Trans-membrane Migration Method Research Articles

Adenosine stimulates human sperm motility via A2 receptors.

The effects of adenosine and its analogues on human sperm motility were studied using a transmembrane migration method. Specific binding sites for adenosine in human sperm were also investigated. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) stimulated human sperm motility with similar efficacies and the maximal amplitudes of motility increases were both about 70%. 3,7-Dimethyl-1-propargylxanthine (DMPX), a potent A2 antagonist, competitively antagonized NECA-induced motility stimulation. Successively higher concentrations of DMPX shifted the dose-response curve of NECA to the right in a nearly parallel fashion. Dipyridamole, an inhibitor of adenosine uptake, does not reduce the ability of adenosine to stimulate human sperm motility. In radioligand-binding studies, adenosine A1 selective analogues, cyclopentyl-1,3-dipropylxanthine and 1-methyl-2-phenylethyl adenosine, have little competitive effect on [3H]NECA binding in human sperm membrane. These results provide evidence that adenosine enhances human sperm motility via adenosine A2 receptors on the surface of sperm membranes.

β-Adrenoceptor antagonists and human sperm motility

Several beta-adrenoceptor blocking agents have been evaluated for spermicidal activity using a transmembrane migration method. The rank order of potency of the active compounds was: penbutolol greater than (+)-propranolol greater than bufuralol greater than (-)-alprenolol greater than oxprenolol greater than metoprolol. Atenolol, pindolol, practolol, tolamolol were without activity. The observed potencies of spermicidal activity are believed to be unrelated to beta-blocking activities, and we have shown that whilst they are not predictable from lipid solubility or nonspecific membrane properties of the compound alone, both these aspects appear to play a role in this pharmacological activity.

Effects of lithium and haloperidol on human sperm motility in-vitro.

Two psychotropic drugs, lithium and haloperidol, were evaluated for their in-vitro effects on sperm motility using a transmembrane migration method. Sperm motility was measured either immediately after semen had been mixed with the drug or after a 2 h incubation period at 37 degrees C. Lithium inhibited human sperm motility in a dose-dependent manner with an EC50 of 10 mM when the semen-lithium mixture had been incubated. Sperm motility was increased to 127% of control when semen had been incubated with 0.027 microM haloperidol; this concentration was within the therapeutic range.

Comparative effects of (+)-propranolol and nonoxynol-9 on human sperm motility in-vitro

Using the modified transmembrane migration method to measure sperm motility, it was shown that the surfactant nonoxynol-9 alone, was twice as potent as (+)-propranolol alone as a spermicidal agent. Addition of (+)-propranolol to nonoxynol-9 shifted the dose-response curve to the left of the curves for either component alone, and a surprising synergistic action was evident. These observations may form the basis for the development of a new advantageous topical contraceptive combination product.

A modified transmembrane migration method for evaluating the spermicidal potency of some nonoxynol compounds.

The transmembrane migration technique, a simple method in-vitro for quantitatively assessing the effects of a drug on human sperm motility, has been evaluated. The original method has been modified to include a preincubation step, and the incubation time has been reduced to 90 min. In semen samples possessing sperm concentrations of less than 75 x 12(6) spermatozoa mL-1 the volume of the lower reservoir has been reduced to 1 mL. This modified method has been used to compare the spermicidal potency of the widely employed non-ionic surfactant nonoxynol-9, with nonoxynol-5 and nonoxynol-15 (containing, respectively, fewer and more ethylene oxide units per molecule). The rank order of spermicidal potency of the compounds evaluated was nonoxynol-9 = nonoxynol-5 greater than nonoxynol-15.

Evaluation of human sperm motility by means of transmembrane migration method and computer assisted semen analysis: a comparison study

Comparing motility parameters with a trans-membrane migration method and a Hamilton-Thorn HTM-2000 computer assisted semen analyzer, we found that trans-membrane migration ratio (TMMR) correlated best with critical motility which indicated the fraction of fastest and straightest sperm in a semen sample. We also found that TMMR correlated better with progressive velocity than with track speed. It is concluded that nonprogressive sperm were not included in the estimation of TMMR and the trans-membrane migration method is most suitable for studying drug effect on straight and rapid sperm motility.

Two plasminogen activators, streptokinase and urokinase, stimulate human sperm motility.

