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Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility
FERTILITY AND STERILITYt VOL. 71, NO. 5, MAY 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility Ahmed Nassar, M.D.,* Mary Mahony, Ph.D.,† Mahmood Morshedi, Ph.D.,† M.-H. Lin, M.D.,† Chartchai Srisombut, M.D.,† and Sergio Oehninger, M.D.† Eastern Virginia Medical School, Norfolk, Virginia, and Tanta University, Tanta, Egypt
Objective: To assess the effect of pentoxifylline on human sperm functions that are crucial to fertilization. Design: Prospective, controlled study. Setting: Academic tertiary care institute. Patient(s): Healthy male sperm donors. Intervention(s): The effects of pentoxifylline (3.6 mM) on hyperactivated motility, sperm binding to the zona pellucida, and sperm protein tyrosine phosphorylation were evaluated. Main Outcome Measure(s): Hyperactivated motility was assessed by computer-assisted motion analysis, and tight binding of sperm to homologous zonae pellucidae was examined using the hemizona assay. Sperm protein phosphorylation was evaluated using indirect immunofluorescence with an antibody to phosphotyrosine (PY20). Result(s): Pentoxifylline significantly stimulated hyperactivated motility at 1 hour and 4 hours; it also significantly increased sperm binding to the zona pellucida and enhanced sperm tail tyrosine phosphorylation at 4 hours under capacitating conditions. There was a statistically significant correlation between hyperactivated motility and sperm tail protein phosphorylation. Conclusion(s): Pentoxifylline stimulates sperm functions that are essential to achieving fertilization under in vitro conditions in sperm obtained from fertile men. The enhancement of hyperactivated motility is associated with the stimulation of sperm tail tyrosine phosphorylation, suggesting a causal relation and the involvement of a modulatory effect after cyclic adenosine monophosphate– dependent phosphorylation of intermediate proteins. (Fertil Sterilt 1999;71:919 –23. ©1999 by American Society for Reproductive Medicine.) Key Words: Hyperactivated motility, human sperm, pentoxifylline, sperm–zona pellucida binding, tyrosine phosphorylation Received February 27, 1998; revised and accepted December 1, 1998. Reprint requests: Sergio Oehninger, M.D., The Jones Institute, 601 Colley Avenue, Norfolk, Virginia 23507 (FAX: 757-446-8998; E-mail: sergio@jones1 .evms.edu). * Department of Dermatology and Andrology, Tanta University. † The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. 0015-0282/99/$20.00 PII S0015-0282(99)00013-8
The process of capacitation renders the spermatozoon able to interact successfully with the zona pellucida (ZP) and to undergo the acrosome reaction, which are crucial steps leading to fertilization. Several cellular changes occur during capacitation, including the removal or modification of surface proteins, the efflux of cholesterol from the membranes, changes in oxidative metabolism, the achievement of a hyperactivated pattern of motility, and an increase in the phosphotyrosine content of several proteins. In addition to tyrosine phosphorylation of specific proteins, modifications of other cellular regulators occur, such as an increase in calcium uptake, a decrease in calmodulin binding to proteins, and an increase in both intracellular pH and
the concentration of cyclic adenosine monophosphate (cAMP) (1). Impaired sperm motion parameters, a reduced capacity to bind to the ZP, and a reduced ability to undergo an agonist-induced acrosome reaction have been associated with male factor infertility. Many substances have been proposed to stimulate human sperm functions in vitro. These include poorly defined biologic materials such as serum, peritoneal fluid, and follicular fluid, and well-defined agents such as progesterone, adenosine analogues, and methylxanthines (2). The underlying mechanisms for the stimulation of sperm functions in vitro by some of these substances have not been completely elucidated. 919
Methylxanthines such as caffeine and pentoxifylline are known to inhibit phosphodiesterase activity in living cells, leading to a subsequent increase in intracellular cAMP levels. Cyclic adenosine monophosphate, which seems to be provided mainly by the glycolytic pathway, stimulates human sperm respiration and motility (3). Another possible mechanism of enhancing motility may be through a modulation of cellular calcium transport, leading to modifications of intracellular calcium levels. However, pentoxifylline does not appear to stimulate sperm motion parameters through the stimulation of calcium influx into human spermatozoa (4). Cyclic adenosine monophosphate appears to be involved not only in the control of sperm motility but also in the regulation of the acrosome reaction (2). The precise mechanism of action of cAMP is still a matter of debate and remains to be elucidated. Pentoxifylline has been shown to enhance sperm motility, to induce the acrosome reaction, to enhance sperm binding to the homologous ZP, to increase the success rate of IVF treatment, and even to improve the pregnancy rate among couples who have had failure of fertilization in previous IVF attempts (2–7). The objectives of this study were to assess the effects of pentoxifylline on capacitation-dependent events such as hyperactivated motility and sperm-ZP binding and to evaluate the relation between these functional processes and protein tyrosine phosphorylation.
MATERIALS AND METHODS Preparation of Pentoxifylline A stock solution of 30 mg/mL of pentoxifylline (Sigma, St. Louis, MO) in Ham’s F-10 medium (GIBCO, Grand Island, NY) supplemented with 0.5% human serum albumin (Irvine Scientific, Irvine, CA) was prepared each week and kept refrigerated at 1°– 6°C until used. Pentoxifylline from this stock solution was added to the experimental tubes to achieve a final concentration of 1 mg/mL (3.6 mM). This concentration was used because Yovich et al. (7) showed improved pregnancy rates with this methylxanthine when spermatozoa were treated before IVF in couples with severe male factor infertility. Further, in previous dosedependency experiments, we demonstrated that the maximum effect of pentoxifylline on sperm motility characteristics and on the penetration of cervical mucus in vitro occurred when spermatozoa were incubated with a dose of 1 mg/mL for 30 minutes (4).
