The relationship between DNA fragmentation and the intensity of morphologically abnormal human spermatozoa

Objective:To determine the relationship between teratozoospermia and sperm DNA fragmentation(SDF)in the human ejaculate.Methods:This retrospective study included 100 normozoospermic men as a control cohort(abnormal forms>14%),210 patients with a high level of abnormal forms(≤4%)and 65 patients presenting with a moderate level of abnormal forms(>4%to≤14%)based on the World Health Organization definitions.Sperm morphology was assessed using bright field microscopy.Sperm DNA fragmentation was assessed using the sperm chromatin dispersion assay.Non-parametric analyses were conducted to examine the relationship between abnormal sperm morphology and sperm DNA fragmentation;receiver operating characteristic(ROC)analyses were conducted to assess sensitivity and specificity of this relationship.Results:A correlation analysis revealed that the higher the proportion of abnormal spermatozoa in the ejaculate,the higher the level of SDF(Spearman's Rho=-0.230;P<0.001).Significant differences in the proportion of SDF were found when all cohorts were compared(P<0.001);these significant differences were also retained when the different cohorts were compared pairwise.ROC analysis showed a moderate but significant predictive value for SDF to differentiate patients with different levels of teratozoospemia.Conclusions:Although analysis of a more continuous range of values for teratozoospermia would help further clarify any causal relationship with SDF,there is clearly a synergistic or coincident affiliation between these variables that needs to be acknowledged by the clinician when interpreting the spermiogram.

Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice

Infertility represents a significant health concern,with sperm quantity and quality being crucial determinants of male fertility.Oligoasthenoteratozoospermia(OAT)is characterized by reduced sperm motility,lower sperm concentration,and morphological abnormalities in sperm heads and flagella.Although variants in several genes have been implicated in OAT,its genetic etiologies and pathogenetic mechanisms remain inadequately understood.In this study,we identified a homozygous nonsense mutation(c.916C>T,p.Arg306*)in the coiled-coil domain containing 146(CCDC146)gene in an infertile male patient with OAT.This mutation resulted in the production of a truncated CCDC146 protein(amino acids 1-305),retaining only two out of five coiled-coil domains.To validate the pathogenicity of the CCDC146 mutation,we generated a mouse model(Ccdc146^(mut/mut))with a similar mutation to that of the patient.Consistently,the Ccdc146mut/mut mice exhibited infertility,characterized by significantly reduced sperm counts,diminished motility,and multiple defects in sperm heads and flagella.Furthermore,the levels of axonemal proteins,including DNAH17,DNAH1,and SPAG6,were significantly reduced in the sperm of Ccdc146^(mut/mut) mice.Additionally,both human and mouse CCDC146 interacted with intraflagellar transport protein 20(IFT20),but this interaction was lost in the mutated versions,leading to the degradation of IFT20.This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility,potentially by disrupting axonemal protein transportation.These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.

Human circBOULE RNAs as potential biomarkers for sperm quality and male infertility

Reliable molecular biomarkers to predict fertility remain scarce.The current study investigated the potential of testis-specific circBOULE RNAs as biomarkers for male infertility and sperm quality.Using reverse transcription-PCR and real-time reverse transcription-PCR assays,we identified seven circular RNAs from the human BOULE gene in human sperm.We observed that the expression level of circEx3-6 was significantly reduced in asthenozoospermia,while the expression levels of both circEx2-6 and circEx2-7 were decreased in terato-zoospermia,compared with the controls.Furthermore,we demonstrated that the expression level of circEx2-6 was negatively correlated with the sperm DNA fragmentation index,and the expression level of circEx2-7 was correlated with both fertilization and cleavage rates in those treated with the assisted reproductive technologies.Further functional analyses in a transgenic fly model supported the roles of circBOULE RNAs in sperm development and human male fertility.Collectively,our findings support that sperm circBOULE RNAs may serve as diagnostic biomarkers for assessing sperm motility and DNA quality.Therefore,clinical application and significance of sperm circBOULE RNAs in the assisted reproductive technologies warrant further investigation.

Low Level of Cystic Fibrosis Transmembrane Conductance Regulator Is Associated with Human Sperm Autophagy and Vitality

Low sperm motility is one of the main causes of male infertility. Cystic fibrosis transmembrane conductance regulator (CFTR, an anion channel protein) is related to the progressive motility of sperm. CFTR disruptor CFTRinh-172 or forskolin (FSK) in this study were used to treat human sperm separately, and the rates of sperm autophagy and progressive motility, mitochondrial membrane potential (MMP) and ATP concentration, and the expression levels of related factors were detected to explore their relationship. It was showed that sperms treated with CFTRinh-172 or FSK reduced the levels of cAMP, CFTR and PKA, but increased sperm autophagy rate, expression levels of AMPK and LC3B. However, reactive oxygen species content had no significant difference. It was indicated that low level of CFTR performed with cAMP and its downstream effectors such as PKA and AMPK to regulate mitochondrial structure and function, leading to increased autophagy rate and reduced vitality of sperm.

