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Biol Reprod 2003, 10.1095/biolreprod.102.013052
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BIOLOGY OF REPRODUCTION 69, 529–534 (2003)
DOI: 10.1095/biolreprod.102.013052
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Decrease in Order of Human Sperm Lipids During Capacitation1

Nicholas L. Cross2

Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma 74078


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of the sperm sterols, cholesterol and desmosterol, is an obligatory step in the capacitation of human sperm. Because sterols can increase the order of membrane phospholipids, it has been suggested that the importance of sterol loss is that it decreases membrane lipid order. The present study tested the hypotheses that sterol loss decreases sperm membrane lipid order during capacitation and that lipid disorder is a sufficient stimulus for capacitation. Steady-state fluorescence anisotropy of the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, decreased during capacitation, indicating a decrease in lipid order. The decrease was dependent on the loss of sperm sterols, suggesting that it reflected diminished sterol-mediated phospholipid ordering. However, the lipid-fluidizing agents, benzyl alcohol and 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, did not cause sperm capacitation or overcome inhibition by cholesterol. In summary, loss of sperm sterols caused a significant decline in lipid order during capacitation; however, decreased bulk lipid order was not sufficient to trigger the subsequent events that complete capacitation.

acrosome reaction, gamete biology, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Freshly ejaculated mammalian sperm are not immediately capable of undergoing an acrosome reaction and fertilizing an egg (for review, see [1]). They require a period of several hours in the female reproductive tract or in suitable media in vitro to acquire this ability. The changes that occur during this period are collectively termed capacitation. Events that have been implicated in capacitation include loss of sperm sterols (for review, see [2]), altered distribution of phospholipids in the plasma membrane (for review, see [3]), loss of molecules from the cell surface (for review, see [4]), membrane hyperpolarization [5], production of reactive oxygen species [6], elevated concentrations of calcium and cAMP [6–8], protein phosphorylation [9–14], and increased intracellular pH [15–19].

Loss of sperm sterols begins soon after sperm are removed from seminal plasma and is obligatory for capacitation of human sperm [20]. Incubation with exogenous sterols to maintain a high level in sperm inhibits progesterone (P4)- and calcium ionophore-induced acrosome reactions of human sperm [21], the zona pellucida-induced acrosome reaction of mouse sperm [22], and the fertilization of rat, mouse, and rabbit eggs [23–25].

Sperm lose sterols more rapidly than they become acrosomally responsive [20], suggesting that sterol loss is an early event in the capacitation process. In fact, sterol loss has been positioned upstream from the rise in intracellular pH [19] that is required for acrosomal responsiveness [16, 18]. Sterol loss is also upstream from tyrosine phosphorylation of a set of sperm proteins [22].

How sterols inhibit capacitation is unknown. In freshly ejaculated sperm, ß-OH sterols are abundant in the sperm plasma membrane [26, 27], and most models propose that they act there. It has long been suggested that the critical event upon sterol loss is an increase in phospholipid fluidity or bilayer permeability [28]. This model was developed from well-studied interactions of cholesterol with phospholipids in model systems. Cholesterol orients perpendicularly to the plane of a bilayer and, above the phospholipid transition temperature, increases the order and decreases the rate of motion of phospholipid acyl chains. As a result, cholesterol reduces the average molecular surface area, increases the bilayer thickness, and reduces the bilayer permeability (for review, see [29]). Cholesterol's ability to order saturated phospholipids also contributes to the formation of lipid rafts, which are sterol-rich regions in membranes that have distinctive protein compositions and that may modify signaling pathways [30, 31]. Phospholipid order also directly affects the activities of some membrane proteins [32 and references therein]. We recently showed that the essential structural feature that is required for sterols to inhibit capacitation is planarity of the fused ring structure [33]. Planarity of sterols is required to create phospholipid order (for review, see [34]), so the present experiments tested the hypotheses that loss of sperm sterols during capacitation causes a decrease in lipid order and that the loss of order plays an essential role in capacitation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

