Calyculin A, protein phosphatase inhibitor, enhances capacitation of human sperm*

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1 1 FERTILITY AND STERILITY Vol. 59, No.1, January 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U.S.A. Calyculin A, protein phosphatase inhibitor, enhances capacitation of human sperm* Satoru Furuya, M.D.t Yoshihiro Endo, M.D.:!: Kazuoki Osumi, B.S. Mikiko Oba, M.S.II Shiro Nozawa, M.D.t Shuetu Suzuki, M.D.t'll Keio University School of Medicine, and Mitsubishi Yuka BCL, Tokyo, Niiza-Shiki Central General Hospital, Saitama, and Morinaga Milk Industry Co., Ltd., Kanagawa, Japan Objective: To examine the effects of protein phosphatase inhibitors on capacitation and protein phosphorylation in human sperm. Design: The chlortetracycline (CTC) fluorescence assay was used to monitor capacitated sperm treated with or without protein phosphatase inhibitors. Capacitation was confirmed by the ability of sperm to undergo the acrosome reaction in response to Ca ionophore A23187 or mouse zonae pellucidae. 32P-Iabeled sperm phosphoproteins were analyzed by one-dimensional gel electrophoresis to detect the effects of protein phosphatase inhibitors on protein phosphorylation. Results: The treatment of sperm with calyculin A resulted in the following: [1] the rapid appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece, in a dose-dependent manner in the 1 to 100 nm range; [2] an accelerated ability to undergo the acrosome reaction; and [3] an enhanced phosphorylation of sperm phosphoproteins in a dose-related fashion in the 1 to 100 nm range. A similar stimulatory effect was observed only with a 100-fold higher concentrations of okadaic acid, another protein phosphatase inhibitor. Conclusion: Our results strongly suggest that protein phosphorylation and dephosphorylation may be involved in the regulation of human sperm capacitation. Fertil Steril 1993;59: Key Words: Human sperm, capacitation, protein phosphatase inhibitor, calyculin A, okadaic acid, protein phosphorylation, chlortetracycline fluorescence assay Received June 11, 1992; revised and accepted September 1, * Supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture (project to S.S.), Tokyo, Japan; and by the World Health Organization Special Programme of Research, Development, and Research Training in Human Reproduction (project to S.S.), Geneva, Switzerland. t Department of Obstetrics and Gynecology, Keio University School of Medicine. :\: Niiza-Shiki Central General Hospital. Mitsubishi Yuka BCL. II Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd. '\I Reprint requests: Shuetu Suzuki, M.D., Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. Mammalian sperm must undergo physiological changes before fertilizing eggs. The definition of capacitation has remained controversial because some investigators include the acrosome reaction in it, whereas the other does not (1). In this study, capacitation and the acrosome reaction are considered to be two separate phenomena. In mouse sperm, it has become possible to evaluate the capacitation state using the fluorescent probe chlortetracycline (CTC) (2, 3). Recently, capacitation and the acrosome reaction in human spermatozoa have also been monitored by a CTC fluorescence assay (4). One of four CTC fluorescence patterns, the clear perimeter pattern, appeared to be correlated with capacitated human sperm (4). 216 Furuya et al. Calyculin A stimulates capacitation

2 Another pattern, the acrosome reaction pattern, was shown to be diagnostic of acrosome-reacted human sperm (4). Therefore, an increase in the clear perimeter and acrosome reaction patterns may provide an empirical monitor of the capacitation and the acrosome reaction process, respectively. In mammalian sperm capacitation and acrosome reaction, the receptor-coupled intracellular signal transduction pathways have been suggested to proceed through a series of sequential changes in enzymes and second messengers (5-7). Recently, De Jonge et al. (8, 9) have provided evidence for the involvement of the diacylglycerol/protein kinase C pathway and the adenylate cyclase/cyclic adenosine 3':5' monophosphate (camp) pathway in the human sperm acrosome reaction. Although the molecular mechanisms involved in the regulation of capacitation and the acrosome reaction are still