Effects of phosphodiesterase inhibitors caffeine and pentoxifylline on spontaneous and stimulus-induced acrosome reactions in human sperm

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1 FERTILITY AND STERILITY Copyright CI 1992 The American Fertility Society Vol. 58, No.6, December 1992 Printed on acid-free paper in U.S.A. Effects of phosphodiesterase inhibitors caffeine and pentoxifylline on spontaneous and stimulus-induced acrosome reactions in human sperm Jan Tesarik, M.D.*t Carmen Mendoza, Ph.D.:j: Alfonso Carreras, Ph.D.:j: American Hospital of Paris, Neuilly sur Seine, France; National Institute of Health and Medical Research (lnserm), Unit 355, Clamart, France; and University of Granada Faculty of Sciences, Granada, Spain Objective: To determine whether the phosphodiesterase inhibitors caffeine and pentoxifylline influence the acrosome reaction in the conditions in which they are currently used as sperm movement enhancers. Design: The frequency of acrosome reaction occurring spontaneously in capacitating media or induced by physiological (follicular fluid [FF]) and artificial (ionophore A23187) stimuli was compared in the presence and absence of the phosphodiesterase inhibitors. Setting: Private hospital and research laboratory. Patients, Participants: Patients undergoing routine semen examination before in vitro fertilization (no pathology detected) and healthy sperm donors. Interventions: None. Main Outcome Measure: Percentage of acrosome-reacted sperm determined with the use of fluorescein-labeled Pisum sativum agglutinin as acrosomal stain. Results: Caffeine alone augmented the frequency of acrosome reaction, but this effect was not observed with pentoxifylline alone. However, pentoxifylline increased sperm responsiveness to the acrosome reaction-inducing stimuli, FF and ionophore A Conclusions: The promotion of spontaneous acrosome reaction may counteract the benefits from application of caffeine as motility stimulant. On the other hand, the sensitization to physiological acrosome reaction stimuli is expected to contribute to the improvement of sperm fertilizing ability by pentoxifylline and make this drug a potential candidate for the treatment of acrosome reaction anomalies. Fertil Steril 1992;58: Key Words: Phosphodiesterase inhibitors, motility stimulants, caffeine, pentoxifylline, acrosome reaction, follicular fluid, ionophore A23187, human sperm Among the variety of chemical agents tested for the capacity of improving sperm movement and fertilizing ability, drugs acting on the sperm intracel- Received June 15, 1992; accepted July 13, * Centre for Reproductive Biology and Medicine, American Hospital of Paris, and INSERM, Unit 355. t Reprint requests: Jan Tesarik, M.D., Ph.D., Centre for Reproductive Biology and Medicine, American Hospital of Paris, 63 Boulevard Victor Hugo, Neuilly sur Seine, France. :j: Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences. lular concentration of cyclic adenosine monophosphate (camp) are of particular interest for several reasons. First, camp is known to be a key element of sperm movement regulation (1), even though its exact mechanism of action remains to be elucidated. Further, agents augmenting the intracellular camp concentration belong among the relatively rare motility stimulants for which a beneficial effect on the success rate of human in vitro fertilization (lvf) has been clinically demonstrated (2, 3). Finally, camp appears to be involved not only in the control of sperm motility but also in that of the acrosome Vol. 58, No.6, December 1992 Tesarik et al. Phosphodiesterase inhibitors and acrosome reaction 1185

2 reaction (4), another event vital for successful fertilization. Inhibition of phosphodiesterase, the enzyme capable of cleaving cyclic nucleotides, is one possibility of increasing the concentration of camp in sperm cells (5). Caffeine and pentoxifylline are the two phosphodiesterase inhibitors that have been used most frequently as motility stimulants for human sperm. The use of the former has a longer tradition, but the results reported in the literature are rather ambiguous. Even though some authors claim an improvement of sperm movement and fertilizing ability by caffeine treatment (6, 7), others argue against its therapeutic use (8,9); a beneficial effect of this drug on human fertilization has never been unequivocally demonstrated. On the other hand, Yovich et al. (2) have reported a carefully controlled study showing that exposure of spermatozoa to pentoxifylline before their use in IVF improves considerably fertilization outcomes in cases of severe male infertility. However, the beginning ofpentoxifylline use as motility stimulant is relatively recent, and clinical experience is thus still limited. It is evident that beneficial effects of a drug improving sperm movement may be compromised if it affects negatively another important sperm function. Inversely, the clinical usefulness of a motility stimulant may be potentiated by its eventual effects