Preparation of Spermatozoa and Experimental Design Semen was collected by masturbation from nine healthy, fertile male donors attending our artificial insemination program. Semen samples were allowed to liquefy for 30 minutes at room temperature and then were analyzed. The lower limits of normal parameters of samples used in the experi920
Nassar et al.
Pentoxifylline and hyperactivation
ments were as follows: sperm concentration, 50 3 106/mL; progressive motility, 50%; and morphology, 14% according to strict criteria. All ejaculates showed ,1 3 106 leukocytes per milliliter, had cultures that were negative for Mycoplasma, and tested negative for antisperm antibodies by the direct immunobead test. After liquefaction, the semen samples were washed twice (at 400 3 g for 7 minutes and 5 minutes, respectively) using Ham’s F-10 medium supplemented with 0.5% human serum albumin. Each pellet was resuspended in a suitable volume of the culture medium, and the suspension was divided into two equal parts that were subdivided into aliquots of 100 mL each. Each aliquot of the first part was incubated with an equal aliquot of the medium (not treated with pentoxifylline), and each aliquot of the second part was incubated with an equal aliquot of pentoxifylline solution (treated with pentoxifylline), to give a final pentoxifylline concentration of 1 mg/ mL, for a period of 30 minutes in 5% CO2 in air at 37°C. Each aliquot then was centrifuged at 400 3 g for 5 minutes and the pellet was overlaid with 100 mL of culture medium and incubated in 5% CO2 in air at 37°C and at a 45-degree angle for 60 minutes. The spermatozoa, both nontreated and treated, were separated by the swim-up technique and collected in two separate tubes (8). The sperm concentration was adjusted to 5 3 106/mL to evaluate motion parameters. Then the sperm suspension was divided into two parts. The concentration of one part was adjusted to 2 3 106/mL to evaluate sperm protein tyrosine phosphorylation and the concentration of the other part was adjusted to 0.5 3 106/mL to assess sperm-ZP binding capacity. Hyperactivated motility was examined at 1 hour and 4 hours, and sperm-ZP binding and protein tyrosine phosphorylation were examined at 4 hours after swim-up separation under capacitating conditions (Ham’s F-10 medium plus 0.5% human serum albumin at 37°C in 5% CO2 in air).
Motion Parameters Sperm motion was evaluated with the HTM-IVOS motility analyzer (Hamilton-Thorne Research, Danvers, MA) as previously described (9). After mixing of the sperm suspension, 3 mL was loaded into one chamber of the 20-Micron 4-Chamber MicroCell Slide (Conception Technologies, La Jolla, CA), transferred to the motion analyzer, and maintained at 37°C for 2 minutes before data collection was begun. At least 100 sperm were analyzed for each of the control and treatment groups on randomly selected fields. The pertinent settings used during the assessment of sperm motion parameters were as follows: [1] analysis during 0.67 seconds, 30 frames; [2] minimum contrast, 85; [3] minimum size, 4; [4] slow gate, 5 mm/s “slow cells” were not accepted as motile; [5] magnification factor, 2.40; [6] low size gate, 0.36; [7] high size gate, 2.99; [8] low-intensity Vol. 71, No. 5, May 1999
gate, 0.27; and [9] high-intensity gate, 1.55. At the outset of each experiment, we verified that the settings permitted accurate differentiation of motile sperm from nonmotile sperm or debris with the use of the “playback” option. During playback, the motions of sperm in the previous field were replayed; a green dot was located over the head of all motile spermatozoa and a red dot was positioned over the head of all nonmotile spermatozoa. When errors were detected, the settings were adjusted until the problem was corrected. To differentiate hyperactivated sperm from those that were not hyperactivated, the following settings were used in the automatic sorting program for the motion analyzer, as previously determined by Burkman (10): track speed or curvilinear velocity (the velocity derived from all 20 head positions), 100 –500 mm/s; linearity (a measure of the straightness of the trajectory), 0 – 65; maximum lateral head displacement (a measure of the side-to-side movement of the head), 7.5–50 mm; progressive velocity (the velocity based on the first and last head positions only), 20 –500 mm/s; and path velocity (five-point running average), 40 –500 mm/s. As sperm tracks were evaluated, any spermatozoon that had been selected as hyperactivated but was actually crossing the border of the field was excluded from the evaluation (9). Hyperactivated motility was evaluated for both sperm that were treated with pentoxifylline and those that were not at 1 hour and 4 hours after swim-up separation of the motile fraction. The intra-assay coefficient of variation (results of repeated measures of a given sample within a 15-minute period) using this system typically is ,15% under these conditions (unpublished observations).
