Shi YL et al / Acta Pharmacol Sin 2003 Jan; 24 (1): 22-30

Ion-channels in human sperm membrane and contraceptive mechanisms of male antifertility compounds derived from Chinese traditional medicine¹

SHI Yu-Liang², BAI Jun-Ping, WANG Wen-Ping

Key Laboratory of Neurobiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

¹ Project supported by the National Natural Science Foundation of China (No 38388013, 38970831, 39480005, and 39870197).
² Correspondence to Prof SHI Yu-Liang.
Phn 86-21-6437-0080, ext 154. Fax 86-21-6433-2445.
E-mail ylshi@server.shcnc.ac.cn

Received 2002-06-13 Accepted 2002-10-14

KEY WORDS spermatozoa; ion channels; lipid bilayers; spermatogenic cells; male antifertility agents; Chinese herbal medicine

ABSTRACT

Ion channel plays a key role in maturation, capacitation and acrosome reaction of sperm. However, as it is difficult to record channel currents from a mature mammal sperm directly by patch-clamp technique, there were no basic data on the types and properties of the channels in human sperm until the method reconstituting the channels into bilayer was used. By reconstituting the channel proteins isolated from sperm membrane into phospholipid-forming bilayer, we have characterized several kinds of Ca²⁺-, Na⁺-, K⁺-, and Cl^--permeable channels with different conductance and properties in human sperm membrane. To study the channels in spermatogenic cells is another approach to understand the ion-channels in mature sperm. The cell is used as a model to analyze the effects of male antifertility agents on Ca²⁺-channel. To date, several male contraceptives derived from Chinese traditional medicine have been attached worldwide interest, a lot of compounds have been purified from them, and the antifertility effects of some compounds were demonstrated. We studied the effects of gossypol and several compounds isolated from Tripterygium wilffordii on Ca²⁺ channel in mouse spermatogenic cells and found that each of them inhibited the channel and sperm acrosome reaction at a proximate concentration, suggesting that the inhibition of Ca²⁺ channels may be one mechanism of the antifertility effects of these contraceptives.

ESSENTIAL ROLE OF ION-CHANNELS IN SPERM MATURATION AND CAPACITATION AND SPERM-OOCYTE INTERACTION

Ionic fluxes through ion-channels are crucial in sperm maturation, capacitation, and the initiating pro cess of gamete interaction. Modifying the activities of ion-channels is one important way of reproductive hormones to regulate the fertility process and of male antifertility agents to induce contraceptive effects. The role of Ca²⁺ channels in these processes was noticed firstly. It was reported that the existence of extracellular Ca²⁺ was essential in acrosome reaction (AR), a rise in Ca²⁺ concentration in sperm occurred during AR and an inhibition in the maturation, capacitation and AR of sperm was observed in Ca²⁺ channel blockage^[1-3]. Regarding the actions of monovalent cations, it has been known that sperm maturation was modified directly by the changes in the concentration of Na⁺ and K⁺ in the epididymis. The effluxes of K⁺ and H⁺ were observed during AR and the progesterone-evoked AR was inhibited in a Na⁺-free medium^[4]. Chloride ions have been demonstrated to be necessary for AR evoked by progesterone or zona pellucida (ZP). The progesterone-evoked AR was accompanied with Cl^- efflux, and the blockers of GABA_A receptor/Cl^- channel, picrotoxin and bicu-culline have been found to inhibit AR significantly^[5-8]. These facts indicate a crucial participation of ion-channels in maturation, capacitation and sperm-oocyte interaction and the existence of various kinds of ion-channels in human sperm membrane. However, due to the small size, high differentiation, complex geometry, and motility of mammalia sperm, intracelluar recording or patch-clamp technique is very difficult to be used satisfactorily to record the channel current and characterize the types and properties of the ion-channel in intact human sperm. This is why there were no basic data on ion-channels in human sperm until ion channel-bilayer reconstitution method was introduced to the study field.

