抗CATSPER1抗体对精子运动的影响

目的探讨CATSPER1蛋白在精子运动中的功能。方法将正常精液标本与终浓度分别为0.8μg/mL、4μg/mL、20μg/mL的CATSPER1抗体混合孵育,然后于37℃1h、2h、6h用计算机辅助分析系统(CASA)进行精液参数分析。结果在不同时间,前向运动精子(a+b级)、快速前向运动精子(a级)的百分比均较对照组明显减少并且成剂量依赖性,慢速前向运动精子(b级)受抗体抑制的作用不明显。结论 CATSPER1在精子运动过程中发挥重要功能,CATSPER1抗体主要抑制精子快速前向运动能力。     

抗SPAG11抗体对精子运动的影响

目的探讨SPAG11蛋白在精子运动中的功能。方法将正常精液标本与终浓度为20μg／mlSPAG11抗体混合37℃孵育，然后分别于1h、2h、6h用计算机辅助分析系统（CASA）进行精液参数分析。结果精液标本前向运动精子（a＋b级）百分数在2h出现运动能力显著性下降（t=3．087，P=0．027），快速前向运动精子（a级）百分数在1h时出现运动能力明显的下降（t=2．676，P=0．044），慢速前向运动精子（b级）的百分数在不同时间点均无明显的改变。结论SPAG11抗体总体上并不能抑制体外成熟精子的前向运动能力。     

槟榔碱对人体外精子运动能力的影响

目的:研究槟榔碱(Ar)对人体外精子运动能力的影响。方法:优选50例正常男性精液与3组不同浓度(10、50、100μg·mL-1)Ar溶液共同孵育,以精子优选液为对照组,在共同孵育0.5、1、2h后用计算机辅助精子分析(CASA)系统对精子活率(Mot)、(a+b)级前向运动精子百分率([a+b)PM]、曲线运动速度(VCL)、直线运动速度(VSL)进行检测。结果:精液与10μg·mL-1Ar溶液共同孵育1h后的Mot与对照组比较有显著性差异(P<0.01),2h后Mot、VCL与对照组比较均有显著性差异(P<0.01);精液与50、100μg·mL-1Ar共同孵育0.5、1、2h后的Mot、(a+b)PM、VCL、VSL与对照组比较均有显著性差异(P<0.01或P<0.05),表明Ar浓度越高,作用时间越长,精子运动能力越低。结论:Ar能降低正常男性体外精子运动能力,其毒性与浓度时间成正比。     

Lithium inhibits human sperm motility in vitro.

Lithium is a widely prescribed drug used for the treatment of bipolar affective illness. Previous reports on its effects on sperm motility and male fertility are conflicting. The effect of lithium on human sperm motility was examined in vitro using the modified transmembrane migration method. This technique takes account of the dilution of lithium that occurs during the incubation. Lithium inhibits human sperm motility in vitro in concentrations comparable with those reported to be achieved in semen after oral administration.     

依立雄胺对大鼠、犬和人精子活力的影响

目的　用更敏感的指标评价依立雄胺的生殖毒性。方法　将精悬液与不同浓度的依立雄胺共育1h ,2h后 ,借助计算机辅助分析系统录像分析精子活力参数的变化。结果　大鼠精子给予终浓度为0 .6 ,6和 6 0 μmol·L- 1的依立雄胺 1h后 ,精子活率(MOT)较对照组分别下降 19.0 %,18.0 %,16 .0 %;2h后 ,中高剂量组的MOT较对照组分别下降9.0 %,10 .0 %,且高剂量组的前向性降低。Beagle犬给予终浓度 0 .6 ,6和 6 0 μmol·L- 1依立雄胺 1h后MOT较对照组呈下降趋势 ,但无显著性差异 ,2h后MOT较对照组分别下降 31.0 %,2 4 .9%,2 8.3%。人精子体外给药实验中 ,给予终浓度为0 .12 ,0 .2 4和 0 .96 μmol·L- 1的依立雄胺 2h后 ,曲线运动速度、直线运动速度较对照组呈下降趋势 ,但无显著性差异 ,而高剂量组的精子头侧摆幅度、精子尾摆动性及MOT较对照组分别下降 2 8.0 %,5 .0 %,15 .0 %。结论　依立雄胺对精子具有一定的直接毒性 ,这种毒性存在种属差异 ,且不表现为剂量 反应关系。     