The following chemicals were used: methanol and chloroform (EM Science, Gibbstown, NJ), hexane and ethanol (Pharma Products, Brookfield, CT), BSA (Pentex Bovine Albumin, Fraction V, Reagent Grade, catalog number 81-066-7, lot 46; Miles, Inc., Kankakee, IL), Pisum sativum agglutinin (Vector Laboratories, Burlingame, CA), and 1,6-diphenyl-1,3,5-hexatriene (DPH; Molecular Probes, Inc., Eugene, OR). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Sperm Preparation

Except where noted otherwise, human sperm were treated as previously described [35, 36]. Informed consent was obtained from the semen donors, and an institutional review board approved this investigation. Briefly, semen was obtained by masturbation, and motile sperm were selected by centrifugation through a Percoll gradient, washed, and suspended in a medium modified from that described by Suarez et al. [37]: 117.6 mM NaCl, 0.36 mM NaH2PO4, 8.6 mM KCl, 2.4 mM CaCl2, 0.49 mM MgSO4·7H20, 25 mM NaHCO3, 2 mM glucose, 0.25 mM sodium pyruvate, 19 mM sodium lactate, 0.05 mg/ml of streptomycin sulfate, 0.075 mg/ml of penicillin, and 26 mg/ml of BSA. Where indicated, medium was used without BSA (0-BSA medium) or as Hepes/0-BSA medium (0-BSA medium in which 27 mM NaCl and 13 mM NaHCO3 were replaced by 40 mM Na Hepes [pH 7.4]). Sperm were incubated at a concentration of 2 x 106 sperm/ml at 37°C in a chamber containing a humidified atmosphere of 5% CO2/95% (v/v) air. In some experiments, sperm were incubated in medium containing 3.75 µM cholesterol, prepared by injecting an ethanolic solution of cholesterol (7.5 mM) into a 1000-fold volume of incubation medium while vortexing. The solution was agitated at room temperature for 15–60 min, passed through a filter (pore size, 0.22 µm), and then combined with an equal volume of sperm suspension. In these experiments, controls demonstrated that the solvent (0.05% [v/v] ethanol) did not affect sperm viability, acrosomal responsiveness, anisotropy, or sterol content (data not shown).

Capacitation cannot be assessed by fertilization of human eggs, so the ability of sperm to acrosome-react when exposed to P4 was employed. Responsiveness to P4 develops with time of incubation (see below and [20]), and in mouse sperm, it correlates with capacitation [38]. Sperm viability and acrosomal status were assessed as previously described [36]. Briefly, sperm were incubated with Hoechst 33258 (H258; 0.5 µg/ml, 10 min) to label dead cells, then fixed and permeabilized in 95% (v/v) ethanol. The acrosomal contents were labeled with fluoresceinated P. sativum agglutinin, and the sperm were examined by fluorescence microscopy. Spontaneously reacted sperm were defined as H258-negative, acrosome-reacted sperm in suspensions that had not been exposed to P4. The P4-responsive sperm were defined as the number of H258-negative, acrosome-reacted sperm following exposure to P4 (10 min, 1 µg/ml), corrected for the number of spontaneously reacted sperm in matched aliquots of the same sperm suspension.

To expose sperm to the fluidizing agent, benzyl alcohol, 0-BSA medium was used to avoid sequestration of this hydrophobic molecule. Sperm stick avidly to glass and plastic in the absence of protein, so before use, containers were blocked overnight at 4°C with 20 mg/ml of BSA in PBS, then rinsed three times with water and dried. Sperm were exposed to benzyl alcohol in two ways. In some experiments, freshly collected sperm were treated with 0, 10, 25, or 50 mM benzyl alcohol for as long as 7 h to see if it induced capacitation. Sperm were assessed for viability (H258 labeling), motility, and responsiveness to P4 as a measure of capacitation. In other experiments, sperm that had been incubated for 24 h in the absence or presence of 3.75 µM cholesterol in regular BSA-containing incubation medium were exposed to benzyl alcohol. At 24 h, the sperm were washed to remove BSA and cholesterol by centrifugation (10 min, 800 x g) through a 0.75-ml layer of 45% (v/v) Percoll/55% (v/v) Hepes/0-BSA medium. The pelleted sperm were suspended in 7 ml of Hepes/0-BSA medium, centrifuged again, and then suspended in Hepes/0-BSA medium to approximately 2 x 106 sperm/ml. The sperm were returned to 37°C (air atmosphere) for 10 min, and then 25 mM benzyl alcohol in Hepes/0-BSA medium was added. After an additional 10 min, the response of the sperm to P4 was determined as described above but in the absence of BSA.