on other fertilization-related phenomena. One of the best known functions the fertilizing spermatozoon must accomplish before it penetrates the egg is the acrosome reaction (10). The recent findings suggesting the implication of camp in the control of the human sperm acrosome reaction (4) raise concern about the possible effects of phosphodiesterase inhibitors used as motility stimulants on this process. However, this issue has not yet been addressed experimentally. In this study, we analyzed the effects of caffeine and pentoxifylline on the spontaneous acrosome reaction of human spermatozoa as well as on that induced by follicular fluid (FF) and ionophore A23187 representing, respectively, a physiological and an artificial acrosome reaction stimulus. Both phosphodiesterase inhibitors were tested at concentrations at which they are currently used as motility stimulants in assisted reproduction. MATERIALS AND METHODS Source and Preparation of Spermatozoa Sperm samples were obtained from healthy volunteers or from patients attending our infertility clinic for routine semen examination before IVF attempt. In both cases, only samples with normal values of sperm count, concentration, motility, and morphology (11) were selected for this study, resulting in a total of 22 sperm samples. Spermatozoa were washed from seminal plasma and incubated for in vitro capacitation as described (12). Briefly, the seminal plasma was washed out by repeated sperm centrifugation and resuspension of pellet in Tyrode's salt solution (Sigma, La Verpilliere, France) followed by the resuspension of the final pellet in B2 culture medium (Bio Merieux, Marcy l'etoile, France) and incubation for 3 hours at 37 C under a gas phase of 5% CO2 in air. These sperm suspensions were subsequently used in individual experiments. Source and Preparation of Acrosome Reaction Inducing Stimuli Samples of FF were from follicular aspirations performed as part of the oocyte recovery procedure in our IVF program. Details of ovarian stimulation, cycle monitoring, and the technique of follicular puncture were described elsewhere (13). Only fluid free of blood contamination and originating from follicles yielding a healthy appearing preovulatory cumulus-oocyte complex was used. Each FF sample was tested for the absence of sperm toxicity. Fluid from 15 follicular aspirates (8 patients) was pooled, distributed in aliquots, and kept at -20 C until used. Ionophore A23187 (Sigma) was diluted in dimethyl sulfoxide (DMSO) to a concentration of 2 mm. This stock solution was distributed in aliquots and kept at -20 C until used. Sperm Incubation With Phosphodiesterase Inhibitors Caffeine and pentoxifylline (both purchased from Sigma) were both used at a final concentration of 4 mm, which is within the range of concentrations the two drugs are used by different laboratories as motility stimulants. Stock solutions (lox) of caffeine and pentoxifylline were prepared by dissolving the substance in Tyrode's salt solution, and additions to sperm suspensions in B2 medium were made at the end of the 3-hour capacitation period. The corresponding volume of Tyrode's salt solution was added to control samples. Spermatozoa were then incubated for different time periods (see Results) under the same conditions as those used for in vitro capacitation Tesarik et al. Phosphodiesterase inhibitors and acrosome reaction Fertility and Sterility

3 Sperm Treatment With Acrosome Reaction Inducing Stimuli In some experiments acrosome reaction-inducing stimuli, FF or ionophore A23187, were added to sperm suspensions at the same time as the phosphodiesterase inhibitor. The final concentration of FF in the sperm incubation medium was 30% (vol/ vol). Control samples for the FF treatment were incubated in medium supplemented with 1 % human serum albumin to avoid the eventual nonspecific effect of FF proteins to be misinterpreted as a specific biological action of this material. The incubation time for FF was 5 hours. In the case of ionophore A23187, the concentration was 10 JlM and the incubation time 30 minutes. Controls for the ionophore treatment were incubated with DMSO alone added at the same concentration as that introduced to the experimental samples as solvent for the ionophore. Acrosome Reaction Evaluation At the end of incubation, spermatozoa were washed once in Tyrode's salt solution, smeared on microscope slides, and allowed to air dry. The smears were then fixed for 20 seconds in 100% methanol, and sperm acrosomes were stained with fluorescence isothiocyanate-iabeled Pisum satiuum agglutinin (Sigma) using the previously described method (12). The acrosomal status was evaluated in 200 spermatozoa in each sample, and the percentage of acrosome-reacted sperm was determined. Statistics All statistical analyses were performed with the use of StatView II (Abacus Concepts, Berkeley, CA) statistical package. Percentages of acrosome-reacted sperm were transformed by arc sine before analysis. Mean values for different experimental