生理学报 1999年第5期第51卷

中国科学院上海生理研究所；上海；200031马学海；施玉樑

关键词：Ca2+通道；人精子；脂双层；重组

摘要：跨膜离子流动在启动配子间的相互作用， 如顶体反应过程中有重要作用。但由于精子太小,无用论细胞内记录或膜片箝技术都难以对哺乳动物精子膜的离子通道进行研究。 本工作将通道蛋白重组于脂双层，在电压箝位下记录了人精子膜的Ca²⁺通道电流。由12个健康人的精子分离的膜蛋白通过融合重组于脂双层后， 在CaCl₂溶液中(cis 50//trans 10 mmol/L)记录到两类单通道活动，它们的平衡电位都接近Ca²⁺的Nernst电位理论值;都对nifedipine和verapamil敏感；单位电导分别为40和25 pS， 但前者的平均开放时间无电压依赖性, 后者(25 pS)在维持电压从－20 mV增至100 mV时,通道平均开放时间增大。在两例不育者精子样本中亦发现可通透Ca²⁺的阳离子通道， 但通道活动显示某些动力学异常。The maturation and capacitation of mammalian sperm and gamete reaction are complex processes, in which the transmembrane flux of ions is essential^［1］. So the characterization of the ion channels in sperm plasma membrane is a basic step to fully understand the detailed mechanism involved in the sperm physiology and then to develop some clinic therapy method for medical purpose^［2～4］. In recent years, some studies have been made using reconstitution of sperm membrane proteins into the lipid bilayer and patch-clamp technique^{［3,5～7］}, showing that there is a variety of types of ion channels on boar sperm. Our previous work^［8,9］, by means of reconstitution of human sperm membrane proteins into the lipid bilayer and voltage clamp technique, recorded single channel current for the first time, and gave evidences for the existence of K⁺,Na⁺, Cl^－ and Ca²⁺ channels in human spermatozoa.

It is believed that acrosome reaction, a required step in the preparation of sperm cell for fertilization of the egg, is triggered by a rapid influx of calcium that is mediated by calcium channels located on the sperm membrane^{［10～13］}. That is why Ca²⁺ channel is the focus in sperm membrane research^{［3,5,13～17］}. In the present work, by the same method as mentioned above, more data about the characteristics of Ca²⁺ channels in human spermatozoa are demonstrated. Two infertile human sperm samples are also investigated.

1MATERIALS AND METHODS

1.1Collection of human semenFresh semen was obtained from 12 healthy and 2 infertile human donors at the age of 25 to 45. The 12 semen samples devoted by healthy donors liquefied within 30 min after extrusion. The parameters of the sperms were in a normal range, e.g. the average ratio of the active spermatozoa is 64% (range from 30% to 88%), and the average sperm concentration is 87.5 million/ml (range from 20 to 255). However, for the two infertile samples, the liquescent time was more than 1 h, sperm concentration was below 15 million/ml and the sperm motility is lower (5% and 15% respectively). The fresh semen collected by masturbation were placed in a refrigerator of 0℃, ready to be treated.

1.2Preparation of human sperm membrane protein The procedure was based on the method described by Yan et al^［8,9,18］. In brief, spermatozoa were crudely separated from seminal plasma by centrifugation at 5000 r/min first for 5 min. After washing by suspending the sperm pellet in 0.01 mol/L phosphate buffered saline of pH 7.4 three times, spermatozoa were collected and resuspended in 0.01 mol/L Tris-HCl buffer containing 0.5 mol/L KCl, 1 mmol/L EDTA, 0.2 mmol/L PMSF, 5% glycine, pH 8.3. Triton X-100 (final concentration 1%,v/v) was then added to the suspension of sperm, and stirred at 4℃ for 12 h. Insoluble materials were removed by centrifugation at 15000 r/min for 40 min. The supernatant was stored at -70℃, and the protein concentration was determined by CBB (coomassie brilliant blue) testing method (i.e. Bradford method) ranging 5～10 mg/ml. After the extraction, the final remaining pellet showed a perfect sperm outline under electron-microscope,but the spermatozoon plasma membrane and the inner and external acrosome membrane were solubilized similar to that in boar sperm^［19］. So the isolated protein must be mainly from the plasma membrane not contaminated by intracellular proteins^［8,9］.

1.3Planar lipid bilayer experiments and data acquisition As described previously^{［8, 9］}, the lipid bilayers were formed by painting a solution of phosphatidylcholine (25 mg/ml) and cholesterol (5 mg/ml) in n-decane on an orifice of 0.5～0.7 mm diameter in the Teflon partition, which was separated into two 3 ml lucite compartments (cis- and trans-compartment). The reconstitution was achieved by adding the isolated sperm membrane protein to the cis-compartment (final protein concentration: ～5 μg/ml) and stirring for 1 min. Between the two compartments in the buffer solution existed an osmotic gradient,which was used to promote the fusion of sperm membrane protein into the bilayer. Both sides were connected to the inputs of a patch-clamp amplifier (CEZ-2300, Nihon Kohden) in voltage-clamping mode via Ag/AgCl electrodes. Current signals (filtered at 10 kHz) were digitized with an audio PCM (Pulse Code Modulation) processor (501 ES, Sony) and recorded on videotape with a video cassette recorder (VCR-N85, NEC) for further analysis. Current was defined as positive when cations flowed into the trans-compartment. All measurements were made at room temperature (20～25℃). When a steady channel current was observed after the addition of protein sample, channel currents at different holding potentials (－100 to +100 mV) were measured and the current-voltage (I-V) relationship was obtained. The original records stored on videotape were trans～ferred using Digidata-1200 A/D system and pCLAMP 6.0.2 software (Axon Instrument, Inc.) to an IBM-AT compatible computer for analysis. I-V data were plotted using SigmaPlot software. Every I-V relation graph in this paper shows a regression line of three experiments obtained at the same experimental condition.

Some reports showed that the channel-like activity could be induced by Triton. However, there should not be possible effect on channel formation of Triton because of its much lower final concentration (＜＜0.01%) in the bilayer compartment^［8,9］.

1.4Chemicals and solutionsBuffer solutions contained 5 mmol/L HEPES (N-［2-hydroxyethyl］ piperazine-N′-［2-ethanesulfonic acid］) and 2 mmol/L Tris (Tris (hydroxymethyl)-aminomethane)-HCl, pH 7.4. Phosphatidylcholine was a product of Avanti Polar. Cholesterol, PMSF (phenylmethyl-sulfonyl),verapamil and nifedipine were products of Sigma-Aldrich, and n-decane was from Merck-Schuchardt. All other reagents were of analytical grade.

2RESULTS

2.1Two channel events with different unit conductance

Since a high Ca²⁺ concentration always disturbs the stability of lipid bilayer, a lower concentration solution system (50//10 mmol/L cis to trans CaCl₂) was used in order to obtain steady bilayer membrane. In this case, the successful rate to get available bilayer was near 25%. Each time when steady current was recorded, the sperm membrane protein preparation was added into the cis