Fluorescence Anisotropy

Bovine serum albumin produces interfering fluorescence, so it was removed by centrifuging sperm through Percoll as described above and suspending them in Hepes/0-BSA medium to approximately 10 million sperm/ml. Sperm were exposed to 0.25 µM dye as described by Plasek and Jarolim [39]. A stock solution of 0.2 mM DPH in acetone was prepared and stored in the dark at -20°C. On the day of use, DPH was diluted to 1.0 µM in water while vortexing. Acetone was removed by heating to 65°C for 7 min with stirring. The solution was then mixed with an equal volume of twice-concentrated Hepes/0-BSA medium (pH 7.4). Finally, it was combined with an equal volume of sperm suspension. After 20 min at 37°C, the fluorescence anisotropy was measured. Low ambient lighting was used whenever DPH was exposed, because it is extremely photolabile.

Steady-state anisotropy was measured using a PTI Quantamaster spectrofluorimeter (Lawrenceville, NJ) in L-configuration (excitation wavelength, 358 nm; emission wavelength, 427 nm; slit widths, 3 nm). A 400-nm long-pass filter in the emission path blocked scattered light, and a 12% transmission neutral-density filter was placed in the exciting path to eliminate photobleaching. The solution was magnetically stirred and maintained at 37.0 ± 0.1°C and monitored continuously with a Teflon-coated thermistor. Fluorescence was recorded for 2–4 sec, with an integration time of 0.25 sec. Each treatment group included two or three samples of DPH-labeled sperm and one or two samples of sperm without DPH. The fluorescence of labeled sperm was approximately 10-fold greater than light scattering from unlabeled sperm, which was subtracted. The steady-state anisotropy, r, was calculated from

where IVV and IVH are the fluorescence emission intensities measured with the polarizer polarization-axis vertical and the analyzer polarization-axis vertical or horizontal, respectively. The value G was determined in separate experiments and corrects for unequal response of the instrument to vertical and horizontal polarized light; it is G = IHV/IHH, where IHV and IHH are the fluorescence emission intensities measured with the polarizer polarization-axis horizontal and the analyzer polarization-axis vertical or horizontal, respectively.

Sterol Assays

Sperm sterol content was determined either immediately after motile sperm were prepared from semen or after 6 or 24 h of incubation. Sperm were collected by centrifugation (10 min, 800 x g), washed in 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), and suspended in PBS. The sperm concentration after washing was determined with a fluorescent DNA-binding dye, CyQuant (Molecular Probes, Eugene, OR), according to the manufacturer's directions. To convert fluorescence units to sperm/ml, the sperm concentrations in two samples were determined in a hemocytometer, and the ratio of fluorescence to sperm concentration was calculated.

To assay sterol content, {alpha}-cholestane was added to each tube of washed sperm as an internal standard, and lipids were extracted with chloroform and methanol as previously described [33]. The extracted material was dissolved in hexane and analyzed by gas chromatography using a Perkin-Elmer Autosystem XL with Turbochrom 4.1 (Perkin-Elmer, Norwalk, CT) for control and analysis and a DB-17 column (inner diameter, 0.53 mm; length, 30 m; J & W Scientific, Folsom, CA). The carrier was helium (18 ml/min), and the flame ionization detector was supplied with hydrogen (45 ml/min) and air (450 ml/min). Preliminary experiments determined that the ratios of sterols to {alpha}-cholestane were not altered by the extraction procedure. To assure that the washing procedure removed soluble sterol from the sperm suspension, blank samples were prepared with sterol in incubation medium but lacking sperm. This protocol assays free, unesterified sterols; in the present study, the term sterol means the unesterified form.