Sperm Protein Tyrosine Phosphorylation Sperm were washed with culture medium, smeared onto glass slides, air-dried, fixed with 100% methanol for 30 seconds, and stored at 270°C until used. The proportion of protein tyrosine–phosphorylated spermatozoa was determined using the fluorescent probe fluorescein isothiocyanate–labeled mouse monoclonal antiphosphotyrosine (PY-20; ICN, Irvine, CA) at 1 mg/mL using indirect immunofluorescence according to previously described techniques (11–13). This was performed after staining with Hoechst 33258 (Sigma) to exclude the analysis of nonviable sperm. Because almost no fluorescence was observed in the sperm head regions under control or treatment conditions but the tails showed variable and intense fluorescence, spermatozoa were categorized according to sperm tail staining into two patterns: lighted (with green fluorescence, including partial and complete lighting of the tail) and nonlighted (without any green fluorescence) (12). The specificity of the antiphosphotyrosine antibody was determined at the onset of each experiment by sperm preincubation with O-phospho-L-tyrosine (Zymed, San Francisco, FERTILITY & STERILITYt
CA) at a final concentration of 20 mM. After incubation with the antibody-phosphotyrosine mixture, spermatozoa exhibited absence of staining in the principal piece region of the tail (negative control). Sperm tail protein phosphorylation was evaluated for aliquots of both treated and nontreated sperm at 4 hours of incubation after swim-up separation under capacitating conditions. At least 200 sperm were evaluated per sample. Control and test duplicate slides were read blindly and the results were averaged.
Sperm–Zona Pellucida Binding Sperm–ZP binding was evaluated with the hemizona assay using matching hemizonae from microbisected human oocytes as detailed previously (14). After separation by the swim-up techniques, the sperm concentration was adjusted to 0.5 3 106/mL. Matching hemizonae (5 oocytes or 10 matching hemizonae per experiment) were incubated for 4 hours (under capacitating conditions) with 50 mL of pentoxifylline-treated (test) and nontreated (control) sperm samples, respectively. Then the hemizonae were rinsed vigorously in Ham’s F-10 medium to dislodge slightly bound spermatozoa. Sperm–ZP binding was evaluated using an inverted microscope with phase-contrast optics, and the average number of sperm that were tightly bound per hemizona was determined. The intra-assay variability of the technique was ,12% (9, 12).
Statistical Analysis Statistical evaluation was performed with the use of the GraphPAD (San Diego, CA) InStat software package (version 1.11a). The paired t-test was used to evaluate the differences between pentoxifylline-treated and nontreated spermatozoa for hyperactivated motility, sperm-ZP binding, and sperm tail protein phosphorylation. Correlations between these parameters were performed using Pearson’s correlation coefficient. Results were expressed as means 6 standard error of the mean. P,.05 was considered statistically significant.
RESULTS Pentoxifylline significantly increased sperm hyperactivated motility at 1 hour (20% 6 3% versus 12.5% 6 2% for pentoxifylline-treated versus nontreated samples, P,.002) and at 4 hours (10% 6 1.7% versus 6.5% 6 1.8% for pentoxifylline-treated versus nontreated samples, P,.002) after swim-up separation during incubation under capacitating conditions. Sperm tail protein phosphorylation measured at 4 hours after swim-up separation also was enhanced by pentoxifylline in a statistically significant fashion (13.1% 6 1.5% versus 8.5% 6 1.3% for pentoxifylline-treated versus nontreated samples, P,.0001). Sperm binding to the ZP also was significantly increased by pentoxifylline (73 6 12.6 sperm per hemizona versus 48 6 9.2 sperm per hemi921
zona for pentoxifylline-treated versus nontreated samples, P 5 .002). Hyperactivated motility was significantly correlated with sperm tail protein phosphorylation in pentoxifylline-treated and nontreated samples (r 5 .9420, P 5 .0002 and r 5 .9476, P 5 .0001, respectively). However, hyperactivated motility and sperm tail protein phosphorylation did not correlate significantly with sperm-ZP binding (P..5).
DISCUSSION The investigation of the effects of cAMP and calcium on sperm flagellar motility has received much attention. Cyclic adenosine monophosphate stimulates sperm motility by activating a cAMP-dependent protein kinase (15). This, in turn, is believed to induce enhanced sperm tail protein phosphorylation of a defined number of specific proteins, with subsequent enhancement of sperm motility (16). A major axonemal protein described in humans, dogs, and sea urchins, namely axokinin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP (15). In sea urchin sperm, the addition of cAMP and cAMP-dependent protein kinase partially restored the motility of the phosphoprotein-phosphatase–treated (inhibited) sperm (17). These and other data provide strong support for the possibility that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system (15–17). In the present study, pentoxifylline significantly increased the hyperactivated motility of human sperm and their capacity to bind tightly to the homologous ZP. The patterns of hyperactivated motility are expressed under capacitating conditions (10). Sperm-ZP binding leading to a physiologically induced acrosome reaction such as that observed during the conditions of the hemizona assay reflects multiple sperm functions and is an end point of capacitation (14). Therefore, this methylxanthine was shown to be a strong stimulant of sperm functions crucial to fertilization. Sperm tail protein phosphorylation correlated positively and significantly with hyperactivated motility. This finding is in agreement with a preliminary report of Yunes et al. (18). However, no correlation was found between sperm-ZP binding and sperm tail protein phosphorylation or hyperactivated motility. Collectively, these results indicate that phosphorylation of the tyrosine residues of sperm tail proteins could be one of the mechanisms through which hyperactivated motility occurs. This stimulation probably is dependent on the stimulation of a cAMP-dependent protein kinase. Although hyperactivation is considered to be an integral part of capacitation, it has been suggested that the two processes may not be coupled (19, 20). Our results suggest a temporal correlation between increased protein tyrosine phosphorylation and the development of hyperactivated motility in human spermatozoa. These results are in agreement with those of Leclerc et al. (21) and Tomes et al. (22), who 922