Antioxidative Protective Effect of Icariin on the FeSO_4/H_2O_2-damaged Human Sperm Based on Confocal Raman Micro-spectroscopy

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H202-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman mi- cro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect oficariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the ＂Raman fingerprint＂ of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H202. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive fimctions.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

GENERATION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SOLUBILIZED MEMBRANE FRACTION OF HUMAN SPERMATOZOA

Human sperm membrane antigens extracted by deoxycholate (DOC) were used to immunizeBALB/c mice.Hybrid cell lines secreting sperm-specific monoclonal antibodies were generatedby cell fusion in a semi-solid medium and screened by indirect immunofluorescent assay usinglive and methanol-fixed sperm.Out of 850 hybrid clones from cell fusion,28 were shownto secrete sperm-specific antibodies which reacted with the acrosome,equatorial segment,whole surface plasma membrane or tail of spermatozoa.Finally,seven hybrid cell lineswere established and shown to secrete monoclonal antibodies which had no cross-reactivitywith arty human tissues other than testis and sperm.The majority were also shown toinhibit fertilization of mouse oocytes in vitro and human sperm penetration of zona-freehamster ova.Western blot analysis revealed that some of these antibodies reacted withsperm membrane antigens of distinct molecular size.

scAAV9-IGF-1对SOD1-G93A小鼠抗凋亡通路的作用

目的 探索以自我互补双链腺相关病毒9为载体介导的人胰岛素样生长因子-1 (scAAV9-IGF-1)对SOD1-G93A转基因小鼠抗凋亡通路的作用。方法 采用肌萎缩侧索硬化(ALS)的动物模型即转基因SOD1-G93A突变型及野生型(wild type-SOD1,WT-SOD1)小鼠,在其出生后60 d龄时,雌性同窝阳性SOD1-G93A转基因小鼠采用随机的方法分配到治疗组及溶剂对照组,治疗组全身多点肌肉注射scAAV9-IGF-1,溶剂对照组多点肌肉注射AAV9-GFP,同年龄WT-SOD1作为阴性对照组。在肌肉注射40~50 d后,利用PCR技术检测腰髓中IGF-1含量的变化,同时检测抗凋亡通路因子Bcl-xl、Bcl-2的mRNA含量变化,通过免疫组化染色观察抗凋亡通路因子Bcl-xl、Bcl-2在小鼠腰髓前角神经元中的表达。结果 PCR技术检测显示scAAV9-IGF-1处理后,腰髓中IGF-1的mRNA含量显著高于GFP对照组,抗凋亡通路因子Bclxl、Bcl-2的mRNA含量均高于溶剂对照组(均P<0.05),而与WT组差异无统计学意义。免疫组化染色结果显示,治疗组中抗凋亡通路蛋白Bcl-xl,Bcl-2在小鼠腰髓前角神经元中的表达多于溶剂对照组,并且与WT阴性对照组相当。结论 scAAV9-IGF-1可以激活SOD1-G93A转基因小鼠模型中的抗凋亡通路,其通过上调Bcl-xl,Bcl-2的mRNA水平,进而增加Bcl-xl、Bcl-2的蛋白表达,从而产生抗凋亡的作用。

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

Aim： To investigate the distribution of cation channel of sperm 1 （CATSPER1） protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Methods： Human ejaculated sperm from normozoospermic donors （n = 12） and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors （n = 12） were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 μg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. Results： CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. Conclusion： CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.

Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus

Aim： To simultaneously determine the localization of histones and protamines within human sperm nuclei. Methods： Immunofluorescence of the core histones and protamines and fluorescence in situ hybridization of the telomere region of chromosome 16 was assessed in decondensed human sperm nuclei. Results： Immunofluorescent localization of histones, protamine 1 （PRM1） and protamine 2 （PRM2） along with fluorescence in situ hybridization localization of chromosome 16 telomeric sequences revealed a discrete distribution in sperm nuclei. Histones localized to the posterior ring region （i.e. the sperm nuclear annulus）, whereas PRM1 and PRM2 appeared to be dispersed throughout the entire nucleus. Conclusion： The co-localization of the human core sperm histones with the telomeric regions of chromosome 16 is consistent with the reorganization of specific non-protamine regions into a less compacted state.

Human sperm immobilization effect of Carica papaya seed extracts: an in vitro study

Aim: To examine if the seed extracts of Carica papaya, which showed anfispermatogenic/sperm immobilizationproperties in animal models, could cause human sperm immobilization in vitro. Methods: Chloroform extract, ben-zene chromatographic fraction of the chloroform extract, its methanol and ethyl acetate sub-fractions and the isolatedcompounds from the sub-fractions i. e., ECP 1 & 2 and MCP 1 & 2, of the seeds of Carica papaya were used at con-centrations of 0.1%, 0.5%, 1% and 2%. Sperm motility was assessed immediately after addition of extracts and ev-ery 5 minutes thereafter for 30 minutes. Results: There were dose-dependent spermicidal effects showing an instantfall in the sperm motility to less than 20% at 2% concentration. Isolated compounds ECP 1 & 2 were more effective in-ducing a motility of less than 10%. Many of the spermatozoa became vibratory on the spot. Total inhibition of motilitywas observed within 20-25 rain at all concentrations of all products. Scanning and transmission electron microscopyrevealed deleterious changes in the plasma membrane of the head and mid-piece of spermatozoa. Sperm viability testand the number of abnormal spermatozoa after completion of incubation suggested that the spermatozoa were infertile.The effects were spermicidal but not spermiostatic as revealed by the sperm revival test. Conclusion: The results re-veal spermicidal activity in vitro of the seed extracts of Carica papaya.

Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

Comparison of Three Different Techniques of Human Sperm DNA Isolation for Methylation Assay

Summary： Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method （method A）, traditional phenol-chloroform method （method B）, and TianGen kit method （method C）. Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C （P〈0.01）. PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 （DDX4） and copy number variations （CNVs） than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reli- able results and could be an optimal technique for extracting sperm DNA for methylation assay.

Evaluation on the Morphology and Membrane Integrity of Immotile Human Sperm

Objective To determine the membrane integrity in the head and tail regions of individual spermatozoon, and observe sperm morphology for samples with totally immotile sperm. Methods Ten infertile men with immotile sperm were enrolled into this study （group A）. The membrane integrity in the head and tail regions of individual spermatozoon of immotile sperm was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test （HOS-EY test）. Sperm morphology was observed by light, scanning and transmission electron microscopy. Ten semen samples from normospermic donors were used as the control （group B）. Results The percentage of sperm with intact both head and tail membranes in group A was significantly lower than that in group B （P〈0.01）, whereas the value of sperm with defective head membrane but intact tail membrane in group A was significantly higher than that in group B （P〈0.01）. Abnormal sperm morphology in group A had a high incidence, and immotile sperm with viability and normal morphology could be observed in some cases. Most sperm had multiple ultrastructural defects. Conclussion Some immotile sperm had intact tail membrane but defective head membrane. Immotile sperm with viability and normal morphology could exist in some cases though abnormal sperm were in a great proportion. Carefully evaluating immotile sperm membrane integrity and morphology should benefit the treatment of patients with immotile sperm.

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

<abstract>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, respectively). However, when correlating sperm energy consumption with the zinc content, a significant positive relation (r=0.323; P=0.01) was observed. Conclusion: Poorly motile sperm are actually wasting the available energy. Moreover, our data clearly support the 'Geometric Clutch Model' of the axoneme function and demonstrate the importance of the outer dense fibers for the generation of sperm motility, especially progressive motility.

Diagnostic and Analysis of Human Sperm Characteristics Using Fourier Transform Infrared Spectroscopy

A spectroscopic method for human sperm evaluation and characterization using Fourier Transform Infra Red (FTIR) is presented. The high sensitivity of FTIR to changes in chemical structure and arrangement of molecules and proteins makes it a powerful diagnostic tool. Our experimental results show that a simple MIR (400 cm-1?- 4000 cm-1) transmission spectrum of a human sperm is very fast and can be used to determine the level of structure, compare to conventional LAB tests. No sample preparations are required, the semen has to be put on a special ZnSe substrate and inserted into the measurement compartment of the FTIR. Furthermore, this method can distinguish between immature sperm cell to white blood cell which by using a microscope is difficult and re-quires experience.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

A patch-clamp study on human sperm Cl^- channel reassembled into giant liposome

Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)

Effect of rhTNF-α on Mitochondrial Transmembrane Potential and Motility of Human Sperm in vitro by Flow Cytometry and Computer Aided of Semen Analysis

To evaluate effect of recombined human tumor necrosis factor （rhTNF- α） on mitochondrial transmembrane potential and motility of human sperm in vitro Methods Semen samples for study were obtained from 40 health men （average age 26 ± 1.2 years） with normal semen analysis. Sperm suspension with computer aided of semen analysis （CASA） technique; 2） were stained in the presence of 10 μg/ml Rh123 and PI, mitochondrial transmembrane potential of those was analyzed by flow cytometry （FCM）. Results Significant differences were found between experimental groups and control groups on viability, straight line velocity, curvilinear velocity, average path velocity, progressive motility of human sperm and number of sperm with normal mitochondrial transmembrane potential （P〈0.01） expect final concentration 30 pg/ml group （P〉0. 05）. Sperm motility lowed with increasing rhTNF-α concentration and incubating time （P〈0. 01）. Number of sperm with normal mitochondrial transmembrane potential decreased with increasing rhTNF-α concentration and incubating time （P〈0.01）. Conclusion rh TNF-α can decrease human sperm motility function in vitro, which can interfere the function of human sperm mitochondrial transmembrane potential and may inhibit sperm mitochondrial enzymatic activities.

The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa： differences from the zona pellucida

The acrosome reaction （AR）, an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca2＋ into the spermatozoa through voltage-dependent Ca2＋ channels and store-operated channels. Maitotoxin （MTx）, a Ca2＋-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida （ZP）, possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 （rhZP3）, mouse ZP and two MTx channel blockers （U73122 and U73343）, we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca2＋ （[Ca2＋]~） in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 （inactive analogue）, can block phospholipase C （PLC）. Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.