Statistics

Means were compared by analysis of variance with Bonferroni posttests using InStat (GraphPad, Inc., San Diego, CA) or Tukey posttests using Systat (Systat, Inc., Evanston, IL), with P < 0.05 indicating significance. Percentage data were transformed before analysis (arcsin [%/100]1/2). The value of 1 x 10-7 was substituted for values of zero, and 99.99 was substituted for 100. Where appropriate, repeated-measures tests were used to accommodate variations among ejaculates. The time course of the effect of benzyl alcohol on sperm fluorescence anisotropy was analyzed with GraphPad Prism (GraphPad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fluorescence Anisotropy

Preliminary experiments determined that r was not affected by varying DPH from 0.125 to 0.25 µM, varying the sperm concentration from 2.5 million to 7.5 million/ml, or varying the time in DPH from 10 to 35 min (data not shown). The effect of the measurement protocol (washing, exposure to dye, stirring, and illumination) on sperm was minor. Sperm were inspected for viability (staining with H258) and the incidence of live, acrosome-reacted sperm. For uncapacitated sperm, the incidence of dead sperm increased from 0.5% ± 0.3% (mean ± SEM, n = 6) to 4% ± 1% (P < 0.05) during measurement. No change was observed in the incidence of dead capacitated sperm or the incidence of acrosome-reacted sperm in either uncapacitated or capacitated groups (data not shown).

When sperm were incubated for 6 or 24 h under capacitating conditions (Fig. 1A), r decreased significantly, indicating diminished lipid order (Table 1). The decrease in r was prevented when sperm were incubated for 24 h with 3.75 µM cholesterol (Table 1), a treatment that maintains elevated sperm cholesterol and prevents capacitation (Fig. 1B) [20, 33].



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  		FIG. 1. A) Changes in the incidence of dead (D), spontaneously acrosome-reacted (S), and capacitated sperm (P; those that acrosome-react when exposed to P4). Sperm were assayed shortly after ejaculation (T0) and after incubation in vitro for 6 h (T6), 24 h (T24), or 24 h with 3.75 µM cholesterol (T24+Ch). Data are shown as the mean ± SEM (n = 5–8). B) Cholesterol (Ch) and desmosterol (De) content of sperm during incubation in vitro. Data are shown as the mean ± SEM (n = 4–7)


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  		TABLE 1. Anisotropy of DPH-labeled sperm.a

The incidence of dead sperm and of spontaneously reacted sperm increased slightly during the 24-h incubation for capacitation (Fig. 1A). To determine if those changes might account for the decrease in r at 24 h, fluorescence anisotropy was measured in suspensions of sperm that were frozen at -20°C to kill sperm and cause acrosomal loss. The sperm were 100% dead (H258-positive), and 58% ± 7% (mean ± SEM, n = 3) had disrupted acrosomes, as assessed by P. sativum agglutinin labeling. Their value of r was not significantly different from that of living sperm (not shown), indicating that sperm death or acrosome loss was not the cause of the capacitation-associated decrease in r at 24 h. Consistent with this conclusion, r was decreased at 6 h, before the numbers of dead or spontaneously reacted sperm increased significantly (Fig. 1A and Table 1).

Fluidizing Agents

To test whether lipid fluidizing agents could bypass the requirement for loss of sperm sterols, sperm were incubated in 0-BSA medium with a well-studied fluidizing agent, benzyl alcohol (e.g., [40]). Bovine serum albumin was omitted to prevent sequestration of the hydrophobic benzyl alcohol and minimize loss of sperm sterols. Benzyl alcohol caused a dose-dependent decrease in r (Fig. 2). Interpolation of the results indicated that a decrease in r equivalent to that observed during capacitation would occur at approximately 20 mM benzyl alcohol. When sperm were continuously exposed to 25 mM benzyl alcohol, r decreased within a few minutes and remained low during the 65-min period of measurement (Fig. 3).



