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BOR - Papers in Press, published online ahead of print November 14, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064253
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BIOLOGY OF REPRODUCTION 78, 390–399 (2008)
DOI: 10.1095/biolreprod.107.064253
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Characterization of Sperm Plasma Membrane Properties after Cholesterol Modification: Consequences for Cryopreservation of Rainbow Trout Spermatozoa1

Karin Müller 2 3, Peter Müller 4, Gwenaëlle Pincemy 5, Anke Kurz 6, and Catherine Labbe 2 5

Leibniz-Institut für Zoo-und Wildtierforschung im Forschungsverbund Berlin e.V.,3 0315 Berlin, Germany Humboldt-Universität zu Berlin,4 Institut für Biologie, 10115 Berlin, Germany Institut National de la Recherche Agronomique,5 IFR 140, UR1037, 35000 Rennes, France Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V.,6 6321 Bernau, Germany

ABSTRACT

During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.

electron spin resonance, fluidity, gamete biology, methyl beta-cyclodextrin, permeability, phospholipid translocation, sperm

INTRODUCTION

Cryopreservation of cells is an artificial process which induces severe membrane damage in spermatozoa, from lower vertebrates such as fish to mammals [1–4]. The organization of membrane lipid and protein is modified and the cellular homeostasis is altered, leading to losses in sperm function. At low temperatures, coexistence of fluid and gel phase areas is responsible for packing defects in the plasma membrane [5–6], and redistribution of membrane proteins eventually induces protein aggregates [7] leading to an irreversible loss of some membrane functions. Osmotic constraints due to cryoprotectant addition/removal and water crystallization add more physical damage to the plasma membrane [8–9]. Membrane permeability to water and cryoprotectant as well as membrane fluidity are often proposed as key parameters in the ability of cells to withstand these constraints induced by freezing-thawing. However, how artificial changes in membrane permeability and fluidity can affect cell fitness for cryopreservation has been barely explored at the experimental level with spermatozoa [10].

Cholesterol is known to regulate lipid chain order [11], resulting in the control of many membrane properties and functions including membrane thickness, fluidity, permeability to water and other molecules, and lipid phase transition [12–14]. Because of this cholesterol influence on plasma membrane properties, many studies have explored the hypothesis of a cholesterol-induced stabilization of spermatozoa with regard to cryopreservation. In descriptive studies, it was surmised that species with the highest cholesterol content had the most cryoresistant spermatozoa (mammals [15]; fish [16]), although this hypothesis was challenged in poultry species [17]. Inter-individual comparisons of spermatozoa within the same species also led to contradictory interpretations on the role of cholesterol: a positive relation between plasma membrane cholesterol content and cryoresistance was observed in goats [18], while an inverse relation was shown in fowl [19], rainbow trout [20], and stallions [18]. No dependence between both parameters was found in boars [18], or humans [21].

The use of the methyl derivative of beta-cyclodextrin (MbCD) as an effective modulator of cell membrane cholesterol level [22–24] allowed more experimental studies to investigate the role of cholesterol. In boars, sperm exposure to MbCD improved cryosurvival, and cholesterol depletion was therefore proposed as favorable to sperm cryopreservation [25]. On the contrary, improvement of sperm cryopreservation in bulls [26–27], stallions [28–29], and rams [30] was achieved with cholesterol-loaded MbCD, indicating that an increase in cholesterol may be favorable in these species. Such discrepancies make the role of cholesterol in membrane strengthening upon cryopreservation difficult to determine. The overall differences in sperm characteristics and the overall importance of cholesterol during mammalian sperm capacitation and acrosome reaction [31–35] is one reason for this difficulty. Thus, any attempt to decipher whether cholesterol-induced changes in biophysical properties of the plasma membrane do have a major influence on cryopreservation outcomes might be challenged because of the cholesterol interfering with major sperm functions related to mammalian fertilization.

