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a Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, sperm capacitation/acrosome reaction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanisms that render sperm acrosomally responsive are not well understood. Events that have been implicated in this process include loss of sperm sterols [4], membrane hyperpolarization [5], protein phosphorylation [6–12], increased intracellular pH [13–16], loss of molecules from the cell surface [17], and elevated intracellular free-calcium concentration [18, 19].
Of the events detected in sperm as they capacitate, the loss of sterols is one of the few that are known to be obligatory. Sterol loss begins when sperm are transferred from seminal plasma to incubation medium [20], and it is required for subsequent increases in intracellular pH [16] and protein tyrosine phosphorylation [21]. Sterols exit the sperm and bind to sterol-accepting components in the medium [22]. Efflux is believed to be driven by the relative chemical activities of the sterols in the cell versus the medium, and perhaps is accelerated by lipid transfer activity in the medium [23]. There may be unrelated obligatory processes, but a reasonable hypothesis based on present information is that sterol loss represents the initial event of capacitation and either directly or indirectly triggers the events that follow.
In this light, it is important to know the intracellular environment of sperm sterols and the factors that control their distribution and efflux. The major sterol in the sperm of most species is cholesterol. Other sterols, particularly desmosterol, are also present [4]. Both cholesterol and desmosterol inhibit capacitation [24]. In sperm, as in somatic cells, cholesterol is abundant in the plasma membrane [25–27]. The phospholipid sphingomyelin is also abundant in the sperm plasma membrane [25–27], and its affinity for cholesterol may have important ramifications for sperm function. The importance of sphingomyelin to cholesterol homeostasis in somatic cells has been demonstrated by removing sphingomyelin from the plasma membrane with exogenous sphingomyelinase (SMase). This increases esterification of cholesterol in the endoplasmic reticulum [28–30], synthesis of sterols [29], and efflux of cholesterol to extracellular acceptors [31, 32]. In addition, association of cholesterol with sphingolipids may produce low-density lipid "rafts" in the plasma membrane that alter the lateral distribution of proteins [33].
The aim of the present study was to determine the role of sphingomyelin in sperm capacitation by assessing the effect of exogenous SMase on the development of acrosomal responsiveness.
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MATERIALS AND METHODS |
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The following chemicals were used: methanol and chloroform (EM Science, Gibbstown, NJ), BSA (Pentex Bovine Albumin, Fraction V, Reagent Grade, catalog no. 81-066-7, lot 46; Miles, Inc., Kankakee, IL), Pisum sativum agglutinin (Vector Laboratories, Burlingame, CA), and phosphatidylcholine-specific phospholipase C (Roche Molecular Biochemicals, Indianapolis, IN). Ceramide analogues were obtained from Biomol (Plymouth Meeting, PA). Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). Thin-layer chromatographic plates were purchased from Analtech (Newark, DE). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Sperm
Except when noted otherwise, human sperm were washed, incubated in vitro, and exposed to P4 as described elsewhere [34, 35]. Informed consent was obtained from the semen donors, and the institutional review board approved this investigation. Briefly, semen were obtained by masturbation, and motile sperm were selected by centrifugation through a Percoll gradient, washed, and suspended in a solution modified from that described by Suarez et al. [36]: 117.6 mM NaCl, 0.36 mM NaH2PO4, 8.6 mM KCl, 2.4 mM CaCl2, 0.49 mM MgSO4·7H2O, 25 mM NaHCO3, 2 mM glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 0.05 mg/ml streptomycin sulfate, and 0.075 mg/ml penicillin. The solution also contained BSA at either 3 or 26 mg/ml (26B medium). In some experiments, the medium was supplemented with cholesterol as described elsewhere [20].