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demonstrated a relation between protein tyrosine phosphorylation and sperm motility. As stated by Tomes et al. (22), these results do not exclude a possible role for tyrosine phosphorylation in additional events, such as sperm binding to the ZP or the acrosome reaction. However, we did not detect any immunofluorescence on the sperm head region using the antibody to tyrosine PY20, and we did not observe any relation between sperm protein phosphorylation and the results of the hemizona assay. Confirming these findings, Carrera et al. (13) demonstrated that capacitation is associated with tyrosine phosphorylation in a time-dependent manner, that this phosphorylation is inhibited by the specific inhibitor genistein, and that most of the tyrosine-phosphorylated proteins are localized to the principal piece of the sperm flagellum. Within this context, Aitken et al. (23) reported on a causal association between reactive oxygen species generation, tyrosine phosphorylation, and sperm function (assessed by sperm-oocyte fusion). Consequently, detection of sperm head tyrosine phosphorylation under different capacitation conditions or using other better-defined probes may be necessary to confirm a relation between protein tyrosine phosphorylation and binding to the ZP, the ZP-induced acrosome reaction, and other human sperm functions that depend on specific head membrane domains (5, 24). We conclude that pentoxifylline enhances two capacitation-dependent sperm functions, hyperactivated motility and tight binding to the ZP, which are essential processes leading to fertilization. These findings, confirmed by studies performed in samples with poor sperm motion parameters, may encourage the use of this methylxanthine in assisted reproduction. Enhancement of hyperactivated motility was significantly associated with tyrosine phosphorylation of sperm tail proteins, providing further evidence of a relation between cAMP and tyrosine kinase signaling pathways in spermatozoa. Abnormalities of other intracellular sperm regulators (e.g., calcium) have been associated with deficiencies of the acrosome reaction in patients with severe teratozoospermia (25). The characterization of the signal transduction cascades involved in the control of crucial sperm functions, such as the initiation and maintenance of motility and hyperactivation, may benefit our understanding of the sperm dysfunctions that are present in some infertile men with asthenozoospermia. References 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill J, editors. The physiology of reproduction. 2nd ed. New York: Raven Press, 1994:189 –317. 2. Tesarik J, Mendoza C, Carreras A. Effect of phosphodiesterase inhibitors, caffeine and pentoxifylline, on spontaneous and stimulus-induced acrosome reactions in human sperm. Fertil Steril 1992;58:185–90. 3. Garbers DL, Lust WD, First NL, Lardy HA. Effect of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 1971;10:1825–31. 4. Nassar A, Mahony M, Blackmore P, Morshedi M, Ozgur K, Oehninger S. Increase of intracellular calcium is not a cause of pentoxifylline-
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5. 6. 7. 8.
9. 10. 11. 12.
13.
14.
induced hyperactivated motility or acrosome reaction in human sperm. Fertil Steril 1998;69:748 –54. Centola GM, Cartie RJ, Cox C. Differential responses of human sperm to varying concentrations of pentoxifylline with demonstration of toxicity. J Androl 1995;16:136 – 42. Yogev L, Amit A, Gamzu R, Lessing JB, Botchan A, Paz G, et al. Pentoxifylline improves sperm binding to the zona pellucida in the hemizona assay. Fertil Steril 1995;64:146 –9. Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril 1990;53: 715–22. Oehninger S, Acosta R, Morshedi M, Philput C, Swanson RJ, Acosta AA. Relationship between morphology and motion characteristics of human spermatozoa in semen and in the swim-up sperm fractions. J Androl 1990;11:446 –52. Mahony M, Oehninger S, Clark G, Acosta AA, Hodgen GD. Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa. Contraception 1991;44:657– 65. Burkman LJ. Discrimination between nonhyperactivated and classical hyperactivated motility patterns in human spermatozoa using computerized analysis. Fertil Steril 1991;55:363–71. Glenney JR, Zokas L, Kamps MP. Monoclonal antibodies to phosphotyrosine. J Immunol Methods 1988;109:227– 85. Mahony M, Gwathmey T. Stimulation of protein tyrosine phosphorylation during capacitation-dependent hyperactivated motility of macaque spermatozoa [abstract]. In: Proceedings of the 23rd Annual Meeting of the American Society of Andrology; March 26 –29, 1998; Long Beach, CA: 1998;27. Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopt F, et al. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol 1996;180:284 –96. Oehninger S, Sueldo C, Lanzendorf S, Mahony M, Burkman L, Alexander N, et al. A sequential analysis of the effect of progesterone or specific sperm functions crucial to fertilization in vitro in infertile patients. Hum Reprod 1994;9:1322– 8.
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15. Tash JS, Hidaka H, Means AR. Axokinin phosphorylation by cAMP dependent protein kinase is sufficient for activation of sperm flagellar motility. J Cell Biol 1986;103:649 –55. 16. Bracho GE, Fritch JJ, Tash JS. Identification of flagellar proteins that initiate the activation of sperm motility in vivo. Biochem Biophys Res Commun 1998;242:231–7. 17. Murofushi H, Ishiguro K, Takahashi D, Ikeda J, Sakai H. Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation. Cell Motil Cytoskeleton 1986;6:83– 8. 18. Yunes R, Doncel GF, Acosta AA. Phosphotyrosine containing proteins and hyperactivated motility in human sperm [abstract]. In: Proceedings of the 50th Annual Meeting of the American Fertility Society; San Antonio, Texas: November 5–10, 1994;53. 19. Neil JM, Olds-Clarke P. A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required. Gamete Res 1987;18:121– 40. 20. Suarez SS, Vincenti L, Ceglia MW. Hyperactivated motility induced in mouse sperm by calcium A23187 is reversible. J Exp Zool 1987;224: 331– 6. 21. Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 39, 59 monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 22:684 –92. 22. Tomes CN, Carballada R, Moses DF, Katz DF, Saling PM. Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation. Mol Hum Reprod 1998;4:17–25. 23. Aitken RJ, Paterson M, Fisher H, Buckingham D, van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci 1995;108:2017– 25. 24. Burks DJ, Carballada R, Moore HDM, Saling PM. Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. Science 1995;269:83– 6. 25. Oehninger S, Blackmore P, Morshedi M, Sueldo C, Acosta A, Alexander N. Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia. Fertil Steril 1994;61:349 –54.