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  		FIG. 2. Effect of benzyl alcohol on the anisotropy, r, of DPH in sperm membranes. Sperm were incubated for 25 min at 37°C with benzyl alcohol, then DPH was added and anisotropy determined 15–30 min later. Data are shown as the mean ± SEM (n = 3). Values with similar letters are not significantly different



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  		FIG. 3. Effect of benzyl alcohol on the anisotropy, r, of DPH in uncapacitated sperm. Anisotropy was measured (0 min), and then 25 mM benzyl alcohol was added to one group (open circles). Data are shown as the mean ± SEM (n = 3). When curves were fit by linear regression over the period of 10–65 min, the slopes were not significantly different from zero for either group. The elevations (anisotropy) of the control and treated curves were significantly different (P < 0.05)

In one set of experiments, freshly prepared sperm were incubated for 1 or 7 h with benzyl alcohol at 10, 25, or 50 mM, concentrations covering the range from no apparent affect on sperm to deleterious effects on motility and viability (Table 2). Nevertheless, benzyl alcohol did not induce acrosome reactions or make sperm responsive to P4 (Table 2). In another experiment, sperm were incubated for 24 h with 3.75 µM cholesterol, washed free of cholesterol and BSA, and then exposed to 25 mM benzyl alcohol. After 10 min, the response to P4 was assessed; benzyl alcohol did not render the cholesterol-inhibited sperm responsive to P4 (Table 3). The fluidizing-agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate (A2C; 10 or 20 µM) also failed to promote capacitation (data not shown).


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  		TABLE 2. Effect of benzyl alcohol on sperm


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  		TABLE 3. Effect of benzyl alcohol on the ability of cholesterol-inhibited sperm to acrosome-react in response to P4


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present experiments determined whether lipid order changes during capacitation and whether a decrease in lipid order can induce capacitation. The experiments employed steady-state fluorescence anisotropy, a technique that assesses the range of motion of a membrane-associated fluorescent probe during its fluorescence lifetime (<10 nsec [41]). Fluorescence anisotropy reflects both the average orientation of the fatty acyl chains (i.e., their tendency to orient perpendicular to the plane of the bilayer) and the rate of their motion during the excited state of the probe [42]. In a general sense, lipid order measures lipid "fluidity," but it should be distinguished from other uses of the term. Acyl chain order is not a measure of lateral diffusion (e.g., as measured by recovery of fluorescence after photobleaching), although in some cases, acyl chain order may affect it. Changes in the lateral diffusion of proteins during capacitation have been reported [43], but because protein diffusion is subject to other controls (e.g., diffusion barriers and interactions with the cytoskeleton), the involvement of lipid order is unknown. Lateral diffusion of lipids does not change during capacitation of hamster and mouse sperm [44, 45].

Fluorescence anisotropy decreased during incubation under capacitating conditions. As previously reported, sperm start to become acrosomally responsive at approximately 6 h and are maximally responsive at approximately 24 h (Fig. 1A) [20, 21]. The loss of sperm sterols proceeds more rapidly, and in these experiments, it was almost complete by 6 h (Fig. 1B) [20]. Anisotropy was significantly decreased at 6 h, raising the possibility that it was related to the loss of sperm sterols. To test this idea, sperm were incubated for 24 h in cholesterol-enriched medium. Sperm cholesterol was elevated compared to 24-h control sperm (Fig. 1B), and the decrease in r was prevented. It is therefore likely that the decrease in r during capacitation is caused, either directly or indirectly, by the loss of sperm sterol. The simplest explanation is that the decrease in r results from the physical effect of sterols leaving sperm membranes, causing membrane lipids to become less ordered. Cholesterol and desmosterol are equally effective at inhibiting capacitation and have approximately the same ability to confer order on egg phosphatidylcholine [33]. The ordering effect of sterols is not the same for all phospholipids [31], however, so it is presently unclear whether cholesterol and desmosterol have the same ability to order sperm phospholipids.

