Lorophyn suppository contains other ingredients which might potentiate the sperm-immobilizing activity of phenylmercuric acetate. It should be pointed out that ...
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THE INHIBITORY EFFECT OF SOME IONOPHORES ON HUMAN SPERM MOTILITY Hong C.Y., Huang J.J., Chiang B.N. & Wei Y.H.'k Departments of Medicine and gcBiochemistry Taiwan Veterans General Hospital, Taipei and National Yang-Ming Medical College, Taipei, Taiwan, R.O.C.
ABSTRACT The inhibitory effect of five ionophores, namely, A23187, nonactin, nigericin, monensin and m-chlorocarbonyl cyanide phenylhydrazone, on human sperm motility were measured with a trans-membrane migration method. The concentrations of A23187 and nigericin that decreased sperm motility to 50% of control were 20 PM (10.5 mg/l) and 8 FM (5.8 mg/l), respectively. Because these two ionophores were more potent than previously reported membrane-active sperm-immobilizing agents, we propose that ionophores could be a new category of vaginal contraceptive if a pharmaceutical preparation that is safe to be administered in vivo can be developed. INTRODUCTION The ionophores are compounds which form lipid soluble complexes with specific cations and act as vehicles for transporting these cations across biological membrane (1). By changing the trans-membrane ion gradients, they may produce profound effect on cellular functions. We have recently used a trans-membrane migration method for studying the influences of physiological and pharmacological substances on human sperm motility (2). It was concluded that sperm-immobilizing agents stopped sperm movement by acting on sperm membrane (3). Although the definite nature of membrane change that renders sperm immotile is not clear, there is evidence that these drugs may bind to the lipid bilayer of cellular membrane and change the ionic transport across the membrane. Calcium ion has recently been proposed as a regulator of sperm function and an increased intracellular calcium concentration is considered to be detrimental to the motility of ejaculated human sperm (4). Our recent finding that A23187, a calcium ionophore, has a potent inhibitory effect on human sperm motility is compatible with such a theory (5). Submitted for publication December 12, 1985 Accepted for publication March 7, 1986
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A study was undertaken to see if other ionophores have spermimmobilizing effect. Ionophores are mixed with freshly ejaculated human semen and the changes in sperm motility are assessed with the trans-membrane migration method (2). Such an approach is similar to the situation when a vaginal contraceptive is applied. Our previous studies on vaginal contraceptives will be compared and the discussion will be focused on whether ionophores could be developed into a new category of vaginal contraceptive. MATERIALS
AND METHODS
Five ionophores were tested, they were A23187 nonactin, nigericin, monensin and m-chlorocarbonyl cyanide phenylhydrazone (CCCP). Among them, A23187 is a calcium ionophore that facilitates the transport of calcium ion across cellular membrane; nonactin binds potassium ion and allows penetration of potassium ion through lipid biological membrane; nigericin acts as a potassium ionophore that exchanges potassium ion for hydrogen ion; monensin is closely related to nigericin but exchanges potassium ion for sodium ion; CCCP is a proton ionophore that uncouples oxidative phosphorylation and therefore inhibits the synthesis of ATP (6). All these five chemicals were purchased from Sigma (USA). A23187, monensin, CCCP and nigericin were dissolved in ethanol and then diluted to the desired concentration with phosphate buffered saline (Dubecco A, pH 7.3). The final concentration of ethanol in test solution was less than 1.6% (v/v) which did not affect sperm motility measured with the trans-membrane migration method (3). Nonactin is insoluble in ethanol; it was therefore dissolved in chloroform (GR grade, Merck, FRG) to make 1 mg/ml solutions and adequate volumes of the solution were pipetted into the bottom of glass tubes. The tubes were heated at 60°C on sand plate and chloroform evaporated with nitrogen gas. Semen was then pipetted into each tube for dissolving the residues. Freshly ejaculated human semen were collected from healthy donors. Each semen sample was divided into several aliquots which were then mixed with ionophores. Only semen samples with a sperm concentration higher than 20 million/ml and a basal motility better than 20% were used (7). Sperm motility was determined with a trans-membrane migration method which measured the percentage of sperm that moved from semenionophore mixtures into phosphate buffered saline during 2 Motility of sperm in semen aliquot hours incubation at 37°C. mixed with solvent was used as a control while those in semen-ionophore mixtures were expressed as percentages of control. At least five semen samples were used for studying EC50 was the concentration of the effect of each ionophore. ionophore that decreased sperm motility to 50% of control.
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A supplementary experiment was performed to test the effect of oligomycin on human sperm motility. Oligomycin is not an ionophore, it inhibits the synthesis of ATP without changing the flow of electrons (6). Procedures for studying this ATP synthesis inhibitor were similar to those for CCCP and other ethanol soluble ionophores.
RESULTS
The figure shows that only A23187 and nigericin have a concentration-dependent inhibitory effect on human sperm motility. The EC50 for A23187 and nigericin to inhibit sperm Although monensin motility is 20 JIM and 8 PM, respectively. also inhibits sperm motility, it cannot decrease sperm motility to 50% of control at concentration as high as 100 JIM.
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Figure. Effect of nigericin ( 0 ), nonactin ( 0 ), CCCP human sperm motility. Each least five samples.