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Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility Ahmed Nassar, M.D.,* Mary Mahony, Ph.D.,† Mahmood Morshedi, Ph.D.,† M.-H. Lin, M.D.,† Chartchai Srisombut, M.D.,† and Sergio Oehninger, M.D.† Eastern Virginia Medical School, Norfolk, Virginia, and Tanta University, Tanta, Egypt
Objective: To assess the effect of pentoxifylline on human sperm functions that are crucial to fertilization. Design: Prospective, controlled study. Setting: Academic tertiary care institute. Patient(s): Healthy male sperm donors. Intervention(s): The effects of pentoxifylline (3.6 mM) on hyperactivated motility, sperm binding to the zona pellucida, and sperm protein tyrosine phosphorylation were evaluated. Main Outcome Measure(s): Hyperactivated motility was assessed by computer-assisted motion analysis, and tight binding of sperm to homologous zonae pellucidae was examined using the hemizona assay. Sperm protein phosphorylation was evaluated using indirect immunofluorescence with an antibody to phosphotyrosine (PY20). Result(s): Pentoxifylline significantly stimulated hyperactivated motility at 1 hour and 4 hours; it also significantly increased sperm binding to the zona pellucida and enhanced sperm tail tyrosine phosphorylation at 4 hours under capacitating conditions. There was a statistically significant correlation between hyperactivated motility and sperm tail protein phosphorylation. Conclusion(s): Pentoxifylline stimulates sperm functions that are essential to achieving fertilization under in vitro conditions in sperm obtained from fertile men. The enhancement of hyperactivated motility is associated with the stimulation of sperm tail tyrosine phosphorylation, suggesting a causal relation and the involvement of a modulatory effect after cyclic adenosine monophosphate– dependent phosphorylation of intermediate proteins. (Fertil Sterilt 1999;71:919 –23. ©1999 by American Society for Reproductive Medicine.) Key Words: Hyperactivated motility, human sperm, pentoxifylline, sperm–zona pellucida binding, tyrosine phosphorylation Received February 27, 1998; revised and accepted December 1, 1998. Reprint requests: Sergio Oehninger, M.D., The Jones Institute, 601 Colley Avenue, Norfolk, Virginia 23507 (FAX: 757-446-8998; E-mail: sergio@jones1 .evms.edu). * Department of Dermatology and Andrology, Tanta University. † The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School. 0015-0282/99/$20.00 PII S0015-0282(99)00013-8
The process of capacitation renders the spermatozoon able to interact successfully with the zona pellucida (ZP) and to undergo the acrosome reaction, which are crucial steps leading to fertilization. Several cellular changes occur during capacitation, including the removal or modification of surface proteins, the efflux of cholesterol from the membranes, changes in oxidative metabolism, the achievement of a hyperactivated pattern of motility, and an increase in the phosphotyrosine content of several proteins. In addition to tyrosine phosphorylation of specific proteins, modifications of other cellular regulators occur, such as an increase in calcium uptake, a decrease in calmodulin binding to proteins, and an increase in both intracellular pH and
the concentration of cyclic adenosine monophosphate (cAMP) (1). Impaired sperm motion parameters, a reduced capacity to bind to the ZP, and a reduced ability to undergo an agonist-induced acrosome reaction have been associated with male factor infertility. Many substances have been proposed to stimulate human sperm functions in vitro. These include poorly defined biologic materials such as serum, peritoneal fluid, and follicular fluid, and well-defined agents such as progesterone, adenosine analogues, and methylxanthines (2). The underlying mechanisms for the stimulation of sperm functions in vitro by some of these substances have not been completely elucidated. 919
Methylxanthines such as caffeine and pentoxifylline are known to inhibit phosphodiesterase activity in living cells, leading to a subsequent increase in intracellular cAMP levels. Cyclic adenosine monophosphate, which seems to be provided mainly by the glycolytic pathway, stimulates human sperm respiration and motility (3). Another possible mechanism of enhancing motility may be through a modulation of cellular calcium transport, leading to modifications of intracellular calcium levels. However, pentoxifylline does not appear to stimulate sperm motion parameters through the stimulation of calcium influx into human spermatozoa (4). Cyclic adenosine monophosphate appears to be involved not only in the control of sperm motility but also in the regulation of the acrosome reaction (2). The precise mechanism of action of cAMP is still a matter of debate and remains to be elucidated. Pentoxifylline has been shown to enhance sperm motility, to induce the acrosome reaction, to enhance sperm binding to the homologous ZP, to increase the success rate of IVF treatment, and even to improve the pregnancy rate among couples who have had failure of fertilization in previous IVF attempts (2–7). The objectives of this study were to assess the effects of pentoxifylline on capacitation-dependent events such as hyperactivated motility and sperm-ZP binding and to evaluate the relation between these functional processes and protein tyrosine phosphorylation.