When sperm were to be exposed to SMase or phospholipase C, the solutions for the Percoll gradient, washing, and incubation with the enzyme did not contain BSA (to prevent the enzyme possibly releasing hydrolysis products from lipid bound to BSA) but contained 40 mM HEPES, replacing an equal amount of NaCl (H-medium). Aliquots (0.5 ml) of sperm suspensions (107 sperm/ml in H-medium) containing 0.1, 0.2, or 0.3 U of Bacillus cereus SMase per milliliter were incubated at 37°C in an air atmosphere, with agitation at 10-min intervals. In other experiments, sperm were similarly treated with 2 U phospholipase C per milliliter. After 30 min, sperm were washed by layering them over 1 ml of 26B medium and centrifuging (800 x g, 5 min). The supernatant was aspirated, the pellet was collected in 100 µl, and 40 µl were combined with 1.16 ml of 26B medium, resulting in a suspension of approximately 1 x 106 sperm/ml. Aliquots of 75 µl were dispersed to the wells of a 96-well plate (catalog no. 25880-96; Corning Glass Works, Corning, NY) and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air.
The possibility that SMase contained contaminating protease activity was assessed using a fluorescent casein substrate (EnzChek-green protease assay kit; Molecular Probes, Eugene, OR).
Sperm viability and acrosomal status were assessed as previously elsewhere [35]. Briefly, sperm were incubated with Hoechst 33258 (H258, 0.5 µg/ml, 10 min) to label dead cells and then fixed and permeabilized in 100% (v/v) ethanol. The acrosomal contents were labeled with fluoresceinated P. sativum agglutinin, and the sperm were examined by fluorescence microscopy. One hundred sperm were inspected in duplicate or triplicate samples. Spontaneously reacted sperm were defined as H258-negative, acrosome-reacted sperm in suspensions that had not been exposed to P4. P4-Responsive sperm were defined as the number of H258-negative, reacted sperm following exposure to P4 (10 min, 1 µg/ml) minus the number of spontaneously reacted sperm in matched aliquots of the same sperm suspension.
Assaying Sperm Cholesterol, Desmosterol, and Sphingomyelin Content
To extract lipids, sperm were washed by centrifuging (800 x g, 5 min), suspending in 5 ml of PBS (138 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 1.0 mM CaCl2, and 0.5 mM MgSO4), centrifuging again (800 x g, 10 min), and then suspending to approximately 1.1 ml with PBS. The sperm concentration was determined using a hemacytometer. Aliquots of sperm suspension (1.0 ml each) were overlaid with nitrogen gas and frozen (-20°C) for as long as 3 days. To each frozen aliquot was added 1.5 µg of the internal standard stigmastadienone (from a solution of 100 µg/ml in chloroform) and 3.75 ml of a mixture of 1:2 (v:v) chloroform/methanol [37]. After 1 h at room temperature, the insoluble material was sedimented (1000 x g, 10 min), and the supernatant was partitioned by sequentially adding, with mixing, 0.26 ml each of chloroform and water for each 1 ml of supernatant. Following centrifugation (1000 x g, 10 min), the lower phase was recovered, mixed with an equal volume of benzene, evaporated to dryness at approximately 37°C under flowing N2, and dissolved in chloroform. To measure the content of cholesterol and desmosterol, lipid extracts were analyzed using a Tracor 565 gas chromatograph fitted with a DB-17 column (inner diameter, 0.53 µm; length, 30 m; J & W, Folsom, CA) and using helium as the carrier. The inlet was maintained at 300°C, the column at 260°C, and the flame ionization detector at 250°C. Peak areas were measured with a Spectra-Physics SP4290 Integrator (Spectra-Physics, Mountain View, CA) and converted to mass using standard curves prepared from mixtures of known amounts of stigmastadienone, cholesterol, and desmosterol. This protocol assays unesterified cholesterol and desmosterol. Except when noted otherwise, all statements concerning those sterols refer to the unesterified forms.