FUNCTIONAL RECONSTITUTION OF ION-CHANNEL PROTEIN IN BILAYER MEMBRANE

Ion-channels are the kind of membrane proteins and when incorporated into lipid bilayer membrane they provide the pathway for ion transmembrane transport. Thus, when ion-channel proteins or fragments of biomembrane are incorporated into artificial bilayer membrane that separated two water phases, the channel-mediated currents can be recorded and characterized by patch-clamp amplifier. That is the ion channel-bilayer reconstitution method being progressively developed and perfected in the recent years^[9,10]. By the method, we have recorded and analyzed several kinds of single events mediated by the channels formed by pore-formers derived from the toxins of snakes and fish^[11-13], antibiotics^[14,15], lectin^[16,17], type II ribosome-inactivating protein^[18], and chloroplast ATP synthase CF₀-CF₁^[19,20]. All the electrophysiological data involving the type and property of the channels in human sperm membrane are resulted from the studies using the channel-bilayer reconstitution method.

Procedures to form artificial phospholipid bilayer exploit the several conformation that phospholipid molecules can adopt in water. These conformations arise as a consequence of the amphiphilic nature of lipids, composed of a polar head group and a non-polar hydrocarbon tail. The two-compartment system with a septum to suppport the bilayer was used firstly. Various reconstitution techniques were introduced to incorporate channel proteins into bilayers. The recent implementation of the method makes use of glass patch-clamp pipettes to assemble the bilayer film at the tip of the electrode. A direct application of patch-clamp technique to reconstitution consists of the formation of giga-seals against the surface of giant liposome containing ion-channels^[9] (Fig 1).

Fig 1. Strategy for isolated patch recording of ion-channels into giant liposomes. The giant liposomes were formed by dehydration-rehydration or freeze-thaw procedures^[9].

TYPES AND PROPERTIES OF ION-CHANNELS IN HUMAN SPERM MEMBRANE

Ca²⁺ channels Using the two compartment system, the ion-channel proteins in human sperm were reconstituted into phospholipid artificial bilayer and single channel currents were recorded in a CaCl₂ solution system. We found that there existed two kinds of Ca²⁺-permeable channels with different conductance at least. One with open-subconductance was voltage-dependent and its open probability (P_o) increased upon depolari-zation. Another was non-voltage dependent. The reversal potentials of them were close to the Nernst potential of Ca²⁺ electrode and both were completely in hibited by nifedipine and verapamil although with different efficacious concentration. These results^[21] indicated that the recorded channel currents were carried by Ca²⁺ (Fig 2). Meanwhile, the infertile human sperm samples were also investigated and an abnormal activity in Ca²⁺ channels was observed, ie, the channels with an extremely long open time and no returning to their previous close state. The abnormal channel activity had never been detected in the samples derived from health donors, suggesting that there were some variations in the channel protein conformation of infertile sperm^[22].

Fig 2. Single channel currents recorded in CaCl₂ solutions and their depression by nifedipine. A, Channel events observed at seven different holding voltages with "-c" indicating the closed state of the channel. B, Current-voltage relationship obtained from three same experiments. C, Depressive effects of Ca²⁺ channel blocker nifedipine (50 mol/L) on Ca²⁺ channel current. The concentration gradient was 50 (cis) to 10 (trans) mmol/L CaCl₂^[21].