Rainbow trout (Oncorhynchus mykiss) spermatozoa do not have an acrosome, and they bear a very simple structure with only one mitochondrion [36]. Fertilization occurs through a micropyle which allows sperm to penetrate the egg envelope, the chorion. Trout spermatozoa are very sensitive to cryopreservation with a poor maintenance of plasma membrane integrity [4, 37], and it is assumed that an improvement of freezing ability could be readily assessed on these cells. Therefore, we hypothesized that we could use these spermatozoa to investigate on how sperm cryopreservation ability is improved by cholesterol-induced changes in the plasma membrane properties. The objective of this study was to explore the extent to which the biophysical properties of the plasma membrane could be modulated by cholesterol changes in rainbow trout spermatozoa, and to explore the relevance of these membrane properties for how well sperm cells tolerate the stress of freezing-thawing. However, the cholesterol changes had to be restricted to a range that precisely maintained fresh spermatozoa function. For fertilization, dilution of sperm in water initiates motility, a process relying on potassium efflux and activation of several ion channels [38–39]. It has been shown in other cell types that the function of those ion channels is cholesterol-dependent [40–41] and related to cholesterol-rich lipid rafts [42]. Subsequent binding to a carbohydrate epitope within the egg micropyle area is mediated by a specific ganglioside on the sperm surface [43] which is also a typical component of membrane rafts. Hence, both motility initiation and fertilization are at risk of being altered by cholesterol treatments.

Therefore, we modified the cholesterol content of rainbow trout sperm membranes with special attention to maintaining the function of fresh sperm. The comparatively large cell number in most trout ejaculates (above 150 x 109 spermatozoa) provided a unique opportunity to test several treatments on the same sperm sample. We characterized the impact of enhancement and removal of cholesterol on several membrane properties of trout sperm cells: (i) resistance of plasma membrane to osmotic stress, (ii) membrane fluidity assessed with probes localized in different regions of the plasma membrane, and (iii) kinetics of transbilayer phospholipid movement. The relevance of these membrane properties for sperm cells to tolerate freezing-thawing was explored with regard to the fertilization ability of the cells after cryopreservation.

MATERIALS AND METHODS

Sperm Cells

The sampling on animals was conducted in accordance with the guiding principles of the French regulation on laboratory animals, by people with the level 1 agreement delivered by the Direction of the French Veterinary Services (DSV). Fresh semen was obtained from 2-year-old male rainbow trout (Oncorhynchus mykiss) originating from a spring spawning strain and held at 13°C under seasonal photoperiod. Sperm was collected by gentle hand stripping from anesthetized males and diluted with 1 volume sperm medium (SpM: 110 mM NaCl, 23.8 mM KCl, 1.1 mM MgSO4 x 7 H2O, 1.8 mM CaCl2 x 2 H2O, 10 mM Bicine, 10 mM Na HEPES; pH 7.8, 300 mOsm/kg) which maintained the immobilized state of the spermatozoa. After centrifugation (250 x g, 10°C, 20 min), sperm concentration was adjusted to about 10 x 109 cells/ml with SpM.

The sperm of each male was split into three samples: one for cholesterol reduction, one for cholesterol enhancement, and one as a control (treated as the other samples, but in SpM). Each treatment was applied in triplicate on the same sample (three different incubation tubes per treatment). Results are the mean of data from sperm of six different males.

In preliminary experiments, we observed that cholesterol modification of sperm strictly depended on the number of spermatozoa present in the incubation medium. For this reason, sperm concentration was always carefully standardized and the treatments were expressed as a ratio of MbCD or cholesterol quantity to the cell number.

Cholesterol Reduction

One volume of the sperm suspension was mixed with 1 volume of 50 mM methyl-beta-cyclodextrin (MbCD, Sigma C4555) in SpM and 8 volumes of SpM. The MbCD/spermatozoa ratio was 5 µmol/109 cells. The final incubation volume (0.5 to 15 ml) had no influence on the extent of cholesterol modification. For testing different MbCD/spermatozoa ratios (0.5–20 µmol/109 spermatozoa), the initial 50 mM MbCD concentration was changed accordingly. After incubation for 15 min at 18°C, cholesterol exchange was arrested by a tenfold dilution with cold (4°C) SpM. After centrifugation, the pellet was resuspended in one volume SpM and stored at 4°C until use.