To assay the sphingomyelin content, lipid extracts were spotted onto thin-layer chromatographic plates (0.25-mm silica gel H, 20 x 20 cm) and developed in a mixture of chloroform/methanol/acetic acid/water (25:15:4:2, v:v:v:v; [38]) until the solvent reached within 1 cm of the top of the plate. Adjacent lanes contained pure phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. The plate was allowed to dry and then stained with iodine vapor to reveal the location of the sphingomyelin. These regions were scraped from the plate. Sphingomyelin was eluted from the silica gel [38], and phosphate was assayed [39]. Sperm-free blanks were carried through the same procedure.
Exposing Sperm to Ceramide Analogues
Ceramide analogues were dissolved at 50 mM in anhydrous dimethylsulfoxide at 37°C and stored at -20°C. Just before use, the solution was warmed to 37°C and diluted to its final concentration in medium containing BSA at 3 mg/ml while vortexing, and sperm were added to a final concentration of 1 x 106 sperm/ml. All sperm suspensions contained 0.1% (v/v) dimethylsulfoxide. After 8 and 24 h, sperm viability and acrosomal status were determined as described above. The BSA concentration was 3 rather than 26 mg/ml to reduce the amount of nonspecific extracellular binding of the ceramide analogue.
Statistics
Percentage data were transformed before analysis (arcsine [%/100]
). Zero values were changed to 1 x 10-7 before transformation. Data were analyzed by ANOVA using a randomized block design, with the experiment date used as the block and P < 0.05 indicating significance. Post hoc multiple comparisons were made using the method of Bonferroni. Systat (Systat, Inc., Evanston, IL) and InStat (GraphPad Software, San Diego, CA) software were used for the analyses.
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RESULTS |
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Sphingomyelinase accelerated the development of acrosomal responsiveness (Fig. 1A). Concentrations of SMase from 0.1 to 0.3 U/ml had similar effects on sperm, so the results using all concentrations of SMase were pooled. In control sperm suspensions, the maximum number of P4-responsive sperm was reached at 18 h. (In Figure 1A, the incidence of responsive sperm appears to be rising at later times, but the apparent further increase at 24 h was not significant [P = 0.2]. Previous work [40] has shown that extending incubation for as long as 48 h does not increase the incidence of responsive sperm.) Sperm treated with SMase reached maximum P4-responsiveness much earlier (8 h), and responsiveness did not change significantly during the ensuing 16 h. The number of SMase-treated sperm responding to P4 at 8 h was not significantly different from the number of control sperm responding to P4 at 24 h (P = 0.08). The accelerating effect of SMase was also apparent when comparing the times at which the half-maximal incidences of P4-responsive sperm were reached: 4 h for SMase-treated sperm, and 10 h for control sperm (Fig. 1).
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Sphingomyelinase also greatly increased spontaneous acrosome reactions and cell death. In control sperm suspensions, H258-negative, spontaneously reacted sperm appeared gradually during incubation (Fig. 1), representing approximately 5% of sperm after 24 h. In contrast, 18% of the SMase-treated sperm were spontaneously reacted after 24 h. In addition, SMase caused many sperm to die at the later time points (Fig. 1C). Whereas control sperm suspensions had only 13% dead at 24 h, 40% of the SMase-treated sperm were dead at this time.
The specificity of SMase was tested in three ways. First, sperm were exposed to SMase that had been immersed for 10 min in a boiling water bath; the denatured enzyme did not accelerate capacitation (data not shown). Second, SMase was tested for contaminating protease activity using a fluorescent casein substrate. When assayed according to the manufacturer's directions (10 mM Tris buffer [pH 7.8], room temperature), protease activity was detected at a level equivalent to 4.6 U of trypsin per 1000 U of SMase. The activity was inhibited by a mixture of protease inhibitors (0.01 µM benzamidine, 21 µM leupeptin, and 1.5 µM aprotinin). However, when SMase was assayed for protease activity under the conditions employed with sperm (H-medium [pH 7.4], 37°C), no protease activity was detected. Furthermore, the protease inhibitors described above did not alter the effect of SMase on acrosomal function or cell viability (assayed at 8 h of incubation; data not shown). If SMase contains protease activity, it is not responsible for the effects on sperm reported here. Finally, sperm were exposed to phosphatidylcholine-specific phospholipase C in place of SMase. No effects on cell viability or acrosomal function were noted at 8 or 24 h, except for a very small but statistically significant increase in spontaneously reacted sperm at 24 h (from 1% [0% to 3%] to 3% [2% to 5%], mean [95% confidence limits], n = 3).