poorly understood, it is commonly accepted that protein phosphorylation may be associated with these events. The stimulation of protein phosphorylation may result not only from activation of protein kinases but also from inhibition of protein phosphatases. Little attention has been paid to the role of protein phosphatases because of the lack of specific inhibitors of protein phosphatases. Recently, two protein phosphatase inhibitors, calyculin A and okadaic acid, have been introduced and recognized as being very useful reagents in the investigation of the role of protein phosphatases in various cell functions (10,11). The regulatory mechanisms involved in mediating human sperm capacitation are not known in detail at the present time. In the present study, we therefore investigated the effects of the protein phosphatase inhibitors, calyculin A and okadaic acid, on human sperm capacitation using CTC fluorescence assay and the capacitation-associated changes in protein phosphorylation. MATERIALS AND METHODS Preparation of Sperm Samples Human sperm ejaculates were obtained from healthy fertile adult donors by masturbation after 3 to 4 days of abstinence. Only donors displaying normal semen analysis were used in the present study (sperm count 40 X 10 6 cells/ml; semen volume > 1.5 ml; motile sperm> 60%). After liquefaction at 37 C for 15 to 30 minutes, the ejaculate was mixed with the same amount of modified Krebs'-Ringer's bicarbonate medium of Biggers, Whitten, and Whittingham (BWW) (12) containing 3 mg/ml human serum albumin (HSA; Sigma Chemical Company, St Louis, MO) and washed by centrifugation at 600 X g for 10 minutes. The sperm pellet was overlayed with 1 ml BWW containing 3 mg/ml HSA and incubated in a test tube inclined at 45 degrees for 15 minutes, enabling the motile sperm to swim up out of the pellet (4, 13). The sperm samples obtained from the upper layer showing >90% motility were used in the following experiments. Capacitation and Induction of the Acrosome Reaction Sperm were incubated in BWW containing 3 mg/ml HSA in the presence or absence of calyculin A at 37 C in a humidified atmosphere of 5% CO 2 in air for up to 3 hours to examine the effect of calyculin A on capacitation. Next, the sperm incubated in the presence or absence of 100 nm calyculin A for 15 minutes were treated with one of the acrosome reaction inducers, 10 JLM Ca ionophore A23187 for an additional 3 hours or treated with another inducer, acid-solubilized mouse egg zonae pellucidae (ZP) (10 zonae/jll) for an additional 2 hours (4) to examine the effect of calyculin A on the ability of sperm to undergo the Ca ionophore- or zona-induced acrosome reaction. In addition, the sperm incubated in the absence of calyculin A for 180 minutes were treated with A23187 or zonae as described previously (4). Mouse egg zonae were solubilized as described by Endo et al. (14). Chlortetracycline Fluorescence Assay Human sperm capacitation and the acrosome reaction were monitored by CTC fluorescence assay (3,4). Five microliters of CTC stock solution (500 JLM CTC [Sigma Chemical Company] in 20 mm Tris, 130 mm NaCI, 5 mm cysteine [Sigma Chemical Company], ph 7.8) was added to 5 JLL of sperm suspension on a slide warmed to 37 C, followed by fixation with 0.1% glutaraldehyde. The samples were examined by microscope (Nikon, Tokyo, Japan) equipped with epifluorescence, using an excitation of 405 nm and an emission of 430 nm. The capacitated sperm were assessed by the appearance of the clear perimeter pattern, specifically, distribution of fluorescence over the entire head exhibiting a bright perimeter and bright midpiece (4). The acrosome-reacted sperm featured the acrosome Vol. 59, No.1, January 1993 Furuya et ai. Calyculin A stimulates capacitation 217