不同有机溶剂对人精子体外运动参数的影响比较

目的研究常用有机溶剂对正常男性精子体外运动活力的影响。方法取正常男性精子,采用上游优化法处理,制备成精子悬液并分成4组,前3组分别加入二甲亚砜,使二甲亚砜终浓度分别为0.5%,1%和2%,pH值均为7.5,渗透压为290 mosm.(kg.H2O-1),D组加入等量Ham’s F10培养液作对照。分别于孵育15,30,45和60 m in后采用CASA进行分析。参照上述方法,考察有机溶剂甘油、聚乙二醇、吐温80对精子体外运动活力的影响。甘油终浓度分别调至1%,5%和10%,聚乙二醇终浓度分别调至1%,5%和10%,吐温80终浓度分别调至1%,5%和10%。结果2%二甲亚砜作用于精子60 m in能导致精子全部死亡,10%甘油作用60 m in后对精子运动参数抑制率达73.8%,10%聚乙二醇400作用于人精子60 m in可致其全部死亡,10%吐温80作用60 m in后对精子运动参数抑制率达95.5%。加入不同浓度二甲亚砜、甘油、聚乙二醇400和吐温80的各组前向运动精子百分率、曲线运动速度、直线运动速度和平均路径速度均较空白对照组显著降低(均P<0.05)。结论高浓度有机溶剂体外可抑制正常男性精子存活率和运动活力。     

The Hepatotoxicity and Testicular Toxicity Induced by Arecoline in Mice and Protective Effects of Vitamins C and E

Arecoline is a major alkaloid of areca nuts which are widely chewed by southeast Asian and it manifests various toxic effects in different organs of human and animals. In this work, mature mice were treated by vitamins C plus E, arecoline, or both daily for four weeks. The results showed that arecoline significantly increased the levels of serum alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and significantly decreased the levels of reduced glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in the liver tissues. Additionally, the body weight, testis weight, sperm counts, motility and normal sperms also were significantly decreased. The supplement of vitamins C and E can bring the activities of ALP and GPT to normal levels and partially restore the sperm counts compared to the arecoline-treated group but have no other positive effects. In conclusion, the vitamins C and E partially attenuated the arecoline-induced hepatotoxiciy but basically had on protective effects against the arecoline-induced testicular toxicity.     

Localization of cyclooxygenase-2 in mice vas deferens and its effects on fertility upon suppression using nimesulide: a preferential cyclooxygenase-2 inhibitor

Accumulating evidence on constitutive expression of cyclooxygenase-2 (COX-2), one of the isoforms of enzyme cyclooxygenase (COX) the other isoform being cyclooxygenase-1 (COX-1), questions the safety profile of non-steroidal anti-inflammatory drugs (NSAIDs). This COX-2 isoform which is induced not only during inflammation but also by factors such as cytokines, steroid hormones and mitogenic stimuli is constitutively expressed in brain, kidney and reproductive organs. Present NSAIDs, particularly COX-2 inhibitors is no longer considered safe since suppression of COX-2 in tissues which it is constitutively expressed may lead to adverse effects. Though intense expression of COX-2 in vas deferens is proved, lack of information with respect to its function has attracted a wide scope for research as to whether COX-2 in vas deferens contributes to male fertility. In the present study, the authors investigated the localization of COX-2 as well as COX-1 in mice vas deferens and also assessed the activity of COX-2 and total prostaglandin (PG) levels in vas deferens. Further they suppressed the expression of COX-2 using a preferential COX-2 inhibitor nimesulide and analyzed the sperm from vas deferens for any defects. COX-2 was intensely expressed in the epithelial cells of mice vas deferens and nimesulide was able to effectively suppress most of COX-2 expression. A decrease in PG levels was observed initially but interestingly, the levels tend to rise on sustained suppression of COX-2. The motility of sperm was affected severely after 6h of nimesulide administration that suggested a crucial role of COX-2 towards fertility of mice sperm.     