The stimulatory effects of two plasminogen activators, namely streptokinase and urokinase, were measured with a trans-membrane migration method. Both drugs induced maximal motility increase at a concentration of 200 international unit/ml; the amplitude of maximal motility increase ranged from 17% to 19% of control. Although their stimulatory effects were much less than those of calcium regulating agents, the clinical application of these two drugs for improving the successful rate of artificial insemination deserves further investigation because the action site is seminal plasma rather than sperm.

Measurement of testosterone in seminal plasma, saliva and serum by solid-phase enzymeimmunoassay.

When testosterone concentrations in serum, saliva and seminal plasma were measured with a solid-phase enzymeimmunoassay in which antitestosterone antibody was bound to CNBr-Sepharose-4B, we found the ratios of testosterone in saliva and seminal plasma to that in serum were 2.5 +/- 0.2% and 5.19 +/- 0.42%. Testosterone level in saliva could be an index of free serum testosterone. In addition, we found a correlation between sperm motility and testosterone level in seminal plasma when sperm motility was measured with a transmembrane migration method. This enzymeimmunoassay could be applied to clinical studies of testosterone.

Fluorescence Supravital Stain of Human Sperm: Correlation with Sperm Motility Measured by a Transmembrane Migration Method/Supravital-Fluoreszenz-Färbung von menschlichen Spermatozoen: Korrelation zur Spermatozoenmotilität (Messung mittels Transmembran-Mi

We developed a supravital stain of human sperm with fluorescent dyes. Either Hoechst 33,258 or fluorescein isothiocyanate could be used, the former stained sperm head while the later stained the whole sperm. Sperm vitality assessed with any of these two fluorescent dyes correlated well with that determined by eosin-nigrosin counterstain. When sperm vitality was compared with sperm motility measured with a transmembrane migration method, we found that many vital sperm were immotile because sperm vitality was higher than sperm motility in tested samples.

Effects of Ferulic Acid on Fertile and Asthenozoospermic Infertile Human Sperm Motility, Viability, Lipid Peroxidation, and Cyclic Nucleotides

The capacity of human sperm fertilization principally depends on sperm motility and membrane integrity. Reactive oxygen species, such as superoxide anion and hydrogen peroxide, are known to impair sperm motility and membrane integrity by inducing membrane lipid peroxidation (LPO). Ferulic acid (FA), an effective constituent in various medicinal herbs, has recently been shown to scavenge oxygen free radicals and increase the intracellular cAMP and cGMP. The aim of this study is to investigate the effects of FA on human sperm motility, viability, lipid peroxidation, and cyclic nucleotides in fertile and asthenozoospermic infertile individuals in vitro. The sperm samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Washed spermatozoa were incubated at 37°C in Ham's F-10 medium with 0, 0.1, 0.2, 0.4, 0.8, or 1.6 mM of FA. Samples were analyzed for viability, determined by eosin-Y dye exclusion method at 0, 1, 2, 3, 5, and 6 h of incubation; motility, determined by the trans-membrane migration method within 2 h of incubation; LPO, determined by thiobarbituric acid (TBA) method at 3 h of incubation and the intracellular cAMP and cGMP, determined, respectively, by 3H-cAMP and 125I-cGMP radioimmunoassay at 3 h of incubation. The results showed: in both fertile and infertile spermatozoa, the viability, trans-membrane migration ratio (TMMR) and the levels of intracellular cAMP and cGMP in FA-treated spermatozoa were significantly higher than those of spermatozoa in control groups, while TBA-reactive substances contents in treated spermatozoa were significantly lower than those in control spermatozoa. The effects of FA on these processes were concentration dependent. These data suggested that FA is beneficial to sperm viability and motility in both fertile and infertile individuals, and that reduction of lipid peroxidative damage to sperm membranes and increase of intracellular cAMP and cGMP may be involved in these benefits. It is possible that FA may be used for cure of asthenozoospermic infertility.Copyright © 1997 Elsevier Science Inc.

Possible role of nitric oxide on fertile and asthenozoospermic infertile human sperm functions.