MATERIALS AND METHODS Preparation of Pentoxifylline A stock solution of 30 mg/mL of pentoxifylline (Sigma, St. Louis, MO) in Ham’s F-10 medium (GIBCO, Grand Island, NY) supplemented with 0.5% human serum albumin (Irvine Scientific, Irvine, CA) was prepared each week and kept refrigerated at 1°– 6°C until used. Pentoxifylline from this stock solution was added to the experimental tubes to achieve a final concentration of 1 mg/mL (3.6 mM). This concentration was used because Yovich et al. (7) showed improved pregnancy rates with this methylxanthine when spermatozoa were treated before IVF in couples with severe male factor infertility. Further, in previous dosedependency experiments, we demonstrated that the maximum effect of pentoxifylline on sperm motility characteristics and on the penetration of cervical mucus in vitro occurred when spermatozoa were incubated with a dose of 1 mg/mL for 30 minutes (4).
Preparation of Spermatozoa and Experimental Design Semen was collected by masturbation from nine healthy, fertile male donors attending our artificial insemination program. Semen samples were allowed to liquefy for 30 minutes at room temperature and then were analyzed. The lower limits of normal parameters of samples used in the experi920
Nassar et al.
Pentoxifylline and hyperactivation
ments were as follows: sperm concentration, 50 3 106/mL; progressive motility, 50%; and morphology, 14% according to strict criteria. All ejaculates showed ,1 3 106 leukocytes per milliliter, had cultures that were negative for Mycoplasma, and tested negative for antisperm antibodies by the direct immunobead test. After liquefaction, the semen samples were washed twice (at 400 3 g for 7 minutes and 5 minutes, respectively) using Ham’s F-10 medium supplemented with 0.5% human serum albumin. Each pellet was resuspended in a suitable volume of the culture medium, and the suspension was divided into two equal parts that were subdivided into aliquots of 100 mL each. Each aliquot of the first part was incubated with an equal aliquot of the medium (not treated with pentoxifylline), and each aliquot of the second part was incubated with an equal aliquot of pentoxifylline solution (treated with pentoxifylline), to give a final pentoxifylline concentration of 1 mg/ mL, for a period of 30 minutes in 5% CO2 in air at 37°C. Each aliquot then was centrifuged at 400 3 g for 5 minutes and the pellet was overlaid with 100 mL of culture medium and incubated in 5% CO2 in air at 37°C and at a 45-degree angle for 60 minutes. The spermatozoa, both nontreated and treated, were separated by the swim-up technique and collected in two separate tubes (8). The sperm concentration was adjusted to 5 3 106/mL to evaluate motion parameters. Then the sperm suspension was divided into two parts. The concentration of one part was adjusted to 2 3 106/mL to evaluate sperm protein tyrosine phosphorylation and the concentration of the other part was adjusted to 0.5 3 106/mL to assess sperm-ZP binding capacity. Hyperactivated motility was examined at 1 hour and 4 hours, and sperm-ZP binding and protein tyrosine phosphorylation were examined at 4 hours after swim-up separation under capacitating conditions (Ham’s F-10 medium plus 0.5% human serum albumin at 37°C in 5% CO2 in air).
Motion Parameters Sperm motion was evaluated with the HTM-IVOS motility analyzer (Hamilton-Thorne Research, Danvers, MA) as previously described (9). After mixing of the sperm suspension, 3 mL was loaded into one chamber of the 20-Micron 4-Chamber MicroCell Slide (Conception Technologies, La Jolla, CA), transferred to the motion analyzer, and maintained at 37°C for 2 minutes before data collection was begun. At least 100 sperm were analyzed for each of the control and treatment groups on randomly selected fields. The pertinent settings used during the assessment of sperm motion parameters were as follows: [1] analysis during 0.67 seconds, 30 frames; [2] minimum contrast, 85; [3] minimum size, 4; [4] slow gate, 5 mm/s “slow cells” were not accepted as motile; [5] magnification factor, 2.40; [6] low size gate, 0.36; [7] high size gate, 2.99; [8] low-intensity Vol. 71, No. 5, May 1999
gate, 0.27; and [9] high-intensity gate, 1.55. At the outset of each experiment, we verified that the settings permitted accurate differentiation of motile sperm from nonmotile sperm or debris with the use of the “playback” option. During playback, the motions of sperm in the previous field were replayed; a green dot was located over the head of all motile spermatozoa and a red dot was positioned over the head of all nonmotile spermatozoa. When errors were detected, the settings were adjusted until the problem was corrected. To differentiate hyperactivated sperm from those that were not hyperactivated, the following settings were used in the automatic sorting program for the motion analyzer, as previously determined by Burkman (10): track speed or curvilinear velocity (the velocity derived from all 20 head positions), 100 –500 mm/s; linearity (a measure of the straightness of the trajectory), 0 – 65; maximum lateral head displacement (a measure of the side-to-side movement of the head), 7.5–50 mm; progressive velocity (the velocity based on the first and last head positions only), 20 –500 mm/s; and path velocity (five-point running average), 40 –500 mm/s. As sperm tracks were evaluated, any spermatozoon that had been selected as hyperactivated but was actually crossing the border of the field was excluded from the evaluation (9). Hyperactivated motility was evaluated for both sperm that were treated with pentoxifylline and those that were not at 1 hour and 4 hours after swim-up separation of the motile fraction. The intra-assay coefficient of variation (results of repeated measures of a given sample within a 15-minute period) using this system typically is ,15% under these conditions (unpublished observations).