3 reaction pattern that demonstrates the lack of fluorescence on the head and fluorescence on the midpiece (4). A total of 100 sperm were scored to determine the percentage of each CTC pattern in the samples. Radiolabeling of Intact Sperm Sperm suspension prepared in phosphate-free BWW were incubated in the presence of 3 mci/ml [32p] orthophosphate for 15 minutes at 37 C with or without 1 to 100 nm calyculin A. The radiolabeled sperm were washed three times with phosphate-free BWW by centrifugation at 12,000 rpm for 3 minutes in a Beckman Microfuge E (Beckman Instruments, Inc., Palo Alto, CA), and the pellets w:ere extracted in 20 ILL of extraction buffer (50 mm Tris-HC1, ph 7.5, 1 % Triton X -100, 1 % sodium deoxycholate, 150 mm NaCl, 1 mm phenyl methyl sulfonyl fluoride, 1 mm ethylene diamine tetraacetic acid, 5 11M sodium vanadate, 10 Ilg/mL aprotinin, 10 Ilg/mL leupeptin, 10 Ilg/mL soy bean trypsin inhibitor, 1 mm dithiothreitol,5 mm benzamidine [Sigma Chemical Company]) for 30 minutes at O C. After centrifugation at 12,000 rpm for 15 minutes, 15 ILL of sperm extract were collected, and 5 ILL of 4X sodium dodecyl sulfate (SDS) sample buffer (15) was added. The samples were then solubilized by incubation in boiling water for 5 minutes before being subjected to one-dimensional gel electrophoresis. 60 <= 50 ~ 0., '" '" S '0:: '" 0...!l " U!? Time (min) Figure 1 Time course of appearance of clear perimeter pattern in the presence of 100 nm calyculin A. Human sperm were incubated for up to 3 hours under capacitation conditions in BWW containing 3 mg/ml HSA in the absence (e) or presence (0) of 100 nm calyculin A. The percent of clear perimeter pattern was estimated using CTC assay. Each point represents the mean of pooled data with nine different experiments of 100 sperm scored per point. Bars are the SEM at each time point. 70 a 60 t3 " ~ S 40 '0:: '" 0. '" ** 30 " U '"!? ** o Calyculin A (nm) Figure 2 Effect of calyculin A concentration on appearance of clear perimeter pattern. Human sperm were incubated under capacitation conditions in the presence of various concentrations of calyculin A. After 15 minutes, the clear perimeter pattern was estimated using CTC assay. Each bar represents the mean of three separate experiments with different donors. Error bars are SEM. ** P < 0.01 compared with control. One-Dimensional Gel Electrophoresis One-dimensional gel electrophoresis was carried out as described by Laemmli (15). 32P-radiolabeled phosphoproteins were loaded on 2.5% stacking gel and a 10% separating gel in the presence of SDS and analyzed by autoradiography. Molecular weight (MW) standards used in electrophoresis were myosin (H-chain) (MW, 200,000), phosphorylase b (MW, 97,400), bovine serum albumin (MW, 68,000), ovalbumin (MW, 43,000), and a-chymotrypsinogen (MW, 25,700). Statistical Analysis of Data Each experiment was performed at least three times. The results were analyzed by X2 analysis. Values were considered statistically significant when P < RESULTS Effect of Calyculin A on Appearance of Clear Perimeter Pattern The treatment of human sperm with one of the protein phosphatase inhibitors, calyculin A, resulted in a rapid appearance of clear perimeter pattern about 15 minutes after the addition in a dose-dependent manner in the 1 to 100 nm range (Figs. 1 and 2). On the other hand, the other protein phos- 218 Furuya et ai. Calyculin A stimulates capacitation

4 Table 1 Representative Time Courses of the Appearance of Clear Perimeter Pattern in Five Different Donors Clear perimeter pattern Donor Concentration Motility Calyculin A o min 15 min 30 min 60 min 120 min 180 min XIO'/mL % loonm % phatase inhibitor, okadaic acid, did not induce a similar rapid appearance of clear perimeter pattern as with calyculin A in the same concentration range (data not shown). The stimulatory effect of calyculin A on human sperm capacitation, in terms of appearance of clear perimeter pattern, was observed in sperm obtained from five donors examined in this study, and interindividual variation in the effect was not significant (Table 1). The stimulatory effect of calyculin A on the appearance of clear perimeter pattern was detected until 3 hours incubation after which there was no significant difference in the percentages of sperm showing the clear perimeter and acrosome reaction pattern between calyculin A treated sperm and the controls (Fig. 1 and data not shown). The reagents tested did not affect viability or motility at these time points. Effect of Calyculin A on the Ability of Sperm to Undergo the Acrosome Reaction Because the data presented in Figures 1 and 2 suggested that calyculin A facilitates the process of human sperm capacitation, we examined the ability of calyculin A-treated sperm to undergo the acrosome reaction (Table 2). Sperm were treated with 100 nm calyculin A for 15 minutes and were further exposed to 10 JlM Ca ionophore A23187 or acidsolubilized mouse egg ZP (10 zonae/jll) to induce the acrosome reaction. The acrosome-reacted sperm, presenting the acrosome reaction pattern in the CTC fluorescence assay, increased significantly in response to Ca ionophore or mouse ZP, whereas calyculin A itself did not induce the acrosome reaction in the absence of these inducers. Sperm treated with calyculin A for 15 minutes showed the same ability Table 2 Effect of Calyculin A on the Ability of Sperm to Undergo the Acrosome Reaction Induction of the acrosome reaction Group Capacitation time Calyculin A min * Values are means ± SEM. t Group 4 is significantly different from group 3 (P < 0.01). :j: Group 6 is significantly different from group 5 (P < 0.01). A23187 Zonae Acrosome reaction pattern * 9 ± ± ± ± 2.1t 16 ± ± 4.4:j: 12.1 ± ± ± Group 8 is significantly different from group 7 (P < 0.01). II Group 9 is significantly different from group 7 (P < 0.01). % Vol. 59, No.1, January 1993 Furuya et al. Calyculin A stimulates capacitation 219

5 to undergo the acrosome reaction in response to the Ca ionophore or mouse zonae as sperm incubated in the absence of any reagents for 3 hours. Therefore, these results support the stimulatory effect of calyculin A on human sperm capacitation. Effect of Calyculin A on Protein Phosphorylation The treatment of sperm with 100 nm calyculin A for 15 minutes resulted in the increased phosphorylation of sperm proteins (Fig. 3). In particular, calyculin A induced the apparent phosphorylation of phosphoproteins of MW 42,000, 45,000, 54,000, 60,000,77,000,88,000,98,000,120,000, and 150,000 (Fig. 3). Calyculin A induced an enhanced phosphorylation of sperm phosphoproteins in a dose-dependent manner in the 1 to 100 nm range (data not shown). On the other hand, 100 nm okadaic acid stimulated neither the appearance of clear perimeter pattern (data not shown) nor protein phosphorylation in human sperm (Fig. 3). Thus, enhanced phosphorylation levels induced by calyculin A may be related to the acceleration of the capacitation process. DISCUSSION We demonstrated the stimulatory effect of calyculin A on the appearance of sperm presenting the CTC fluorescent clear perimeter pattern (Figs. 1 and 2). At 3 hours, the incident of sperm exhibiting the clear perimeter pattern is limited to the same level as in sperm capacitated spontaneously (Fig. 1). Lee et al. (4) have reported that the clear perimeter pattern is an empirical monitor of human sperm capacitation, rather than an actual indicator of fully capacitated sperm cells that are defined as having the ability to undergo the acrosome reaction. The appearance of sperm exhibiting the acrosome reaction pattern increased in calyculin A-treated (15 minutes) sperm to the same extent as in 3 hours capacitated sperm compared with the controls (Table 2) in response to Ca ionophore A23187 or mouse ZP. The CTC acrosome reaction pattern in human sperm has been well defined by indirect immunofluorescence assay (4, 16-18), the triple stain (4), and the ultrastructural studies (18). Thus, the results support the stimulatory effect of calyculin A on capacitation, in terms of the ability of calyculin A-treated sperm to undergo the induced acrosome reaction. We did not detect any significant effects of calyculin A on the sperm motility (data not shown). Be- 2 3 Figure 3 Effect of calyculin A on protein phosphorylation. Human sperm were 32P-radiolabeled for 15 minutes in the presence or absence of 100 nm okadaic acid or 100 nm calyculin A. Radiolabeled phosphoproteins were subjected to one-dimensional gel electrophoresis. Film exposure time was 4 days. Lane 1, control sperm; Lane 2, okadaic acid-treated sperm; Lane 3, calyculin A treated sperm. The experiment was performed three times, and similar results were obtained in each case. The chevrons point to phosphoproteins that are apparently phosphorylated in the presence of calyculin A. cause we determined the motility using manual mode, there was a limitation with respect to evaluation of sperm velocity and hyperactivation. As far as viability is concerned, there was no significant difference between the calyculin A-treated sperm and the controls for at least 5 hours (data not shown). The calyculin A concentrations required for 50% inhibition (IC 50) of the phosphatase type 1 and 2A are 1.6 to 2.0 nm and 0.5 to 1 nm, respectively (10). In this study, 100 nm of calyculin A was needed to stimulate fully human sperm capacitation. The need for higher concentrations of calyculin A in these studies is likely to be related to the limitation of permeability through the plasma membrane. Calyculin A activates the voltage-dependent Ca - channel in intact smooth muscle cells (19). However, the contractile response induced by calyculin A is not because of an increased influx of extracellular Ca because calyculin A caused contraction in the absence of extracellular Ca (19). Similarly, in the present study, we examined the effect of calyculin 220 Furuya et a!. Calyculin A stimulates capacitation

6 A on capacitation of human sperm incubated in Ca -free medium containing 1 mm ethylene glycol tetraacetic acid. The stimulatory effect of calyculin A on the appearance of the clear perimeter pattern was not lost through the removal of extracellular Ca (data not shown). Further investigation of intracellular mobilization of Ca will be absolutely required to determine the involvement of Ca in the calyculin A effect on human sperm capacitation. In mammalian sperm, protein phosphorylation has been shown to be one of the candidates for bringing about changes in the plasma membrane associated with the process of capacitation, as well as that in the flagellar movement (5, 7). Recently, De Jonge et al. (8, 9) demonstrated that dibutyryl camp and protein kinase C activators caused a significant increase in the acrosome reaction of capacitated human sperm. Therefore, protein phosphorylation is likely to be involved in human sperm capacitation and the acrosome reaction. The level of protein phosphorylation depends on the balance of activities of kinases and phosphatases. In the present study, an inhibitor of type 1 and 2A phosphatase, calyculin A, caused a marked increase in protein phosphorylation in parallel with stimulated capacitation within 15 minutes after the addition of the agent to human sperm cells (Fig. 3). It is unknown if the increased gross phosphorylation or specific substrates for calyculin A may be directly related to modifications of the sperm plasma membrane required for capacitation. Also, it should be noted that it would be very difficult to clarify the precise mechanism by which calyculin A regulates a specific process in capacitation because this reagent stimulates the phosphorylation of many proteins. Another well-known protein phosphatase inhibitor, okadaic acid, showed the same effects only at 100-fold higher concentrations on either capacitation or the capacitation-associated changes in protein phosphorylation (Fig. 3 and data not shown). The major differences between calyculin A and okadaic acid is manifested in the inhibition of the type 1 phosphatase (10). Therefore, the existence of the type 1 phosphatase as compared with the type 2A phosphatase might be dominant in human sperm cells, or there may be a difference in the binding ability of okadaic acid receptors (20, 21) or in the permeability of calyculin A through the plasma membrane. We can conclude that the rapid enhanced level of protein phosphorylation induced by calyculin A probably indicates a shift in the phos- phorylation and dephosphorylation balance in human sperm leading to the facilitation of biochemical changes underlying capacitation. Acknowledgments. We thank Yasumasa Tsukitani, Ph.D., Fujisawa Chemical Company, Tokyo, Japan, for his generous gifts of okadaic acid; Keishi Hata, B.S., Mitsubishi Yuka BCL, Tokyo, Japan, for his valuable arrangement for study of one-dimensional gel electrophoresis; and Mrs. Yukari Matsui for her skilled technical assistance. REFERENCES 1. Chang MC. The meaning of sperm capacitation. J Androl 1984;5: Saling PM, Storey BT. Mouse gamete interactions during fertilization in vitro: chlortetracycline as fluorescent probe for the mouse sperm acrosome reaction. J Cell Bioi 1979;83: Ward CR, Storey BT. Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev Bioi 1984;104: Lee MA, Trucco GS, Bechtol KB, Wummer N, Kopf GS, Blasco L, et al. Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay. Fertil SterilI987;48: Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J, editors. 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