conditions were compared by ANOV A and t-test for multiple comparison. RESULTS When washed spermatozoa are incubated in capacitating media, some undergo the acrosome reaction spontaneously, but the rate of the acrosome reaction can be considerably increased by addition of specific acrosome reaction-inducing stimuli. For convenience, we use here the term "spontaneous acrosome reaction" for all reactions occurring during sperm incubation in capacitating medium, supplemented or not with phosphodiesterase inhibitors. The term "induced acrosome reaction" is reserved for the reactions occurring in the presence of one of the known acrosome reaction inducers, FF and ionophore A Effects of Phosphodiesterase Inhibitors on Spontaneous Acrosome Reaction The incidence of the spontaneous acrosome reaction among living spermatozoa remained nearly constant throughout 3 hours of incubation in the control group (Fig. 1). It has to be recalled, however, that the period during which the acrosome reaction was monitored was preceded by a 3-hour incubation for in vitro capacitation so that time 0 in Figure 1 corresponds to samples at the end of this preincubation step. The addition of pentoxifylline to incubation media did not affect significantly the rate of the spontaneous acrosome reaction at any time point examined (Fig. 1). In contrast, caffeine increased significantly (P < 0.01) the acrosome reaction frequency as early as after 1 hour after addition and the difference from controls augmented during further incubation (Fig. 1). Effects of Phosphodiesterase Inhibitors on FF Induced Acrosome Reaction When spermatozoa were incubated for 5 hours with 30% (vol/vol) FF in the absence of phosphodiesterase inhibitors, the acrosome reaction frequency was augmented only slightly, though significantly (P < 0.01), as compared with the control (Fig. 2). However, the response to FF was considerably increased when the incubation with FF was [>_.. '0 al 20 o '" o 10 Caffeine Pentoxirylline Control _--t----t :--: o O------_r r--- -I o Hours of Incubation Figure 1 Changes in the frequency of the spontaneous acrosome reaction during sperm incubation with caffeine and pentoxifylline. Values for caffeine are significantly different from controls (P < 0.01) at all time points after zero. In contrast, values for pentoxifylline are not different from controls (P > 0.05) at any time point studied. Data are means ± SEM of five replicate experiments. Vol. 58, No.6, December 1992 Tesarik et al. Phosphodiesterase inhibitors and acrosome reaction 1187

4 Sham Pentoxifylline Caffeine Component Added Figure 2 Effects of pentoxifylline and caffeine on the frequency of the FF-induced acrosome reaction. Human serum albumin (HSA) substituted for FF in control incubations to assess the frequency of the spontaneous acrosome reaction. Data are means ± SEM of five replicate experiments. carried out in the presence of pentoxifylline (P < 0.01; FF + pentoxifylline versus FF alone). On the other hand, the acrosome reaction frequency in samples incubated with pentoxifylline in the absence of FF was not significantly different from those incubated without any addition (Fig. 2). Caffeine increased the acrosome reaction frequency when added alone, and there was hardly any further increase upon simultaneous addition of FF (Fig. 2). Effects of Phosphodiesterase Inhibitors on Ionophore-Induced Acrosome Reaction Like FF, the frequency of the ionophore-induced acrosome reaction was potentiated considerably in the presence of pentoxifylline (P < 0.01; A pentoxifylline versus A23187 alone), whereas pentoxifylline alone did not increase the acrosome reaction scores in the solvent-control groups (Fig. 3). Caffeine also enhanced the response to the ionophore, though to a lesser extent than pentoxifylline (Fig. 3). DISCUSSION Phosphodiesterase inhibitors of the methylxanthine group are increasingly used in assisted reproduction as enhancers of sperm movement and fertilizing ability. They are generally supposed to act by increasing the intracellular camp concentration (1), even though camp-independent effects may also be involved (14). The actual role of camp in the acrosome reaction is still a matter of debate. Several authors using rodent spermatozoa have reported an enhancement of the acrosome reaction by treatments increasing the intracellular camp concentration (15, 16), but an inhibitory effect of camp on the acrosome reaction has also been observed (17). As to human sperm, De Jonge et al. (4) have recently provided a strong experimental evidence in favor of a role for the camp second-messenger system in the acrosome reaction induction. The authors of that study observed a significant increase in the acrosome reaction frequency after sperm treatment with isobuthylmethylxanthine (another phosphodiesterase inhibitor of the methylxanthine group). This effect was limited to sperm populations incubated in capacitating conditions; noncapacitated sperm did not respond to the drug (4). In this study, we obtained a