Sperm Protein Tyrosine Phosphorylation Sperm were washed with culture medium, smeared onto glass slides, air-dried, fixed with 100% methanol for 30 seconds, and stored at 270°C until used. The proportion of protein tyrosine–phosphorylated spermatozoa was determined using the fluorescent probe fluorescein isothiocyanate–labeled mouse monoclonal antiphosphotyrosine (PY-20; ICN, Irvine, CA) at 1 mg/mL using indirect immunofluorescence according to previously described techniques (11–13). This was performed after staining with Hoechst 33258 (Sigma) to exclude the analysis of nonviable sperm. Because almost no fluorescence was observed in the sperm head regions under control or treatment conditions but the tails showed variable and intense fluorescence, spermatozoa were categorized according to sperm tail staining into two patterns: lighted (with green fluorescence, including partial and complete lighting of the tail) and nonlighted (without any green fluorescence) (12). The specificity of the antiphosphotyrosine antibody was determined at the onset of each experiment by sperm preincubation with O-phospho-L-tyrosine (Zymed, San Francisco, FERTILITY & STERILITYt
CA) at a final concentration of 20 mM. After incubation with the antibody-phosphotyrosine mixture, spermatozoa exhibited absence of staining in the principal piece region of the tail (negative control). Sperm tail protein phosphorylation was evaluated for aliquots of both treated and nontreated sperm at 4 hours of incubation after swim-up separation under capacitating conditions. At least 200 sperm were evaluated per sample. Control and test duplicate slides were read blindly and the results were averaged.
Sperm–Zona Pellucida Binding Sperm–ZP binding was evaluated with the hemizona assay using matching hemizonae from microbisected human oocytes as detailed previously (14). After separation by the swim-up techniques, the sperm concentration was adjusted to 0.5 3 106/mL. Matching hemizonae (5 oocytes or 10 matching hemizonae per experiment) were incubated for 4 hours (under capacitating conditions) with 50 mL of pentoxifylline-treated (test) and nontreated (control) sperm samples, respectively. Then the hemizonae were rinsed vigorously in Ham’s F-10 medium to dislodge slightly bound spermatozoa. Sperm–ZP binding was evaluated using an inverted microscope with phase-contrast optics, and the average number of sperm that were tightly bound per hemizona was determined. The intra-assay variability of the technique was ,12% (9, 12).
Statistical Analysis Statistical evaluation was performed with the use of the GraphPAD (San Diego, CA) InStat software package (version 1.11a). The paired t-test was used to evaluate the differences between pentoxifylline-treated and nontreated spermatozoa for hyperactivated motility, sperm-ZP binding, and sperm tail protein phosphorylation. Correlations between these parameters were performed using Pearson’s correlation coefficient. Results were expressed as means 6 standard error of the mean. P,.05 was considered statistically significant.
RESULTS Pentoxifylline significantly increased sperm hyperactivated motility at 1 hour (20% 6 3% versus 12.5% 6 2% for pentoxifylline-treated versus nontreated samples, P,.002) and at 4 hours (10% 6 1.7% versus 6.5% 6 1.8% for pentoxifylline-treated versus nontreated samples, P,.002) after swim-up separation during incubation under capacitating conditions. Sperm tail protein phosphorylation measured at 4 hours after swim-up separation also was enhanced by pentoxifylline in a statistically significant fashion (13.1% 6 1.5% versus 8.5% 6 1.3% for pentoxifylline-treated versus nontreated samples, P,.0001). Sperm binding to the ZP also was significantly increased by pentoxifylline (73 6 12.6 sperm per hemizona versus 48 6 9.2 sperm per hemi921
zona for pentoxifylline-treated versus nontreated samples, P 5 .002). Hyperactivated motility was significantly correlated with sperm tail protein phosphorylation in pentoxifylline-treated and nontreated samples (r 5 .9420, P 5 .0002 and r 5 .9476, P 5 .0001, respectively). However, hyperactivated motility and sperm tail protein phosphorylation did not correlate significantly with sperm-ZP binding (P..5).
DISCUSSION The investigation of the effects of cAMP and calcium on sperm flagellar motility has received much attention. Cyclic adenosine monophosphate stimulates sperm motility by activating a cAMP-dependent protein kinase (15). This, in turn, is believed to induce enhanced sperm tail protein phosphorylation of a defined number of specific proteins, with subsequent enhancement of sperm motility (16). A major axonemal protein described in humans, dogs, and sea urchins, namely axokinin, has been tentatively identified as a phosphoprotein subject to regulation by cAMP (15). In sea urchin sperm, the addition of cAMP and cAMP-dependent protein kinase partially restored the motility of the phosphoprotein-phosphatase–treated (inhibited) sperm (17). These and other data provide strong support for the possibility that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system (15–17). In the present study, pentoxifylline significantly increased the hyperactivated motility of human sperm and their capacity to bind tightly to the homologous ZP. The patterns of hyperactivated motility are expressed under capacitating conditions (10). Sperm-ZP binding leading to a physiologically induced acrosome reaction such as that observed during the conditions of the hemizona assay reflects multiple sperm functions and is an end point of capacitation (14). Therefore, this methylxanthine was shown to be a strong stimulant of sperm functions crucial to fertilization. Sperm tail protein phosphorylation correlated positively and significantly with hyperactivated motility. This finding is in agreement with a preliminary report of Yunes et al. (18). However, no correlation was found between sperm-ZP binding and sperm tail protein phosphorylation or hyperactivated motility. Collectively, these results indicate that phosphorylation of the tyrosine residues of sperm tail proteins could be one of the mechanisms through which hyperactivated motility occurs. This stimulation probably is dependent on the stimulation of a cAMP-dependent protein kinase. Although hyperactivation is considered to be an integral part of capacitation, it has been suggested that the two processes may not be coupled (19, 20). Our results suggest a temporal correlation between increased protein tyrosine phosphorylation and the development of hyperactivated motility in human spermatozoa. These results are in agreement with those of Leclerc et al. (21) and Tomes et al. (22), who 922
Nassar et al.