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( H ), A23187
( 0
), monensin ( A) on It S.E.M. for at
( A ) and oligomycin point
was mean
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DISCUSSION A number of drugs known to have so-called membrane stabilising effect had been tested with the trans-membrane migration method for their sperm-immobilizing potencies. Among them, imipramine is the most potent one (8). Its EC50 is 0.16 mM, equivalent to 50.7 mg/l. That A23187 and nigericin have an EC50 of 20 PM (10.5 mg/l) and 8 PM (5.8 mg /l,, respectively, indicates that these two ionophores are more potentthan previously reported membrane-active sperm immobilizers. Even when we compare the potencies of A23187 and nigericin with those of currently available vaginal contraceptives, their sperm-immobilizing activities are still impressive. For example, the EC50 for Menfegol, the active ingredient in Neosampoon tablet (Eisai, Japan) and Delfen cream (Ortho, UK) is 69.4 mg/l (9). Although the EC50 for phenylmercuric acetate as an active ingredient in Lorophyn suppository (Eaton, USA) is 0.5 mg/l (9), the EC50 for phenylmercuric acetate per se is 22 PM or 7.4 mg/l only (Hong, unpublished). Lorophyn suppository contains other ingredients which might potentiate the sperm-immobilizing activity of phenylmercuric acetate. It should be pointed out that Lorophyn is no longer allowed for use as a spermicide in the United States due to the possible hazardous effect of mercury to the fetus or breast-feeding infant. It is reasonable to propose that A23187 and nigericin inhibit human sperm motility by changing ionic transport across sperm membrane. A23187 is a calcium ionophore that increases intracellular calcium concentration. Calcium ion has been demonstrated to be detrimental to the motility of ejaculated+human, dog and sea urchin sperm (4). Nigericin actsas a K ionophore, it exchanges extracellular H' for intracellular Kf and thus produces an intracellular pH drop (1). A decreased intracellular pH was found to be associated with poor sperm motility (10). Nigericin i$ a carboxylic ionophore that forms neutral complex with K . Although nonactin is also a K+ ionophore, it is a neutral ionophore that forms charged complex with K+ (1). Monencin is a Na+/Hf exchanger with low affinity for Kt (1). Whether these differences are responsible for the lack of spermimmobilizing activity for nonactin and monencin cannot be answered directly from this study. CCCP is a proton ionophore that eliminates protonic potential difference across mitochondrial membrane and thus acts as an uncoupler of oxidative phosphorylation to inhibit ATP synthesis (6). Oligomycin is also an inhibitor of ATP synthesis (6). That both CCCP and oligomycin have no inhibitory effect on human sperm motility is compatible with our previous finding that mitochondrial inhibitors and
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uncouplers
are not sperm-immobilizers
(7).
It has been claimed that for the development of vaginal contraceptive, safety rather than potency is the main consideration (11). In spite of the fact that the safety of ionophores including their local effect on vaginal tissue and the systemic effect of the absorbed fraction has never been studied, our findings nevertheless provide a new direction for the search of more potent sperm-immobilizing agents. ACKNOWLEDGEMENT This study was supported by grants from National Science Council and the Institute of Biomedical Sciences, Academic Sinica at Taiwan, R.O.C. REFERENCES 1.
Pressman, B.C. and Fahim, M. Pharmacology and toxicology of the monovalent carboxylic ionophores. Rev Pharmacol Toxic01 22:465-490 (1982).
Ann
2.
Hong, C.Y., Chaput de Saintonge, D.M. and Turner, P. simple method to measure drug effects on human sperm motility. Br J Clin Pharmacol 11:385-387 (1981).
3.
Hong, C.Y. and Chiang, B.N. Local anaesthetic effect of antiarrhythmic drugs and human sperm immobilization: mechanism and application of the interrelationship. Br J Clin Pharmacol 17:687-690 (1984).
4.
Hong, C.Y., Chiang, B.N. and Turner, P. Calcium ion is the key regulator of human sperm function. Lancet 2: 1449-1451 (1984).
5.
Hong, C.Y., Chiang, B.N., Ku, J., Wei, Y.H. and Fong, J. C. Calcium antagonists stimulate sperm motility in ejaculated human semen. Br J Clin Pharmacol 19:45-49 (1985).
6.
Mayes, P-A., in Harper's Review of Biochemistry (David W. Martin, Jr., Peter A. Mayes and Victor W. Rodwell, Editors). Lange Medical Publications, California, 1983, Chapter 12, p.134-141.
7.
Hong, C.Y., Chiang, B.N. and Wei, Y.H. Mitochondrial respiration inhibitors and human sperm motility: implication in the development of spermicides. Br J Clin Pharmacol 16:487-490 (1983).
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8.
Sperm immobilizing Hong, C.Y., Chiang, B.N. and Ku, J. potency of amitriptyline and imipramine: measured with transmembrane migration. Arch Androl 12:25-28 (1984).
9.
Hong, C.Y., Chiang, B.N. and Wu, P. In vitro spermicida potencies of the dosage forms and active ingredients of some vaginal contraceptives: measured with a transmembrane migration method. Pharmaceut Med 1:33-39 (1984).
10
Christen, R., Schackmann, R.W. and Shapiro, B.M. Metabolism of sea urchin sperm: interrelationships between intracellular pH, ATPase activity and mitochondrial respiration. J Biol Chem 258:5392-5399 (1983).
11
Coleman, S. and Piotrow, P.T. Spermicides - simplicity and safety are major assets. Population Reports, Series H, No. 5. Baltimore, Johns Hopkins University, Population Information Program, 1979.
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