significant augmentation of the acrosome reaction frequency by treating capacitated sperm with caffeine, whereas pentoxifylline at the same molar concentration failed to produce this effect. However, the pentoxifylline treatment increased considerably the sperm capacity of undergoing the acrosome reaction in response to both natural (FF) and artificial (ionophore A23187) stimuli. There are several possible explanations of the differential effects of the two phosphodiesterase inhibitors. First, both drugs may inhibit sperm phosphodiesterases to a different extent. This may be because of unequal intracellular concentrations of the active substance, subject to different transport and degradation mechanisms, or to differences in enzyme sensitivity. A quantitative analysis of the effects of caffeine on sperm phosphodiesterase activity as related to changes in sperm motility has been reported (18), but a similar study for pentoxifylline is still lacking. 80 '" c::.9 o 40 E o '" 20 o ri1l Solvent Control A23187 Sham Pentoxifylline Caffeine Component Added Figure 3 Effects ofpentoxifylline and caffeine on the frequency of the ionophore-induced acrosome reaction. Solvent control was made with the same concentration ofdmso (0.5%) as that introduced in the experimental group with ionophore A Data are means ± SEM of eight replicate experiments Tesarik et al. Phosphodiesterase inhibitors and acrosome reaction Fertility and Sterility

5 Alternatively, caffeine and pentoxifylline may be equally efficient in inhibiting sperm phosphodiesterases in situ, but the final impact on the acrosome reaction may be modified by different secondary effects of both drugs. In fact, alterations of the ultrastructure of the sperm head because of caffeine treatment have been described (19). Such alterations may signal a destabilization effect of caffeine on the sperm plasma membrane. It is well known that membrane destabilization can augment the susceptibility of sperm cells to the acrosome reaction (10). If the latter alternative were true, it would also help to understand the observed effects of the two phosphodiesterase inhibitors on the acrosome reaction induced by additional stimuli. The augmentation of intracellular camp appears to represent only one component of the acrosome reaction-triggering mechanism. The treatment with a phosphodiesterase inhibitor can thus sensitize spermatozoa for the action of stimuli that activate other components of this complex system. In our study, this component may have been calcium because both FF and calcium ionophores are known to promote calcium entry into human sperm (20). This sensitization would clearly be less apparent when a secondary effect of the phosphodiesterase inhibitor brings the sperm response near the maximum, which may be the case of caffeine. Regardless of the underlying mechanism, the ability of pentoxifylline to potentiate the sperm response to a physiological acrosome reaction inducer without increasing the rate of spontaneous acrosome reaction is of immediate clinical interest. There is experimental evidence that human spermatozoa must synchronize the acrosome reaction with the pace oftheir penetration through the egg coat ifthey are to fertilize (21). In agreement with this concept, the fertilizing ability of human sperm decreases when the acrosome reaction is induced artificially before sperm incubation with the oocyte (22). The results of this study suggest that preincubation of spermatozoa with pentoxifylline can increase their fertilizing ability by rendering them more responsive to the acrosome reaction inducers present in the egg coat without provoking a premature acrosome reaction. This will increase the proportion of spermatozoa ready to undergo a properly timed acrosome reaction upon their interaction with the egg coat and hence capable of fertilization. This pentoxifylline effect is combined with a stimulation of sperm velocity and hyperactivation (23), both effects increasing the sperm fertilizing ability. On the other hand, the increase in the rate of the spontaneous acrosome reaction by caffeine appears less advantageous for the sperm fertilizing ability and may counteract the beneficial effect of caffeine on sperm movement. This might explain the inconsistencies of the clinical results obtained with this motility stimulant described in the literature. On the other hand, the direct stimulation ofthe acrosome reaction by caffeine may be instrumental in the augmentation of penetration rates into zona-free hamster oocytes reported by different groups (6, 7). The findings described in this study were obtained with the use of normal sperm samples. Recent reports have pointed out a relationship between an insufficient acrosome reaction response of sperm to calcium ionophores and infertility (24, 25). It is intriguing to examine whether pentoxifylline can be used to improve the acrosome reaction scores and fertilizing ability in these poor responders. 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