Comparison of chlordecone effects on autoimmunity in (NZBxNZW) F(1) and BALB/c mice

Arecoline, the main areca alkaloid in betel quid (BQ), is reported to have cytotoxic, genotoxic, and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucous fibrosis, leukoplakia, and oral cancer. To clarify the role of arecoline in BQ-induced carcinogenesis, primary human gingival keratinocyes (GK) and human KB epithelial cells were used for studying the molecular mechanisms of arecoline-mediated cell cycle deregulation for comparison. After 24 h of exposure, arecoline (0.2–0.8 mM) inhibited KB cell growth in a dose- and time-dependent manner with a reduction in cell number by 27–37 and 37–58%, respectively, as determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. Incubation of KB cells with arecoline (0.1–0.4 mM) caused late-S and G2/M phases’ cell cycle arrest. Western blot analysis revealed that arecoline induced cyclin Bl, Wee 1, and phosphorylated cdc2 protein levels whereas it declined p21 protein expression in KB cancer cells. Nevertheless, arecoline induced p21, but decreased cdc2 and cyclin B1 protein levels in GK. We demonstrated that higher concentrations of arecoline (0.2–1.2 mM) induced both cell necrosis and apoptosis as detected by DNA fragmentation and Annexin V–PI staining after long-term (48 h) treatment. Our results suggest that differential regulation of S and/or G2/M cell cycle-related proteins in the GK and KB cells play a crucial role in different stages of BQ-mediated carcinogenesis.     

In vitro effects of metal ions (Fe2+, Mn2+, Pb2+) on sperm motility and lipid peroxidation in human semen

The effects of divalent manganese ion (Mn2+), ferrous iron (Fe2+), and lead ion (Pb2+) on human sperm motility and lipid peroxidation were examined. Human semen from healthy male volunteers was incubated with 0, 5, 50, or 500 ppm divalent metal ions, and the sperm motility was determined at 0, 2, 4, 6, or 8 h by microscopy. Malondialdehyde (MDA) levels in seminal plasma was measured by high-performance liquid chromatography after 8 h of exposure. The results showed that 500 ppm Mn2+ or Pb2+ significantly inhibited sperm motility without an accompanying change in seminal MDA levels. Incubation with Fe2+ significantly inhibited sperm motility at 5 ppm, associated with a marked rise in MDA levels. Our results suggested that Fe2+ may induce lipid peroxidation to inhibit sperm motility. In the case of Mn2+ and Pb2+ there is an absence of seminal lipid peroxidation and the observed inhibition of sperm motility at high concentrations is not biologically or environmentally relevant.     

Measuring mouse sperm parameters using a particle counter and sperm quality analyzer: a simple and inexpensive method

This study examined a method for analyzing the count, motility, and morphology of mouse epididymal sperm, optimizing the diluent, incubation time, sample concentration, and temperature, using a particle counter (CDA-500) to count and size sperm and a sperm quality analyzer (SQA-IIC) to measure sperm motility, quantified as the sperm motility index (SMI). The optimal conditions consisted of a 30-min incubation in D-MEM (Dulbecco's modified Eagle's medium; considering cost and availability) at 37 degrees C, with 5 x 10(6)cells mL(-1) in the original solution. Furthermore, the influence of formalin fixation, and the correlation between the automated counter and a manual method were investigated. The sample fixation had no marked effect on the sperm count or morphology assessment. A linear correlation was observed between the manual and automated methods (y=0.920x +0.276; r(2)=0.571; p<0.001; range: (3-6) x 10(6)). The suitability of the proposed method was confirmed using spermatozoa prepared from mice treated with the reproductive toxin diethylstilbestrol (DES). Using sperm from the cauda epididymidis on one side per mouse, we confirmed that measurement of these sperm parameters using the two devices was simple, rapid, inexpensive, and reproducible.     

Matrine compromises mouse sperm functions by a [Ca2+]i-related mechanism

Matrine, a bioactive alkaloid widely used in Chinese medicine, inhibits mouse sperm functions in vitro. In this study, we investigated the reproductive toxicity of matrine to male mice in vivo. C57BL/6J mice were administered with daily doses of 0, 1, 10 and 50 mg/kg matrine by intraperitoneal injection for 30 days. The results showed that matrine did not affect testis size, testis weight, sperm count and sperm viability, but it significantly inhibited total motility, progressive motility, linear velocity, capacitation and the progesterone-induced acrosome reaction of mouse sperm. Furthermore, the intracellular Ca²⁺ concentration ([Ca²⁺]_i), a key regulator of sperm function, was reduced in sperm of matrine-exposed mice. The current and gene expression of the sperm specific Ca²⁺ channel, CatSper, which modulates Ca²⁺ influx in sperm, were decreased in testes of matrine-exposed mice. These results indicate that matrine inhibits mouse sperm functions by a [Ca²⁺]_i-related mechanism via CatSper channel.     

