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.

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.

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.

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.

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.

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.

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)

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.

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

acrosome 反应(AR ) ，为精子的一个绝对要求和卵熔化，通过电压依赖者 Ca2+ 隧道和操作店的隧道要求 Ca2+ 的流入进精子。Maitotoxin (MTx ) ，一个动员 Ca2+ 代理人，被显示了是老鼠精子 AR 的有势力 inducer 与类似于带 pellucida (ZP ) 的药理学，可能为两 inducers 建议一条普通小径。使用的 recombinant 人 ZP3 (rhZP3 ) ，老鼠 ZP 和二 MTx 隧道 blockers (U73122 和 U73343 ) ，我们在人和老鼠精子调查了并且比较导致 MTx 、导致 ZP 的艺术。此处，我们报导 MTx 导致了 AR 并且提高了细胞内部的 Ca2+([Ca2+] 我) 在人的精子，哪个被 U73122 和 U73343 堵住。这二混合物也在老鼠精子禁止了导致 MTx 的 AR。在有我们的以前的建议的争论， AR 由 rhZP3 被触发或老鼠 ZP 没被 U73343 堵住，显示在人和老鼠精子，由生理的 ligands 或由 MTx 的 AR 正式就职通过不同小径发生了。U73122，然而并非 U73343 (不活跃的类似物) ，能堵住 phospholipase C (PLC ) 。另一个 PLC 禁止者， edelfosine，也堵住了导致 rhZP3 、导致 ZP 的艺术。这些调查结果在人和老鼠带证实了一条 PLC 依赖的发信号小径的参予导致蛋白质的 AR。尤其是， edelfosine 也禁止了导致 MTx 的老鼠精子 AR 然而并非人的，建议导致毒素的 AR 在老鼠是 PLC 依赖的并且在人 PLC 独立。

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.

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.

Protein tyrosine phosphorylation of the human sperm head during capacitation： immunolocalization and relationship with acquisition of sperm-fertilizing ability

The occurrence of tyrosine phosphorylation （TP） in the sperm head during capacitation has been poorly investigated, and no data exist on the relationship of its dynamics with the acquisition of sperm fertilizing ability. This study localized TP of head proteins in human spermatozoa during capacitation and explored its relationship with acquisition of the ability to display progesterone （P）-stimulated acrosome reactions （ARs） and to penetrate zona-free hamster oocytes. By immunofluorescence, TP immunoreactivity was revealed in the acrosomal region of formaldehyde-fixed/unpermeabilized samples, whereas it was abolished in fixed/permeabilized samples, in which TP immunoreactivity was high in the principal piece. No TP immunoreaetivity was detectable in unfixed spermatozoa. Head TP immunoreactivity was localized externally to the acrosome, close to the cytoplasmic membrane, as assessed by transmission electron microscopy. The increase in head TP was an early event during capacitation, occurring within 1 h in capacitating conditions. At this time, the P-stimulated ARs were also increased, whereas egg penetration was as poor as in uncapacitated spermatozoa. At 5 h of capacitation, the extent of neither head TP nor the P-induced ARs were greater than that at 1 h, whereas egg penetration had significantly increased. Seminal plasma inhibited head TP, P-induced ARs and egg penetration. None of these inhibitory effects, unlike those on tail TP, were prevented by the cAMP analogue dbcAMP （N,2-O-dibutyryladenosine 3＇,5＇-cyclic monophosphate）. In conclusion, head TP is a subsurface event occurring early during capacitation and is closely related to acquisition of the ability to display P-stimulated ARs, whereas the ability to fuse with oolemma and to decondense is a later capacitation-related event.

Effects of MTG and GSH on Human Sperm Motility and DNA Integrity during Vitrification in the Presence of Trehalose

Limited information exists about the effects of antioxidants on sperm motility and DNA integrity during vitrification in humans. This study compared the effects of reduced glutathione (GSH) and monothioglycerol (MTG) at different concentrations on post-thaw sperm motility and DNA integrity after vitrification in humans using 0.25 M trehalose as a cryoprotective agent, and found that supplementation of MTG or GSH at 0.5 mM resulted in significantly higher (P < 0.05) recovery rates of post-thaw total and progressive motilities. GSH was more powerful than MTG at the same concentration in cryoprotecting sperm motility (38.9% ± 3.6% vs 32.8% ± 2.4% compared to the control 26.8% ± 2.1% in recovery rate of progressive motility), but both had no significant influence on sperm DNA integrity during vitrification. It was concluded that sperm motility is more sensitive to oxidative stress during vitrification than sperm nuclear DNA, and supplementation of MTG or GSH in vitrification medium is beneficial in cryoprotecting sperm motility.

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.

Sensitivity of sperm motility assay for detecting endotoxin effect on human sperm in vitro

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endotoxin, there was a significant reduction in the development rate at all developmental stages with 1-cell and 2-cell mouse embryo assays and at the level of 10 ng/mL, the embryo development was completely arrested. Conclusion: The sperm motility assay could detect high levels of endotoxin effect in vitro, but its sensitivity is low as compared with the 1-cell or 2-cell mouse embryo bioassay.

Pharmacological Investigation of Voltage-dependent Ca^(2+) Channels in Human Ejaculatory Sperm in vitro

The types of the voltage-dependent calcium channels （VDCCs） in human ejaculatory sperm and the effects of calcium channel blocker （CCB） on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine （NIF, inhibitor of L-type VDCC） and ω-conotoxin （GVIA, inhibitor of N-type VDCC） were compared and analyzed statistically. The results showed that NIF （1, 5, 10 μmol/L） could not only significantly affect human sperm＇s shape but also spermatozoa motility after incubated at least 10 rain in vitro （P〈0.001）. GVIA （0.1, 0.5 and 1 μmol/L） could just only significantly affect human sperm＇s progressive motility （a %＋b %） after incubated for 20 min in vitro （P〈0.01）, but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

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.

GOSSYPOL-INDUCED ALTERATIONS OF AMINOPHOSPHOLIPID COMPOSITION IN HUMAN SPERM

Gossypol-induced alterations of aminophospholipid composition in human sperm wereobserved and the effects of some factors on these alterations were investigated.The altera-tions of aminophospholipid composition of human sperm induced by gossypoI included aprogressive decrease in the levels of phosphatidylethanolamine（PE）and phosphatidylserine（PS）when gossypol concentrations ranged from 5-500μmol/L,and a progressive increase inthe level of（LPE）at lower gossypol concentrations（5-50μmol/L）.The conversion of PEinto lysophosphatidylethanolamine（LPE）was strongly enhanced by Ca2+and inhibitedby 0.5mmol/L EDTA,while PMSF,NEM,iodoacetamide and Zn2+had no effect.Thisconversion was weaker in sperm with stripped surface proteins than in normal sperm.In addition,three surface proteins（MW 80,60 and 40 kD）were fixed on the plasmamembrane by gossypol treatment.The significance of gossypol action and the possibilitythat sperm PLA2, PE and.some surface proteins might play an important role in the physio-logical acrosome reaetion of human sperm,as well as in oocyte penetration,are discussed.