A previous attempt to detect a change in the anisotropy of DPH during capacitation of human sperm found none [46], perhaps because of the short incubation time (3 h). On the other hand, using the spin-label 16-doxyl stearate, a decrease was observed in rotational correlation time during capacitation of human sperm, but no change was observed in the structural order parameter or lateral diffusion of the probe [47]. Capacitating boar sperm exhibit increased labeling with merocyanine 540 that may reflect membrane lipid disorder, but this has been correlated with alterations in the phospholipid content of the membrane leaflets [48, 49] rather than with decreased sterol content, as reported here.

Anisotropy decreased approximately 7% during 24-h incubation, but the local effect on membrane structure may be greater. Some regions of the sperm membrane likely are more affected than others by sterol loss, because sperm 3ß-OH sterols are not uniformly distributed. Filipin labeling shows the highest concentration to be in the plasma membrane of the anterior head, with much less in the remaining plasma membrane and intracellular membranes [27]. In many types of cells, DPH penetrates to the intracellular space [50–53], so it probably reports an average signal that includes intracellular membranes. (A more polar derivative, trimethylammonium-DPH, appeared to reside mainly in the sperm plasma membrane, but it produced too low a signal for use in these experiments.) If sterols are predominantly lost from the anterior head during capacitation, the decrease in anisotropy in this region may be considerably more than the cell-averaged decrease. The degree to which the change is greater in the anterior head depends on the relative amount of DPH in this region and on how much the fluidity in the anterior head differs from the fluidity in the other DPH-labeled sperm structures. A second consideration is that lipid microviscosity—a property that might affect downstream regulators of capacitation—is not a linear function of anisotropy. Apparent microviscosity can be estimated by

where r0 is the anisotropy of completely immobilized probe (0.362 for DPH [54]). Substituting values from Table 1 indicates that the microviscosity decreased approximately 15% during 24-h incubation.

If a decrease in lipid acyl chain order is the essential consequence of the loss of sperm sterols, then agents that reduce lipid order should cause capacitation, even in the presence of sperm sterols. Fluidizing agents failed to promote capacitation or the acrosome reaction, however. These results contrast with those of an earlier report that ethanol, which can decrease lipid anisotropy (e.g., [55]), induces acrosome reactions in human sperm [56]. The acrosome reaction was not of normal morphology, however, and we have not been able to reproduce those results (unpublished observations).

The ineffectiveness of fluidizing agents might result from trivial inadequacies in the experimental protocol or from sterols inhibiting a separate function that is required for capacitation. Because 50 mM benzyl alcohol killed sperm, it is possible that sublethal damage occurred at the 25 mM concentration used for these experiments. Acyl chain order may play no role in capacitation, but other evidence makes this seem unlikely. A study of cholesterol and 11 of its structural analogs revealed that all of the sterols were efficient inhibitors of capacitation except for the two that were least able to order phosphatidylcholine (coprostanol and epicoprostanol) [33]. These observations might be reconciled if sterol-phospholipid interactions are critical, but in a way that is not related to the action of the fluidizing agents. Perhaps, benzyl alcohol and A2C do not accurately mimic the effect of loss of sterols from membranes during capacitation. One possibility derives from the heterogeneous lateral distribution of sterols in cell membranes. Sterols preferentially associate with, and impose order on, saturated phospholipids [57]. Sterol-rich lipid domains are not fluidized by A2C, even though A2C fluidizes the bulk lipid phase of the membrane [58]. Perhaps the structure of sterol-rich lipid domains is more important to sperm capacitation than bulk membrane lipid fluidity. Other possibilities exist, however, and additional experiments are required to test this idea.

In summary, the present experiments detected a significant decrease in membrane lipid order during capacitation, resulting from a loss of sperm sterols, but a decrease in bulk lipid order was not sufficient to cause the sperm to become capacitated.


   FOOTNOTES
 
1 Supported by NIH grant HD30763. Back

2 Correspondence: Department of Physiological Sciences, 264 McElroy Hall, Oklahoma State University, Stillwater OK 74078. FAX: 4057448263. ncross{at}okstate.edu Back

Received: 2 November 2002.

First decision: 27 November 2002.

Accepted: 1 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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