The toxic effect of opioid analgesics on human sperm motility in vitro

Opioid analgesics are the most common therapeutic analgesic for acute pain. In this study, the toxicological and pharmacological features of a group of opioid analgesics were characterized by the motility of human sperm. Aliquots of sperm were incubated with various concentrations of opioid analgesics in vitro. Computer-assisted sperm analysis was used to assess sperm motility at 15 minutes, 2 hours, and 4 hours after drug addition to the medium. Butorphanol and dezocine showed marked reduction of motility after incubation with sperm for 15 minutes. Butorphanol was more effective than dezocine in immobilizing sperm. Other opioids studied, such as fentanyl, alfentanil, and sufentanil, showed only partial inhibitory activity. Based on the data reported herein, we have found that butorphanol and dezocine exert a sperm-immobilizing effect. However, fentanyl, alfentanil, and sufentanil exhibit only partial inhibition of sperm motility. Given the increasing use of opioids and their potential effect on sperm motility, these findings are greatly relevant to male reproductive health.     

Effect of organic and inorganic mercury on human sperm motility.

The effects of mercuric chloride and methyl mercuric chloride on the motility of human spermatozoa in vitro were investigated. Organic as well as inorganic mercury compounds decreased the percentage of motile spermatozoa. After 15 min. incubation with 40 microM mercuric chloride a significant decrease in sperm motility was observed. Less than 5% of spermatozoa were motile after 30 min. of exposure to 20 microM methyl mercuric chloride. These effects could not be attenuated by addition of 5 microM sodium selenite. The ultrastructural localization of mercury was demonstrated by autometallography. Silver-enhanced mercury deposits could be demonstrated only in spermatozoa exposed to inorganic mercury. In these cells mercury grains were most abundant in membranes of midpiece and tail.     

Comparative effects of adrenaline and noradrenaline on the synthesis and secretion of soluble protein by isolated hepatocytes.

1. Adrenaline (A) supplementation of the incubation medium of monolayer cultures of hepatocytes at 0.1, 0.2, 0.3, 1 and 10 ng/ml resulted in consistently enhanced levels of secreted and newly synthesised non-secreted proteins; supplementation with 100 and 1000 ng/ml resulted in lower or unchanged levels. These effects were most consistent 6 hr after a medium change. 2. Noradrenaline supplementation of the medium resulted in increased levels of secreted and non-secreted proteins at low concentrations (less than 2.4 ng/ml) at 6 hr after a medium change, but significantly decreased levels of both populations at high concentrations of noradrenaline (NA) (2.4 less than NA less than 1000 ng/ml), at 6-9 hr after a medium change and sustained to 24 hr, the most significant decrease being at 10 ng/ml. 3. All combinations of concentrations of A + NA resulted in non-dose-dependent decreased levels of both the secreted and non-secreted soluble protein fractions. The most significant decreases occurred at concentrations of (1 + 5) and (10 + 10) ng/ml adrenaline + noradrenaline. 4. Medium supplementation with adrenaline, noradrenaline or a combination of the two had no effect on the uptake of [3H]leucine by the cells. 5. The results are discussed in relation to receptor status on the hepatocyte membranes.     

Evaluation of zearalenone and alpha-zearalenol toxicity on boar sperm DNA integrity

This study aimed at investigating the in vitro effects of zearalenone (zen) and alpha-zearalenol (alpha-zen) on motility and nuclear chromatin integrity (NCI) of boar spermatozoa. Mycotoxins were tested, at levels ranging from 10 to 30 microg ml(-1) of diluted semen. Four boars were used for semen collection (eight replicates per boar, four per mycotoxin). After the addition of zen or alpha-zen, semen samples were incubated for 4 h at 38.5 degrees C, 5% CO(2) and 96% humidified air. Motility and NCI were assessed at 0 and 4 h of incubation. No significant differences were noticed in motility among the experimental groups (P > 0.05) for all tested boars. Chromatin instability was significantly higher (P < 0.05) in spermatozoa of only one boar treated with zen and alpha-zen independently of the dose. In conclusion, under our experimental conditions, zen and alpha-zen did not affect the motility of boar sperm, whereas the effects of these toxins on sperm NCI were individual-dependent.     