Monocation channels Tetraethylammonium(TEA)-sensitive K⁺ channels and tetrodotoxin (TTX)-sensitive Na⁺ channels were detected from human sperm membrane by the similar reconstituiton system^[21-23]. For further analysis of the Na⁺-channels, we changed reconstitution method to ressembling the proteins into liposomes and then fusing the liposomes into giant liposomes (diameter >10 m) by dehydra tion-rehydration procedure^[24]. By this method, two kinds of Na⁺ channels with different conductance were observed. Both of them were TTX-sensitive and strongly rectifying and with a reversal potential near the Nernst potential of Na⁺ electrode (Fig 3). In a symmetric solution system (200 mmol/L NaCl), their unit conductance were 60 pS and 140 pS, respectively. The large one exhibited subconductance state between close and full open and burst activity with flicker transition. By constructing the open or close dwell time distribution histogram and fitting it by exponential function, we obtained two time constants for each state, implying that there were two open and two close states in the large Na⁺ channel at least. In addition, another Na⁺-channels with a unit conductance somewhat between the mentioned two channel events was also observed. The I-V curve for the middle one was linear, with a conductance of 70 pS^[24]. By the way, similar to abnormal Ca²⁺-channel activity, no-close property of monovalent cation channels was observed in infertile sperm sample^[22].

Fig 3. Single-channel currents recorded from giant liposome in which channel proteins derived from human sperm membranes were reconstituted. A and B are respectively from two excised patches, demonstrating two Na⁺ channels with different conductance and open probability. C shows current-voltage relationship of the large (, n=9) and the small (, n=3) Na⁺ channels with reversal potential () of -18 mV^[24].

Cl^- channels By the reconstitution method mentioned above, ie, reassembling ion channel into giant liposome, three kinds of Cl^- channels with unit conductance of 74.1 pS, 117.0 pS, and 144.7 pS, respectively were characterized in an N-methyl-D-glucamine (NMDG) solution sysytem^[25]. All of them could be blocked by 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), a Cl^--channel blocker, in a concentration-dependent manner. By constructing open or close dwell time histogram and then fitting it with exponential function, two time constants were obtained in both open and close states. The burst activity and subconductance states were often observed in the three Cl^- channels. Their open was voltage-dependent (Fig 4) as demonstrated by the following facts: 1) The channel P_o showed a maximum at -100 mV and decreased upon depolarization; 2) The channel open frequency was high at negative potential and obviously low at positive potential.

Fig 4. Voltage-dependence of human sperm Cl^- channel. A, Original recordings from a three-channel patch; B, P_o (open probability)-voltage relationship. The unit conductance of channel is 117 pS in bath 100// pipette 200 mmol/L NMDG-Cl solution. The results were obtained from a patch^[25].

SPERMATOGENIC CELL, AN USEFUL MODEL IN ANALYZING CONTRACEPTIVES ACTION ON Ca²⁺-CHANNELS

Spermatogenic cells are the developmental precursors of spermatozoa. Pachytene spermatocytes and round spermatids are two prominent cell types of spermatogenic cells obtained from diploid meiotic and haploid postmeiotic stages of spermatogenesis. These cells with diameter about 20 m in mice are suitable for patch clamp recording. Now, it is accepted that spermatozoa are terminally differentiated cells without the ability to synthesize protein, and the ion-channels expressed during spermatogenesis will retain in mature sperm. So, besides channel-bilayer reconstitution, to characterize the ion-channels in the spermatogenic cells is another method to approach the ion-channels in mature sperm.

Up to date, Ca²⁺-, Cl^--, and K⁺-permeable channels were observed by whole-cell patch clamp recording in mammal spermatogenic cells^[4,26-28]. The K⁺ channels were sensitive to TEA and their density progressively decreased during spermatogenesis^[26]. The Ca²⁺-channels with low voltage-activated, fast activating and inactivating, high Ni²⁺-sensitivety showed the properties of T-type channels^[26-28]. In view of the essentiality of Ca²⁺ in the function of sperm and important role of T-type Ca²⁺ channels in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) during capacitation and the acrosome reaction in sperm^[4], the spermatogenic cell is often used as a model system of mature sperm to study the effects of male antifertility agents on Ca²⁺ channels and hence to explore their contraceptive mechanisms.

