapour phase, 6 cm above liquid nitrogen, for 30 min before the straws were plunged into the liquid. Sperm motility was assessed blind for coded straws by phase-contrast microscopy on a warmed stage (35 degrees C), before freezing and after rapid thawing in a water bath at 37 degrees C (10 s). The highest recovery of both percentage motility (around 50-60%) and progressive motility (around 0.5-1 unit lower than prefreeze) occurred when spermatozoa were frozen and thawed in the presence of 17.5% glycerol. Recovery of motility was greater at the higher sperm concentrations (2 and 5 x 10(6) mL-1). There was no evidence of acrosomal damage or loss after freezing and thawing in high concentrations of glycerol. The only defect detected in spermatozoa subjected to the protocol was a variable tendency to bending of the neck region. This ranged from heads inclined at a slight angle to the tail through to complete flexure.(ABSTRACT TRUNCATED AT 250 WORDS)     

玉米赤霉醇染毒对雄性小鼠睾丸及生精功能的影响

目的研究玉米赤霉醇对小鼠生精功能的影响及可能机制。方法选择健康性成熟的雄性昆明种小鼠40只,随机分为对照组和染毒组(玉米赤霉醇25、50、100mg/kg),连续灌胃染毒35天后脱臼处死。检测睾丸、附睾、精囊腺重量及脏器系数,附睾尾精子数、精子的形态结构及活力,并观察睾丸组织病理学改变。结果玉米赤霉醇各染毒组小鼠的精囊腺系数及精子计数均低于对照组,差异有显著性(P<0.05);50、100mg/kg组精子活动率下降,与对照组比较差异有显著性(P<0.05);睾丸组织有明显的病理改变,生精上皮细胞排列疏松、紊乱,层次减少,成熟精子数明显减少。结论在本实验剂量条件下,玉米赤霉醇对小鼠生精功能有毒性作用。     

Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway)

The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the S?rfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The S?rfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions.