Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay.код для вставкиСкачать
American Journal of Primatology 20115-125 (1990) Capacitation and the Acrosome Reaction in Squirrel Monkey (Saimiri sciureus) Spermatozoa Evaluated by the Chlortetracycline Fluorescence Assay S.D. KHOLKUTE, YE LIAN, W.E. ROUDEBUSH, AND W.R. DUKELOW Endocrine Research Center, Michigan State Uniuersity, East Lansing Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode’s medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 KM to 20p,M) induced acrosomal loss in 60-70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca’ ‘-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swimup” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey. Key words: squirrel monkey acrosome reaction, sperm fluorescence, capacitation INTRODUCTION The importance of capacitation and the acrosome reaction (AR) in mammalian fertilization is well recognized [Yanagimachi, 1981; Bedford, 19831. However, in spite of numerous studies, capacitation per se is poorly understood. It is known that capacitation involves the removal and alteration of sperm membrane components in the anterior portion of the sperm head so that the acrosome reaction can occur. Bioassays for the assessment of capacitation, especially for human spermatozoa, have been developed [Overstreet & Hembree, 1976; Yanagimachi et al., 1976; Overstreet et al., 19801. However, these assays are cumbersome and may not Received for publication July 12, 1989; revision accepted November 6 , 1989 Address reprint requests to Dr. W. Richard Dukelow, Endocrine Research Center, East Lansing, MI 48824. 0 1990 Wiley-Liss, Inc. 116 / Kholkute et al. adequately represent the physiologic events of fertilization [Boldt & Wolf, 19841. Methods have been developed to evaluate AR by a triple stain method [Talbot & Chacon, 1980; Cross et al., 1986,19891 and a n indirect immunofluorescence assay utilizing monoclonal antibodies [Wolf et al., 19851. Ericsson [1967a,bl demonstrated that if ejaculated sperm are incubated for 10 min at 37°C with tetracyclineHCI(6 pg/106 sperm) and then placed in a capacitating environment (as the uterus of a n estrous rabbit), the tetracycline coating is removed over time. Others [Dukelow & Chernoff, 19691 used this technique to study capacitation of human and rhesus spermatozoa after incubation in the reproductive tract of the rat, mouse, rabbit, and hamster. This technique has limitations when used to study time relationships in capacitation. Ericsson [ 1967bl found that tetracycline was removed from sperm in the rabbit oviduct in 6 to 7 h r whereas it has been established that capacitation a t this site requires 9.5 to 10 hr. Later Vaidya et al. El9691 confirmed the removal of tetracycline a t a rate faster than normal capacitation occurs. More recent work has used the halogenated chlortetracycline for a fluorescence assay (the CTC assay) developed for mouse sperm [Ward & Storey, 19841 and extended to human spermatozoa [Lee et al., 19871. The results from the CTC assay indicated that this method not only scores the percent AR spermatozoa but also could be used to monitor the time course of capacitation. The squirrel monkey has been extensively studied as a n animal model for IVF LKuehl & Dukelow, 1975, 1979, 1982; Hutz et al., 1983; Dukelow & Ridha, 19881. Indirect evidence indicates that capacitation in the squirrel monkey occurs after 2-3 h r in vitro [Kuehl & Dukelow, 19821. The present study reports the use of the CTC assay to characterize capacitation and the AR in spermatozoa from this new world primate species. METHODS Capacitation Medium Tyrode’s medium prepared with 3 x distilled water was used for incubation (TALP:NaCl, 92.9 mM; CaC1,.2H,O, 4.0 mM; KCI, 4.0 mM; Na,HPO,, 0.7 mM; MgCl,~GH,O, 0.5 mM; NaHCO,, 25.9 mM; HEPES, 20 mM; glucose, 12.8 mM; sodium pyruvate, 1.3 mM; lactate, 7.6 mM; supplemented with gentamicin sulphate, 1.9 ml, penicillin, 8.1 mg, and streptomycin, 5.4 mg per 100 ml of medium). Two different concentrations (4 and 40 mgiml) of bovine serum albumin (BSA: fraction V, Sigma Chemical Company, St. Louis, MO) were used. pH was adjusted to 7.4 and the osmolality was 280 15 mOsmol. Calcium concentration (2 mM and 6 mM) was varied in some experiments. Calcium-poor medium was prepared by deleting calcium and adjusting the osmolality with NaC1. Medium 199 with 25 mM HEPES buffer, Earle’s salt, and L-glutamine (GIBCO Laboratories, Grand Island, NY) was also used for incubation of spermatozoa. This was supplemented with 20% heat-inactivated fetal calf serum. * Semen Collection Sperm ejaculates were collected from adult male squirrel monkeys by electroejaculation [Kuehl & Dukelow, 19741. Samples contaminated with urine or showing less than 60% active forward motility were discarded. Ejaculates were suspended in prewarmed (37°C) medium. The sperm concentration was adjusted to 4 to 6 x lo6 sperm/ml. The sperm suspension was incubated a t 37°C in 5% CO, and 95% air under a humidified atmosphere. At varying time intervals, aliquots of the sperm suspension (10 p1) were removed and placed on prewarmed glass slides for motility assessment and for studying the CTC pattern. Motility was evaluated for Capacitation of Squirrel Monkey Sperm / 117 percentage of active, forwardly motile sperm (100 spermatozoa scored) by phase contrast microscopy at 200 x . In one experiment, motile spermatozoa were recovered after incubation for 1hr to allow the non-motile cells and debris to settle and then the supernatant was aspirated (“swim-up technique”). CTC Assay The spermatozoa were stained with CTC as described by Lee et al. [19871 with slight modification. The CTC solution was made by dissolving CTC-HC1 (Sigma Chemical Co., St. Louis, MO) at a concentration of 500 pM in a buffer containing 20 mM Tris, 130 mM NaC1, and 5 mM cysteine. pH was adjusted to 7.8. The solution was stored in brown bottles in a light-shielded container a t 4°C. Fresh CTC solution was made daily. At varying time intervals, 10 pl of the sperm suspension was placed on a slide warmed to 37°C and 10 p1 of CTC solution was rapidly added followed 10-15 sec later by addition of 5 pl of 12.5%glutaraldehyde in 2M Tris buffer (pH 7.8). This was then stirred to ensure uniform mixing. Following application of the cover-glass, the slides were held at room temperature in a light-shielded box. The sperm were examined at 400x for CTC fluorescence patterns with a Nikon epi-fluorescence microscope by using a “V” dichroic mirror and a 520-K guard filter combination. A total of 100 spermatozoa were scored at each time interval to determine the percentage of the different CTC patterns in each sample. Preparation of Ionophore A23187 and Induction of Acrosomal Loss Calcium ionophore A23187 (free acid, Sigma Chemicals Co., St. Louis, MO) was dissolved in dimethyl-formamide (DMF) to a final concentration of 5 mM. Aliquots of stock were frozen at - 20°C. Before use, this was thawed and added to the sperm suspension at concentrations of 1, 5, 10, and 20 pM with a maximal DMF content of 0.1% (V/V). To the control group of ejaculates, a similar volume of DMF without ionophore was added. The addition of ionophore or DMF was carried out after 2.5 hr incubation in Ca+ +-TALP containing 40 mg/ml BSA. The samples were incubated for another hour and sperm samples evaluated at 0, 75, 150, and 210 min for CTC fluorescence patterns. In one experiment, ionophore A23187 was added (5 pM) to fresh ejaculates suspended in TALP. Incubation was carried out up to 3.5 hr and CTC patterns were studied a t various time intervals as above. The data were evaluated statistically by Student’s “t” test and a value of Pc0.05 was taken as significant. RESULTS Four distinct fluorescence patterns using CTC were observed in squirrel monkey spermatozoa depending on the time of incubation (Figs. A-D). Since these patterns are similar to that reported for human spermatozoa [Lee et al., 19871the same descriptive terminology has been used. EF stands for early fresh sperm characterized by a bright band in the post-acrosomal region (Fig. 1A).DP stands for dark posterior head (sperm), and as the name suggests, the spermatozoa showing this pattern were identified by a bright anterior portion and a dark post-acrosomal region (Fig. 1B). The CP spermatozoa showed uniform fluorescence over the entire head with a clear perimeter mainly visible at the posterior portion of the head (Fig. 1C).The AR pattern was characterized by lack of fluorescence on the head (Fig. 1D). The mid-piece in all the four CTC patterns showed bright fluorescence. Changes in the CTC fluorescence patterns EF, DP, and CP during various time courses of incubation are shown in Figures 2-4. At 0 hr, 65% of the spermatozoa incubated in the presence of 40 mg/ml BSA showed an EF pattern, which 118 / Kholkute et al. Capacitation of Squirrel Monkey Sperm I 119 EF PATTERN - 801 ----- 2 0 40rng/ml BSA 4 m g / m l BSA TC 199 4 8 6 TIME ( h r ) Fig. 2. Percentage of fluorescence EF pattern in squirrel monkey spermatozoa over a n 8 h r incubation period in TALP medium containing 4 mgiml BSA (- - -) or 40 mgiml BSA ( 0 )and in medium TC-199 fortified with 10% fetal calf serum (*I. Mean of five ejaculates % standard deviation. DP PATTERN ----- 601 1 OJ, 0 I I * , I 2 4 TIME ( hr) 40mg/ml BSA 4mg/ml BSA TC i 9 9 I 6 8 Fig. 3. Percentage of fluorescence DP pattern in squirrel monkey spermatozoa over a n 8 h r incubation period in TALP containing 4 mgiml BSA (- - -) or 40 mgiml BSA ( 0 ) and TC-199 fortified with 10% fetal calf serum (*I. Means t standard deviations (n = 5). Fig. 1. Epifluorescence photomicrographs of squirrel monkey spermatozoa exposed to CTC. A: Fluorescence EF pattern showing bright band of fluorescence in the post-acrosomal region and bright midpiece. B: Fluorescence DP pattern. Note the bright fluorescence on the anterior portion of the head and dark band in the post-acrosomal region. Midpiece is bright. C: Fluorescence C P pattern showing uniform fluorescence over both the head and postacrosomal portion with bright perimeter a t the anterior portion. D Fluorescence AR pattern: Note mild or lack of fluorescence on the head and bright midpiece. ( x 645). 120 / Kholkute et al. CP PATTERN 801 - 4 0 m g / m l BSA 4 mg/ml BSA TC 193 -I,,,,, 0 0 2 4 6 8 TIME ( h r ) Fig. 4. Percentage of fluorescence pattern CP in squirrel monkey spermatozoa over a n 8-hr incubation period in TALP containing 4 mgiml BSA (- - -) or 40 mgiml BSA ( 0 ) and TC-199 fortified with 10% fetal calf serum ( 0 ) .Means -t standard deviations ( n = 5 ) . declined to ~ 2 0 % by 2 hr. The loss of this pattern in 40 mgiml BSA was more rapid than with 4 mgiml BSA especially for the first 2 hr. The changes in the DP pattern following various time intervals were not clear. From the initial 25%, it increased slightly during the first 1 to 1 112 hours of incubation, thereafter declining gradually to <lo% by 8 hr. The increase in C P pattern coincided with the disappearance of the E F pattern a t the two concentrations of BSA. The majority (50%) of the sperm showed this pattern after 2 h r incubation in TALP with 40 mgiml BSA. Under identical conditions with 4 mgiml BSA, 50% of the spermatozoa showed the CP pattern by 4 hr. The maximal CP pattern observed was 70% after 8 h r of incubation. The AR pattern remained consistently low (<15%) starting from time 0 and up to 8 hr incubation. This is expected in a medium designed to promote capacitation but not the acrosome reaction [Byrd & Wolf, 1986; Lee et al., 19871. Since this method fails to differentiate between the normal acrosome reaction and that observed in dying sperm, motility was monitored throughout the incubation period and the percentage of normal reactions was calculated by subtracting the percentage of immotile spermatozoa a t the time of sampling from the mean value of spermatozoa showing the AR pattern. Initial motility was 74 -t 6%, which declined to 63 -+ 6% by 4 hr. By 8 hr, the motility was 48 -+ 7%. The CTC fluorescence patterns observed during various time intervals in TC 199 supplemented with 20% heat-inactivated fetal calf serum (FCS) also are shown in Figures 2-4. The disappearance of the E F pattern and appearance of the CP pattern was comparable t o Ca’ TALP + 4 mgiml BSA. Approximately 50% of spermatozoa showed the CP pattern 3-4 hr after incubation. The AR pattern was always <15% in spite of 8 h r incubation. Motility, however, was maintained better in TC-199 when compared to Ca’ TALP. Fresh active motile spermatozoa were allowed to swim up for 1 h r in 1.5 ml Ca’ +-TALP containing 40 mg/ml BSA. The upper 1ml of medium was then carefully removed leaving the debris. The “swim-up” spermatozoa thus obtained were incubated for a n additional 6 hr. The CTC patterns at various time intervals did not differ significantly from the patterns observed in spermatozoa incubated in the presence of seminal plasma (data not shown). The AR pattern was also 515% after the swim up procedure. + + Capacitation of Squirrel Monkey Sperm I 121 a ::2 0 “ i OJ 0 I 5 1 i 10 20 A 23187 CONCENTRATION (pM) Fig. 5 . Effect of various concentrations ( 1 to 20 KM)of calcium ionophore A23187 on induction of acrosomal loss in squirrel monkey spermatozoa incubated in TALP containing 40 mgiml BSA and 4 mM C a t + . Spermatozoa were incubated for 2.5 hr in medium. Ionophore was added and incubation was carried out for another hour Means 5 standard deviations ( n = 5 ) . Chemically Induced Acrosomal Loss by A23187 The results presented above indicate that squirrel monkey spermatozoa incubated in capacitating medium up to 8 h r do not undergo spontaneous acrosomal loss. The calcium-ionophore A23187, known to induce acrosomal loss in mouse and human spermatozoa, was therefore selected for chemical induction of the acrosome reaction. Sperm suspended in Ca’ +-TALPwith 40 mg/ml BSA were incubated for 2.5 h r (by this time a t least 50% of the spermatozoa were capacitated) followed by 1 hr incubation in various concentrations of A23187 (Fig. 5). Approximately 60% of the sperm lost their acrosome following exposure to 5 pM A23187; however, 1 p.M ionophore induced only 20% AR sperm. Exposure to ionophore concentrations of 10 p.M or 20 pM did not increase the percentage of AR in spermatozoa. Ionophore concentrations of 10 pM and 20 pM also caused marked reduction in sperm motility to less than 20% within a n hour. As in the human [Wolf et al., 19851 squirrel monkey spermatozoa also showed absolute dependency on extracellular calcium a s evident from the lack of AR by A23187 in Ca’ +-free TALP. Increasing the concentration of C a + to 6 mM increased the acrosome reaction in sperm in a dose-dependent manner (Fig. 6). When freshly ejaculated spermatozoa were suspended in C a + +-TALP with 40 mg/ml BSA, and exposed to 5 p.M A23187 at 0 hr, the percent acrosome reaction (14 5 6) observed was not significantly different from the control AR values (12 5 4) under the same incubation conditions (data not shown). + DISCUSSION The results of the present study demonstrate that a finite number of fluorescence patterns occur and changes in those patterns occur over a time course where capacitation and the acrosome reaction might occur in squirrel monkey spermatozoa, a s in the mouse [Ward & Storey, 19841 and human [Lee et al., 19871. This method is simple and rapid and thus can be used to resolve the changes in fluorescence patterns during incubation. The fluorescence E F pattern was dominant in fresh spermatozoa and was 122 / Kholkute et al. n z CALCIUM CONCENTRATION (mM) Fig. 6. Effect of 5 pM calcium ionophore A23187 on induction of acrosomal loss of squirrel monkey spermatozoa incubated in TALP + 40 mgiml BSA containing various concentrations (2 mM, 4 mM, and 6 mM) of calcium. Minimal acrosomal loss could be induced in C a + +-poormedium. Means f standard deviations ( n = 5 ) . found to decrease with time. Furthermore, the disappearance rate was faster in TALP with a higher BSA concentration compared with lower albumin and medium TC199. At 0 hr, 22 t 4% of spermatozoa exhibited the DP pattern which showed an increasing trend for the first 1-2 h r followed by a decline. Nevertheless, even following 8 h r of incubation, 15-20% spermatozoa showed EF and DP patterns. The CP spermatozoa increased from initial low values (12 3%)to 50% following 2 h r incubation in TALP containing 40 mg/ml BSA, while a t lower concentrations of BSA and in TC199 50% spermatozoa required almost 4 h r to show the CP pattern. This suggests that capacitation in squirrel monkey spermatozoa was hastened by increasing the BSA concentration in the medium. This is consistent with the results of Byrd and Wolf  and Lee et al.  in human spermatozoa and also with earlier studies in the mouse [Wolf, 1979; Ward & Storey, 19841. The results of the present studies also demonstrate that squirrel monkey spermatozoa incubated under capacitation conditions do not readily undergo spontaneous loss of the acrosome in vitro. Even after 8 h r incubation, only 15% of the spermatozoa showed the AR pattern. This is similar to that reported for human spermatozoa [Talbot & Chacon, 1981; Plachot et al., 1984; Byrd & Wolf, 1986; Lee et al., 19871 and contrasts with that observed in other species [Talbot et al., 1976; Rogers, 1981; Bedford, 19831. The acrosome reaction in all mammalian sperm studied so far displays a n absolute dependency on extracellular calcium. In the squirrel monkey also, chemical induction of AR did not occur in Ca+ +-poor medium. Furthermore, increasing the concentration of Ca’ from 2 mM to 6 mM significantly increased the chemical induction of the AR. Increased intracellular calcium appears to be the primary signal for induction of AR in capacitated spermatozoa [Singh et al., 1978; Yanagirnachi, 19811; however, the precise mechanism(s) in vivo is still not well understood. It has been reported that exposure to seminal plasma blocks or prevents capacitation in the human [Byrd & Wolf, 19861; in the present study we could not observe such a n effect. This is evident from the similarity of fluorescence CP patterns in spermatozoa incubated in the presence of seminal plasma and spermatozoa harvested by the “swim-up” technique. This may be due to the very low * + Capacitation of Squirrel Monkey Sperm I 123 volume of the squirrel monkey ejaculate and thus the seminal plasma is diluted further when suspended in the capacitation medium. Presence of even dilute seminal plasma appears to prevent attachment to zona-free hamster and salt-stored human eggs [Kanwar et al., 19791. Nonetheless, successful in vitro fertilization of squirrel monkey oocytes has been reported following insemination with sperm suspensions without removing the seminal plasma constituents [Kuehl & Dukelow, 1975, 1979, 1982; Dukelow & Ridha, 19881. In the present study, the maximal A23187-induced acrosome reaction was 70%, indicating that some portion of the population of spermatozoa failed to respond. This further reinforces the view that capacitation is asynchronous [Bedford, 19831. However, whether this is due to variability in sperm morphology [Amelar & Dubin, 19821, wherein substantial numbers of spermatozoa did not respond due to defective or absent acrosome, or is due to some other mechanism(s) needs to be determined. When fresh sperm suspension consisting mainly of E F and DP spermatozoa and incubated under capacitating conditions were exposed to calcium ionophore A23187, no acrosome reactions could be induced. This suggests that for the occurrence of acrosome reaction the spermatozoa must reach the capacitation stage first and then only they can react to ionophore. Exposure of spermatozoa to ionophore A23187 also caused a rapid decline in motility depending on the dose of the ionophore. Sperm motility was reduced to <20%. A similar effect has been reported for human spermatozoa [Byrd & Wolf, 19861; however, the motility decline was reported to be more severe. It has been reported that motility fades within 2 h r after the occurrence of spontaneous AR in eutherian mammals [Fleming & Yanagimachi, 19811. The results of the present study and that of Byrd & Wolf [ 19861, however, suggest that ionophore-induced AR caused a faster decline in motility. CONCLUSIONS 1. Squirrel monkey sperm, subjected to the CTC assay, showed a finite number of fluorescence patterns over a timecourse where capacitation and the acrosome reaction occur, similar to the patterns in human sperm. 2. Increasing the concentration of bovine serum albumin concentration in the medium hastened capacitation. 3. Chemical induction of the acrosome reaction did not occur in calcium-poor medium. Conversely, increasing the calcium concentration significantly increased the chemical induction of the acrosome reaction. ACKNOWLEDGMENTS Thanks are due to Dr. Karen Chou and Mr. Mike Oswalt for their suggestions in the CTC fluorescence assay and to Ms. L.M. 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