The capacity of human sperm fertilization is principally dependent on sperm motility and membrane integrity. Oxygen-derived free radicals, such as superoxide anion, are known to impair sperm motility and membrane integrity by inducing membrane lipid peroxidation (LPO). Nitric oxide (NO), a biologically active free radical, has recently been shown to inactivate superoxide and increase intracellular guanosine-3', 5'-cyclic monophosphate (cGMP). The aim of this study is to investigate the effects of NO on human sperm motility, viability, lipid peroxidation and cGMP in fertile and asthenozoospermic infertile individuals in vitro. Semen samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Washed spermatozoa were incubated at 37 degrees C in Ham's F-10 medium with 0, 25, 50, 100, 200, or 400nM sodium nitroprusside (SNP, Na2 [Fe(CN) 5NO] 2H2O), a nitric oxide releaser. Samples were analyzed for viability, determined by eosin-Y dye exclusion method at 0, 1, 2, 3, 5 and 6 h of incubation; motility, determined by the trans-membrane migration method within 2 h of incubation; LPO determined by malondialdehyde (MDA)-thiobarbituric acid method at 3 h of incubation; and the intracellular cGMP, determined by 125I-cGMP radioimmunoassay at 3 h of incubation. The results showed: in both fertile and infertile samples, viability, trans-membrane migration ratio and the levels of intracellular cGMP in 25-100nM SNP-treated spermatozoa were significantly higher than those in control groups, while MDA contents in treated groups were significantly lower than those in controls. However, when concentrations of SNP increased to 200-400nM, the opposite effects were exhibited. The effects of SNP on these processes were biphasic within 25-400nM. The most effective concentration was 100nM. These data suggested that NO is beneficial to sperm viability and motility in both fertile and infertile individuals, and that reduction of lipid peroxidative damage to sperm membranes and increase of intracellular cGMP may be involved in these benefits.

A possible role of heat shock proteins in human sperm motility.

The testicular spermatogenic stem and seminiferous tubular cells selectively synthesize heat shock proteins (Hsps) during heat stress. Hsps, synthesized from testicular cells and leukocytes, are identical in molecular masses as well as chemical properties. In this study, we induced the Hsps from leukocytes and investigated their in vitro effects on human sperm motility. Semen samples were divided into two parts, washed and unwashed. The whole blood was heated in 43 degrees C for 15 minutes for induction of Hsps. A trans-membrane migration method was used to examine the effect of heated blood plasma on human sperm motility. The main heat-induced proteins of leukocytes were detected by 2-D electrophoresis and Coomassie blue stain. Leukocytes treated by heat produced a large amount of Hsp72 and Hsp80, while only a small amount was observed in that of non-heated leukocytes. The heated blood plasma inhibited motility of washed sperm in a manner that was dose-dependent. In the presence of seminal plasma fluid, however, the inhibitory effects of heated plasma on human sperm motility could not be observed. It was concluded that the heat-induced substance(s) from leukocytes, which being highly possible the Hsps, interfered the mobility of wash human sperm and the inhibition might be antagonized by seminal plasma.

Effects of pentoxifylline in the hamster zona-free oocyte spermatozoa penetration assay and on spermatozoa transmembrane migration motility.

The effects of pentoxifylline on capacitation, acrosome reaction and motility of spermatozoa in vitro were evaluated by the hamster zona-free oocyte penetration assay and the transmembrane migration method, respectively. The improvement in penetration capacity by the addition of pentoxifylline 40 min before coincubation had a trend toward statistical significance (0.05 less than less than 0.1) in normal fertile men and had statistical significance (p less than 0.05) in asthenospermic patients. There was no improvement by the addition of pentoxifylline 16 h before coincubation. Pentoxifylline significantly increased sperm motility in both normal (p less than 0.001) and asthenospermic patients (p less than 0.05) in the transmembrane migration method. This study showed that pentoxifylline could be used as a stimulant of in vitro sperm motility in male infertility.

Effects of Antiepileptic Drugs on Sperm Motility of Normal Controls and Epileptic Patients with Long‐Term Therapy

The in vitro effects of four antiepileptic drugs (AEDs) on human sperm motility were studied with a transmembrane migration method. Sperm motility of epileptic patients receiving chronic AED therapy was also investigated. Sperm motility was measured immediately after semen had been mixed with AED and after a 2-h preincubation at 37 degrees C. Both in vitro and in vivo studies demonstrated that AEDs inhibited sperm motility. When the drug effect was evaluated after the semen-AED mixture had been preincubated for 2 h, sperm motility was inhibited to 50% of control at concentrations of 1.59, 4.23, and 5.00 mM for phenytoin, carbamazepine, and valproate, respectively. Both with and without preincubation, phenobarbital, even up to 12.92 mM, did not inhibit the motility to less than 50% of the control. In the in vivo study, poor sperm motility was noted in epileptic patients with long-term AED therapy despite serum levels within the therapeutic range. Shorter duration of activity of spermatozoa was also observed in these patients. Interference with sperm membrane function by AEDs may be the underlying mechanism.

Astragalus membranaceus stimulates human sperm motility in vitro.