Reproductive toxicity of sodium valproate in male rats

Objectives:

To assess the effects of sodium valproate on rat sperm morphology, sperm count, motility, and histopathological changes in testis.

Materials and Methods:

Male Wistar rats (12 week old) were treated with sodium valpraote and sacrificed at the end of 2^nd, 4^th, 5^th, 7^th, 10^th and 15^th week after the last exposure to sodium valproate. Epididymal sperm count, sperm motility, sperm morphology, and histopathology of testes were analyzed.

Results:

Sperm count and sperm motility were decreased significantly by sodium valproate. The percentage of abnormal sperms increased in a dose-dependent manner. A histopathological study revealed that sodium valproate had caused sloughing of epithelial cells in testes.

Conclusion:

Sodium valproate causes reversible change in sperm motility, sperm count, morphology, and cytoarchitecture of testes.     

In Vitro Effects of Metal Ions (Fe2+, Mn2+, Pb2+) on Sperm Motility and Lipid Peroxidation in Human Semen

The effects of divalent manganese ion (Mn²⁺), ferrous iron (Fe²⁺), and lead ion (Pb²⁺) on human sperm motility and lipid peroxidation were examined. Human semen from healthy male volunteers was incubated with 0, 5, 50, or 500 ppm divalent metal ions, and the sperm motility was determined at 0, 2, 4, 6, or 8 h by microscopy. Malondialdehyde (MDA) levels in seminal plasma was measured by high-performance liquid chromatography after 8 h of exposure. The results showed that 500 ppm Mn²⁺ or Pb²⁺ significantly inhibited sperm motility without an accompanying change in seminal MDA levels. Incubation with Fe²⁺ significantly inhibited sperm motility at 5 ppm, associated with a marked rise in MDA levels. Our results suggested that Fe²⁺ may induce lipid peroxidation to inhibit sperm motility. In the case of Mn²⁺ and Pb²⁺ there is an absence of seminal lipid peroxidation and the observed inhibition of sperm motility at high concentrations is not biologically or environmentally relevant.     

Pilot study of the pharmacokinetics of betel nut and betel quid biomarkers in saliva,urine, and hair of betel consumers

Approximately 600 million people worldwide practise the carcinogenic habit of betel nut/quid chewing. Carcinogenic N‐nitroso compounds have been identified in saliva or urine of betel chewers and the betel alkaloid arecoline in hair from habitual betel quid chewers. However, the pharmacokinetic parameters of these compounds have been little explored. Assessment of betel use by biomarkers is urgently needed to evaluate the effectiveness of cessation programmes aimed at reducing betel consumption to decrease the burden of cancers in regions of high betel consumption. In the search for biomarkers of betel consumption, we measured by liquid chromatography‐mass spectrometry (LC‐MS) the appearance and disappearance of betel alkaloids ( characteristic for betel nuts ), N‐nitroso compounds, and chavibetol ( characteristic for Piper Betle leaves ) in saliva (n=4), hair (n=2), and urine (n=1) of occasional betel nut/quid chewers. The betel alkaloids arecoline, guvacoline, guvacine, and arecaidine were detected in saliva of all four participants and peaked within the first 2 h post‐chewing before returning to baseline levels after 8 h. Salivary chavibetol was detected in participants consuming Piper Betle leaves in their quid and peaked ~1 h post‐chewing. Urinary arecoline, guvacoline, and arecaidine excretion paralleled saliva almost exactly while chavibetol glucuronide excretion paralleled salivary chavibetol. No betel nut related compounds were detected in the tested hair samples using various extraction methods. From these preliminary results, we conclude that betel exposure can only be followed on a short‐term basis (≤8 h post‐chewing) using the applied biomarkers from urine and saliva while the feasibility of using hair has yet to be validated. Copyright © 2015 John Wiley & Sons, Ltd.