STUDIES ON MALE CONTRACEPTIVES DERIVED FROM CHINESE TRADITIONAL MEDICINES

There are a lot of male contraceptives in Chinese traditional medicines, such as seeds of Gossypium herbaceum L, roots of Tripterygium wilfordii Hook f, and whole-plant of Begonia yunnanensis Lévl or Sphagnum teres Aongstr. Up to now, many antifertility compounds were purified and identified from these Chinese traditional medicines, and the contraceptive effects of some of them, for example gossypol purified from cotton seeds, the extracts and compounds isolated from roots of Tripterygium wilfordii Hook f were demonstrated in the studies performed on human and animals. Meanwhile, their antifertility mechanisms were analyzed.

Gossypol Gossypol, a polyphenolic compound, when it was administered via mouth induced infertility in rat, hamster, guinea pig, monkey, and man. The effect could be reversible after withdrawal of the drug^[29]. Although the idea that to use gossypol as a clinical long-term contraceptive drug was disputed due to its side-effects, such as hypokalemia, weakness, and irre versible azoospermia, recent studies showed that the drug could cause contraception and without side-effects at a low dosage when it was cooperated with steroid hormone contraceptives^[30]. Previous data demonstrated that gosspol affected sperm capacitation, motility, AR, and ability to penetrate oocytes^[31]; induced morphological changes in spermatogenic cells and spermatozoa, such as inducing the damage in sperm nucleus and acrosome, even head-tail separation after injecting drugs into epididymis^[29]; reversibly decreasing the amount of tubulin and dynein in spermatocyte and spermatid, hence inhibiting the transformation from round- to elongated-head spermatid^[32]; impairing mitochondrial sheath, probably by uncoupling oxidative phosphorylation or inhibiting lactate dehydrogenase. It was also reported that after treatment with gossypol, the animals caused hypophysectomy-like symptoms, ie, a block of hormone regulation on spermatogenesis^[33].

Cyclic adenosine monophosphate (cAMP) plays a key role in sperm maturation and sperm-oocyte fusion. It was reported that gossypol inhibited adenylate cyclase and cAMP phosphodiesterase, two key enzymes were involved in the synthesis and hydrolysis of cAMP, and resulted in the reduction of intracellular cAMP, and then down-regulated the activities of cAMP-dependent protein kinases and the biosynthesis of androgens^[29,34].

Acrosome reaction, an initial event necessary to sperm-oocyte fusion, was found to be Ca²⁺-dependent. Based on that gossypol concentration-dependently inhibited sperm AR as well as Ca²⁺-uptake and Ca²⁺-ATPase in human spermatozoa membrane vesicles^[31,35], we studied the effects of gossypol on Ca²⁺ channels in mouse spermatogenic cells by whole-cell recording. The results showed that at a similar concentration to inhibit AR (5-80mol/L), gossypol concentration-, time-dependently, and irreversibly inhibited T-type Ca²⁺ channels. The time to block the channels was progressively shortened as the gossypol concentration was increased from 5 to 80 mol/L (Fig 5). Moreover, this drug did not affect the activation of the channels, while increased their inactivation time constant in concentration- and time-dependent manner. It was also observed that the inhibitory effect on the T-type Ca²⁺ current did not correlate with signalings mediated by G proteins and tyrosine phosphorylation, suggesting that the effect may result from a direct action on the channels. Gossypol had no obvious effects on Cl^- currents^[36]. The facts that gossypol inhibited AR and Ca²⁺ channel at a similar concentration, indicated that the gossypol-induced blockade of Ca²⁺ channels could be responsible for its inhibition on AR and hence for the antifertility activity of the drug. These results suggest a possibility to use gossypol locally as an emergency vaginal contraceptive drug.

Fig 5. Gossypol-induced inhibition of T-type Ca²⁺ currents. A, an example showing the original records (every 20 s) in a spermatogenic cell before and after gossypol application and after washing. B, Time course of the current decrease after the application of different concentrations of gossypol. Each point represents the mean±SEM of 3-5 experiments similar to that shown in A^[36].