Cholesterol Enhancement

The cholesterol-MbCD complex (cho-MbCD) was prepared according to the method in [24]: one volume of cholesterol (Sigma C3137) dissolved in chloroform at a concentration of 50 mM was added dropwise to 10 volumes of 50 mM MbCD in SpM continuously stirred at 70°C. After 1 h, water evaporation was compensated for by the addition of distilled water, and the solution was filtered through a 0.22 µm filter. The cholesterol concentration of the complex solution was 4.61 ± 0.45 mM (8 independent preparations).

For cholesterol enhancement of spermatozoa, 1 volume of the sperm suspension was mixed with 1 volume of cho-MbCD and 8 volumes SpM. The cholesterol/MbCD/spermatozoa ratio was 0.4 µmol/5 µmol/109 cells. When a different cholesterol/spermatozoa ratio was needed, the cho-MbCD volume was changed accordingly while the total volume and cell number were unchanged. Cells were incubated for 15 min at 18°C after which spermatozoa were washed as described for cholesterol reduction.

Determination of Cholesterol and Phospholipid Content

Lipids from the washed sperm samples were extracted according to [44]. Total cholesterol was determined enzymatically as described in [45]. Phospholipid content of the sperm lipid extract was measured by colorimetric assay according to [46] after hydrolysis of the total lipid sample with perchloric acid. Although cholesterol and phospholipids were assessed on whole cellular extracts, we consider that the C/P ratio reflects that of the plasma membrane, because (i) cholesterol is known to be localized almost exclusively in the plasma membrane (reviewed in [47]) and (ii) plasma membrane phospholipids account for most of the cell phospholipids considering the shape of rainbow trout spermatozoa (small head of 2.5 µm, long tail of 35 µm with lateral membrane extensions, only one mitochondrion; [36]).

Assessment of Sperm Motility

Sperm concentration was adjusted to about 0.3 x 109 cells/ml with SpM. One microliter was mixed directly on a microscopic slide with 20 µl of the motility activating solution Actifish (IMV, France) supplemented with BSA (5 mg/ml) to prevent spermatozoa from sticking to the glass slide. Actifish induced immediate activation of spermatozoa and the motility lasted for about 30 to 40 sec at 20°C. Percentage of motile spermatozoa was estimated under the microscope (x200, dark field optics); two replicates per sample were measured.

Assessment of Plasma Membrane Resistance to Hypotonic Stress

The resistance of spermatozoa to hypotonic stress was measured by the propidium iodide (PI) assay [48], for which 5 µl of the sperm suspension were diluted in 2 ml distilled water containing 2.5 µg PI. Due to cell swelling under osmotic stress, the integrity of the plasma membrane was affected and PI permeated in the cells. The time course of DNA staining by PI in cells undergoing swelling was monitored at 18°C with a PTI spectrofluorimeter (Bio-Tek Instruments, excitation 535 nm, emission 617 nm). The entrance of PI was quantified by determining the time at which 50% of the cell population was stained, referring to this as the half-time of PI entrance.

Characterization of Membrane Fluidity by Fluorescence Anisotropy

Sperm membrane fluidity was characterized by estimating of the lipid order measuring steady-state fluorescence anisotropy of the lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH), as described by [11]. A 2 mM DPH stock solution in tetrahydrofuran (THF) was diluted in SpM to obtain a 2.5 µM working solution. The sperm suspension was diluted 50 times with SpM to decrease light scattering by the cells during the anisotropy measurements. Twenty microliters (4 x 106 spermatozoa) of this diluted sperm suspension were mixed with 2 ml of the 2.5 µM DPH working solution into a quartz cuve. Maximum DPH fluorescence intensity (excitation 352 nm, emission 440 nm) was reached after 10 min and remained constant during a further 80 min of incubation. Anisotropy (r) was calculated from the polarized DPH fluorescence measured at 20°C with a PTI spectrofluorimeter equipped with light polarizer: r = (I// – GI{perp})/(I// + 2GI{perp}) where I// and I{perp} are the intensities of vertical and horizontal components of emitted light and G is the apparatus grating factor. Three measurements were performed on each sperm sample.