To determine if the effects of SMase resulted from alterations in the content of sperm sterols, the amounts of cholesterol and desmosterol in sperm were measured during the first 8 h of incubation, the time at which the effect on P4-responsiveness is most obvious. (Sterols were not measured at later times, because significant loss of sterol would be expected as the sperm lose plasma membrane during spontaneous acrosome reactions and, perhaps, on cell death.) Four hours after exposure to SMase, sperm contained significantly less cholesterol and desmosterol compared with control sperm (Fig. 2). At 8 h, SMase-treated sperm had less desmosterol than control sperm, but their cholesterol content was not significantly different from that of control sperm. The sum of the two sterols was less in SMase-treated sperm at both 4 and 8 h. Compared with control sperm, the sterol loss in SMase-treated sperm was accelerated by approximately 2 h.
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Loss of sperm cholesterol can be inhibited by adding cholesterol to the incubation medium, thereby preventing sperm from becoming acrosomally responsive [20]. The effect on control sperm is illustrated in Figure 3C, in which exogenous cholesterol reduced the incidence of P4-responsiveness by approximately 90% in 24-h sperm. The EC50 was 0.96 µM. At 24 h, the SMase-treated sperm contained approximately 10% P4-responsive sperm, and the size of this population was little affected by exogenous cholesterol, even in amounts as high as 8 µM.
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Among SMase-treated sperm at 8 h, cholesterol maximally reduced P4-responsiveness by 47%; the EC50 was 1.2 µM (Fig. 3B). Only a few control sperm were P4-responsive at 8 h, but their appearance was inhibited 71% by exogenous cholesterol, with an EC50 of 0.5 µM (Fig. 3A).
Exogenous cholesterol inhibited cell death and spontaneous acrosome reactions, except in 8-h control sperm suspensions, in which these populations were very small even in the absence of exogenous cholesterol.
Sphingomyelinase catalyzes the hydrolysis of sphingomyelin to phosphorylcholine and ceramide. Because ceramides have been suggested to be intracellular signal mediators [41], the possibility that SMase-generated ceramide stimulated acrosomal responsiveness was examined. Sperm were treated with N-hexanoylsphingosine (C6-ceramide), a short-chain, cell-permeable analogue that mimics the effects of endogenous ceramide (see Discussion), or with the inactive negative control N-hexanoylsphinganine (C6dH).
C6-Ceramide caused 10% of the sperm to be P4-responsive at 8 h (Table 1). At 24 h, sperm were not responsive to P4, but 22% of the sperm were spontaneously acrosome-reacted. C6-Ceramide caused significant cell death only after 24 h. The inactive ceramide analogue, C6dH, had no effect on acrosomal function or sperm viability. Increasing the concentration of C6-ceramide to 50 µM caused 84% to 100% of the sperm to be dead at 8 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Under the conditions described here, control sperm require 18–24 h to reach maximum acrosomal responsiveness [40]. During the 24 h after that, the incidence of P4-responsive sperm gradually decreases, as the numbers of spontaneously reacted and dead sperm increase. Exogenous SMase accelerated this sequence, causing the maximum number of P4-responsive sperm to appear by 8 h and substantial numbers of dead and spontaneously reacted sperm at 24 h. No effect was seen when using boiled enzyme or phosphatidylcholine-specific phospholipase C.