Poor sperm motility is an important cause of male infertility. In an attempt to identify Chinese medicinal herbs that might improve human sperm motility in vitro, we screened water extracts of 18 herbs with a trans-membrane migration method which measured the percentage of sperm that moved across the 5 micron pores of a Nucleopore membrane from a semen-drug mixture into phosphate buffered saline during 2 hours incubation. Astragalus membranaceus was the only herb that showed a significant stimulatory effect. At 10 mg/ml, it increased the motility of sperm in semen to 146.6 +/- 22.6% of control. It also increased the motility of washed sperm to 138.2 +/- 13.8% of control. Purification of the active component(s) from this herb as well as its application in assisted reproduction technology await further investigation.

Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy.

Pentoxifylline is a haemorrheologic agent often used in the treatment of peripheral vascular disorders. In this study, we measured sperm motility with a trans-membrane migration method and investigated the effect of this drug in the treatment of male infertility. We found that pentoxifylline increased motility of ejaculated spermatozoa in vitro from both normal and asthenozoospermic samples. After giving pentoxifylline to patients with asthenozoospermia for 3 months, sperm motility significantly increased, but sperm concentration did not increase. From the above results, it can be concluded that pentoxifylline is a useful drug in the treatment of normogonadotropic asthenozoospermia.

Human Follicular Fluid Stimulates the Motility of Washed Human Sperm

Human follicular fluid (hFF) was collected by laparoscopic oocyte pickup during IVF to evaluate the effect of hFF on human sperm motility with a transmembrane migration method. Freshly ejaculated human sperm were washed with phosphate buffered saline (PBS) and mixed with either PBS or hFF. Amplitude of motility increases were 38% and 72% in washed fertile sperm and washed asthenozoospermic sperm when individual control motility was considered to be 100%. The stimulatory effect of hFF was lost when preheated at 100 degrees C for 30 minutes. hFF collected from mature follicles stimulated sperm motility better than that collected from intermediate or immature follicles. hFF did not stimulate the motility of unwashed sperm in freshly ejaculated human semen. A heat labile factor(s) in hFF may stimulate the motility of washed human sperm. Whether this factor could be used to improve the success rate of IVF and artificial insemination awaits further investigation.

The inhibitory effect of anticonvulsants on human sperm motility: measured with a trans-membrane migration method.

The in-vitro effects of four anticonvulsant drugs (phenytoin, phenobarbitone, carbamazepine and valproate) on human sperm motility were studied with a trans-membrane migration method. Sperm motility was measured either immediately after semen had been mixed with the drug or after a 2-hour pre-incubation at 37 degrees C. When the drug effect was evaluated after the semen-drug mixture had been pre-incubated for 2 hours, the concentration of phenytoin, carbamazepine and valproate that inhibited sperm motility to 50% of control (EC50) was 1.59, 4.23, and 5.00 mM, respectively. If sperm motility was immediately measured without preincubation, the EC50 was increased to 8.47 and 100.00 mM for carbamazepine and valproate, respectively, whereas phenytoin could not inhibit sperm motility to less than 50% of the control at the tested concentrations. Both with and without pre-incubation, phenobarbitone, even up to 12.92 mM, could not inhibit sperm motility to less than 50% of the control. The result was compatible with previous findings that drugs with a membrane stabilizing effect may inhibit human sperm motility. This study provided further information regarding the application of human sperm motility as a cellular model for future pharmacological research.

Lithium inhibits human sperm motility in vitro.

Lithium is a widely prescribed drug used for the treatment of bipolar affective illness. Previous reports on its effects on sperm motility and male fertility are conflicting. The effect of lithium on human sperm motility was examined in vitro using the modified transmembrane migration method. This technique takes account of the dilution of lithium that occurs during the incubation. Lithium inhibits human sperm motility in vitro in concentrations comparable with those reported to be achieved in semen after oral administration.

The inhibitory effect of gossypol on human sperm motility: relationship with time, temperature and concentration.

The inhibitory effect of gossypol acetic acid on human sperm motility was studied with a transmembrane migration method. Gossypol decreased sperm motility after it had been incubated with semen for more than 15 min. However, when sperm motility was evaluated immediately after semen had been mixed with gossypol, no inhibitory effect could be found. We consider that the sperm immobilizing potency of gossypol is much less than our previously studied sperm immobilizing agents. It is unlikely that gossypol can be developed as a vaginal spermicide. The importance of time course in the pharmacological study of sperm motility is emphasized in this study.