Pentoxifylline and hyperactivation
demonstrated a relation between protein tyrosine phosphorylation and sperm motility. As stated by Tomes et al. (22), these results do not exclude a possible role for tyrosine phosphorylation in additional events, such as sperm binding to the ZP or the acrosome reaction. However, we did not detect any immunofluorescence on the sperm head region using the antibody to tyrosine PY20, and we did not observe any relation between sperm protein phosphorylation and the results of the hemizona assay. Confirming these findings, Carrera et al. (13) demonstrated that capacitation is associated with tyrosine phosphorylation in a time-dependent manner, that this phosphorylation is inhibited by the specific inhibitor genistein, and that most of the tyrosine-phosphorylated proteins are localized to the principal piece of the sperm flagellum. Within this context, Aitken et al. (23) reported on a causal association between reactive oxygen species generation, tyrosine phosphorylation, and sperm function (assessed by sperm-oocyte fusion). Consequently, detection of sperm head tyrosine phosphorylation under different capacitation conditions or using other better-defined probes may be necessary to confirm a relation between protein tyrosine phosphorylation and binding to the ZP, the ZP-induced acrosome reaction, and other human sperm functions that depend on specific head membrane domains (5, 24). We conclude that pentoxifylline enhances two capacitation-dependent sperm functions, hyperactivated motility and tight binding to the ZP, which are essential processes leading to fertilization. These findings, confirmed by studies performed in samples with poor sperm motion parameters, may encourage the use of this methylxanthine in assisted reproduction. Enhancement of hyperactivated motility was significantly associated with tyrosine phosphorylation of sperm tail proteins, providing further evidence of a relation between cAMP and tyrosine kinase signaling pathways in spermatozoa. Abnormalities of other intracellular sperm regulators (e.g., calcium) have been associated with deficiencies of the acrosome reaction in patients with severe teratozoospermia (25). The characterization of the signal transduction cascades involved in the control of crucial sperm functions, such as the initiation and maintenance of motility and hyperactivation, may benefit our understanding of the sperm dysfunctions that are present in some infertile men with asthenozoospermia. References 1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill J, editors. The physiology of reproduction. 2nd ed. New York: Raven Press, 1994:189 –317. 2. Tesarik J, Mendoza C, Carreras A. Effect of phosphodiesterase inhibitors, caffeine and pentoxifylline, on spontaneous and stimulus-induced acrosome reactions in human sperm. Fertil Steril 1992;58:185–90. 3. Garbers DL, Lust WD, First NL, Lardy HA. Effect of phosphodiesterase inhibitors and cyclic nucleotides on sperm respiration and motility. Biochemistry 1971;10:1825–31. 4. Nassar A, Mahony M, Blackmore P, Morshedi M, Ozgur K, Oehninger S. Increase of intracellular calcium is not a cause of pentoxifylline-
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15. Tash JS, Hidaka H, Means AR. Axokinin phosphorylation by cAMP dependent protein kinase is sufficient for activation of sperm flagellar motility. J Cell Biol 1986;103:649 –55. 16. Bracho GE, Fritch JJ, Tash JS. Identification of flagellar proteins that initiate the activation of sperm motility in vivo. Biochem Biophys Res Commun 1998;242:231–7. 17. Murofushi H, Ishiguro K, Takahashi D, Ikeda J, Sakai H. Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation. Cell Motil Cytoskeleton 1986;6:83– 8. 18. Yunes R, Doncel GF, Acosta AA. Phosphotyrosine containing proteins and hyperactivated motility in human sperm [abstract]. In: Proceedings of the 50th Annual Meeting of the American Fertility Society; San Antonio, Texas: November 5–10, 1994;53. 19. Neil JM, Olds-Clarke P. A computer-assisted assay for mouse sperm hyperactivation demonstrates that bicarbonate but not bovine serum albumin is required. Gamete Res 1987;18:121– 40. 20. Suarez SS, Vincenti L, Ceglia MW. Hyperactivated motility induced in mouse sperm by calcium A23187 is reversible. J Exp Zool 1987;224: 331– 6. 21. Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 39, 59 monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 22:684 –92. 22. Tomes CN, Carballada R, Moses DF, Katz DF, Saling PM. Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation. Mol Hum Reprod 1998;4:17–25. 23. Aitken RJ, Paterson M, Fisher H, Buckingham D, van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci 1995;108:2017– 25. 24. Burks DJ, Carballada R, Moore HDM, Saling PM. Interaction of a tyrosine kinase from human sperm with the zona pellucida at fertilization. Science 1995;269:83– 6. 25. Oehninger S, Blackmore P, Morshedi M, Sueldo C, Acosta A, Alexander N. Defective calcium influx and acrosome reaction (spontaneous and progesterone-induced) in spermatozoa of infertile men with severe teratozoospermia. Fertil Steril 1994;61:349 –54.
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