Two possible explanations for the effects of SMase were investigated. Because loss of the inhibitory sperm sterols is a critical event for capacitation in vitro, the possibility that sphingomyelin controls the rate of sterol loss was examined. We found that SMase accelerated sterol loss during the first 8 h of incubation. In somatic cells, exogenous SMase causes loss of free sterols by increasing their efflux to extracellular acceptors and by increasing the rate of esterification (see Introduction). Whether the accelerated loss of free sterol reported here results from increased efflux or modification of the molecule, however, has not been determined. Regardless of these details, the results support a role for sphingomyelin in determining the distribution of sterols in sperm.
The accelerating effect of SMase on sterol loss was small (
2 h) compared with its effect on sperm capacitation (6 to 10 h), raising the possibility that SMase affects capacitation via additional pathways. Support for this view came from efforts to inhibit capacitation by adding cholesterol to the medium. Supplementing the medium with 1 µM cholesterol normally inhibits capacitation by preventing a net loss of sperm cholesterol [20]. Among SMase-treated sperm, however, as much as 8 µM exogenous cholesterol inhibited capacitation only by approximately 50% at 8 h, and it did not inhibit capacitation at 24 h. These results suggest that augmented sterol loss does not account for all the action of SMase.
The products of sphingomyelin degradation, including ceramide and sphingosine-1-phosphate, trigger cell type-specific responses of proliferation, differentiation, or apoptosis [41]. A ceramide-dependent protein phosphatase [42] and a ceramide-dependent protein kinase [43] have been offered as candidate intermediates in these signaling pathways. The present study examined the possibility that ceramide contributes to the sterol-independent action of SMase. Short-chain, cell-permeable ceramide analogues have been reported to stimulate protein phosphatase 2A, activate mitogen-activated protein kinase, induce apoptosis, and suppress insulin-induced tyrosine phosphorylation [44–48]. C6dH lacks C6-ceramide's activity and, therefore, serves as a negative control [48]. To reduce sequestration by BSA in the medium, BSA was reduced from 26 to 3 mg/ml for these experiments. Human sperm do not normally capacitate in this medium (Table 1), perhaps because the low concentration of a sterol acceptor in the medium reduces the net efflux of sperm sterols. Nevertheless, 25 µM C6-ceramide induced a significant number of sperm to become capacitated at 8 h; C6dH was without effect. By 24 h, the capacitated sperm had died, spontaneously reacted, or regressed to an uncapacitated state.
Two ideas have been offered to explain how ceramides trigger downstream events [49]. First, ceramide may serve as a lipid anchor that causes proteins to attach to membranes. Second, ceramides may cause disorder in the cell membrane bilayer. Generation of ceramides increases the tendency of the lipid bilayer to form a hexagonal II phase [49, 50]. The short-chain ceramide analogue N-acetylsphingosine increases membrane fluidity, as measured by fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene [51]. These structural changes are similar, in some respects, to the lipid disorder and fluidity that are predicted to accompany loss of sterol during sperm capacitation [52]. In cholesterol-poor bilayers, the hydrocarbon chains of phospholipids have greater mobility and lower lateral packing density than in cholesterol-rich bilayers [53]. Evidence of increased lipid disorder in boar sperm membranes under capacitating conditions has been reported [54]. Therefore, ceramides may mimic the loss of sperm sterol from the membrane bilayer by increasing phospholipid disorder.
The present study demonstrates a role for sphingomyelin in determining the distribution of sterols in sperm. Does sphingomyelin hydrolysis, however, play a role in capacitation? This possibility deserves more attention, but the present evidence does not make a compelling case for it. A neutral SMase has been reported in ram sperm plasma membranes [55], but capacitation does not decrease the sphingomyelin content of guinea pig or boar sperm [56–58].
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FOOTNOTES |
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1 Supported by NIH grant HD30763. 
2 Correspondence. FAX: 405 744 5275; ncross{at}okstate.edu
Accepted: May 23, 2000.
Received: February 21, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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