Extract and compounds derived from Triptery-gium wilfordii Hook f The plant containing alkaloids, diterpenoids, triterpenoids, and sesqueterpenes, was used in Chinese traditional medicine to cure rheumatoid arthritis, chronic nephritis, ankylosing spodylitis and various skin diseases. Its male antifertility effect was found as a side effect in treating the diseases^[37]. It was observed that when oral administration with GTW (10 mg×kg^-1 ×d^-1), a water-chloroform extract from the plant, all male rats became infertile in 8 weeks while the histology of the seminiferous tubules and serum testosterone level were unaltered^[37]; oral administration with 20-30 mg×kg^-1 ×d^-1 induced infertile in man without side effects. It has been shown that GTW acts mainly on spermatogenic cells, so its antifertility effect may result from an inhibition of sperm transformation and maturation via its selective action on spermatogenic cells in testis^[38].

Up to now, many compounds including triptolide, tripchlorolide, tripdiolide, demethylzeylasteral, celastrol, and L-epicatechin, etc, have been isolated from GTW^[37]. Some of them have been demonstrated to have an antifertility effect, in which tripchlorolide was the most effective. Previous studies showed that GTW could induced damages of microtubule, microfilament, and plasma membrane, even head-tail separation of sperm, and decrease the motility and the concentration of sperm in the cauda epididymis dramatically^[37]. Following the treatment with these compounds, a reduction of total basic nuclear protein and an increase of total histone/protamine ratio were observed in sperm, indicating an inhibition of nuclear protein transition and a decrease of protamine levels in sperm^[39].

Tab 1. A comparison of the efficacious dosages of some male antifertility compounds to inhibit both sperm AR and spermatogenic cell Ca²⁺ current.

It was reported that celastrol and demethylzeyl-asteral inhibited progesterone-induced AR concentration-dependently^[40,41] (Tab 1). The blocking effects of GTW, demethylzeylasteral, celastrol, and L-epicatechin on mouse spermatogenic cell Ca²⁺ channels were demonstrated in our recent studies^[41,42] (Fig 6). Demethyl-zeylasteral and GTW inhibited the channels concentration-dependently and reversibly with a similar IC₅₀. Both of them enhanced the time constants of activation and inactivation of the channels. The effective concentration of L-epicatechin to inhibit the channels was 1000 times higher than that of GTW and demethylzeylasteral. L-epicatechin prolonged the inactivation of the channel at a higher concentration. The inhibition of Ca²⁺ channels by celastrol was similar to that by gossypol, showing time- and concentration-dependence. From that these compounds inhibited spermatogenic cell Ca²⁺ channels and Ca²⁺ influx-evoked sperm AR at a proximate concentration, it was suggested that the inhibition of Ca²⁺ channels might be one mechanism of antifertility effect of the compounds. Previous studies showed that the dosages of the immuno-suppressive effect of GTW and these compounds were 5-10 times higher than those of their antifertility effects^[37]. The further studies to elevate safety factor and eliminate side-effect of these drugs are performing in some laboratories.

Fig 6. GTW and demethylzeylasteral-induced inhibition of T-type Ca²⁺ currents in mouse spermatogenic cells. A, Change of the Ca²⁺ current after 10 g/mL GTW application and washing. Insert: whole-cell original recording. B, Concentration-response relationships. Peak currents were normalized with respect to the control. Each point represents the mean±S.E.M. of 5 experiments^[42].

In conclusion, ion-channel plays a crucial role in sperm maturation, capacitation, and AR. Several kinds of Ca²⁺-, Na⁺-, K⁺- and Cl^--channels have been successfully identified by channel protein-lipid bilayer reconstitution method. Mouse spermatogenic cell, as a model of mature sperm, has been used to analyze the effects of male contraceptive drugs on Ca²⁺-channels. The antifertility effects of gossypol and some compounds isolated from Tripterygium wilfordii Hook f have been demonstrated and the inhibition of T-type Ca²⁺ channels could be responsible for their antifertility activity.

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