Characterization of Membrane Fluidity by Spin-Labeled Lipids

The mobility of spin-labeled (sl) lipid analogues 1-palmitoyl-2-(4-doxylpentanoyl) phosphatidyl-choline (sl-PC), N-(4-doxylpentanoyl)-trans-sphingenyl-1-phosphocholine (sl-SM), and 25-doxyl-cholesterol (sl-chol) within the plasma membrane was recorded. Sl-PC and sl-SM were synthesized as described by [49]. Sl-chol was synthesized according to the protocol in [50]. Aliquots of sl-PC and sl-SM in chloroform were dried and resuspended by vigorous vortexing with SpM at 4°C; 7.2 nmol of labeled lipid in SpM was added to the cell pellet containing about 2 x 109 sperm cells. Considering a membrane phospholipid content of about 375 nmol per 109 cells [51], the molar concentrations of sl-PC and sl-SM in the trout sperm membrane were about 1%. After 5 min at 18°C, cells were washed by addition of 20 volumes SpM and then were centrifuged. To reoxidize the label, 5 µl potassium hexaferricyanide (final concentration 10 mM) was added to the cell pellet. Subsequently, cells were inserted into an electron spin resonance (ESR) capillary and ESR spectra recorded.

From the spectra, a rotational correlation time {tau}c of the label moiety was calculated according to the formula given in [52, 53].


Formula 78

with {Delta}H being the width of the central line (in G) and I0 and I–1 being the amplitudes of the central and high-field line, respectively.

For incorporation of sl-chol into membranes, 25 nmol sl-chol and 25 nmol cholesterol in chloroform were dried and vigorously resuspended with 50 µl of a 4 mM MbCD-solution in SpM. After addition of 50 µl SpM, this label-solution was added to about 2 x 109 sperm cells. After incubation for 15 min at 4°C, labeled cells were washed, and the cell pellet was inserted into a capillary for ESR measurements. From the spectra of sl-chol, the outer hyperfine splitting (see Fig. 4) was estimated as a parameter for analog mobility.


Figure 4
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  		FIG. 4. Changes in mobility of spin-labeled lipid analogues in trout sperm membranes after cholesterol modification. Left: ESR-spectra of spin-labeled cholesterol (sl-chol), phosphatidylcholine (sl-PC) and sphingomyelin (sl-SM) in the plasma membrane of non-modified (control, C/P = 0.45 ± 0.03), cholesterol enriched (+ CHO, C/P = 0.88 ± 0.04) and cholesterol reduced (– CHO, C/P = 0.22 ± 0.02) trout spermatozoa at 4°C. For better comparison, the low and mid field peaks of sl-PC and sl-SM spectra at the different conditions were enlarged and superimposed. Right: Temperature dependence of membrane fluidity as revealed by outer hyperfine splitting (sl-chol, arrows in the left panel) and rotational correlation time (sl-PC and sl-SM) of nonmodified (closed circles), cholesterol-reduced (open circles), and cholesterol-enriched (triangles) trout spermatozoa. Data are the mean ± SD of 3 measurements (see statistics in the text).

ESR spectra were recorded at different temperatures using an ECS 106 spectrometer (Bruker, Karlsruhe, Germany) with the following parameters: microwave power, 20 mW; modulation amplitude, 2 G; accumulation 8 times.

Translocation of Spin-Labeled Lipid Analogues Across the Membrane

Transbilayer movement of analogues in sperm membranes was measured at 15°C by the back exchange technique [49]. After cholesterol modification, a sperm pellet containing about 15 x 109 cells was incubated with 5 mM diisopropylfluorophosphate to minimize phospholipid hydrolysis [54–55]. The translocation experiment was initiated by mixing one volume of sl analog (sl-PC, sl-SM, and 1-palmitoyl-2-(4-doxylpentanoyl)phosphatidylserine (sl-PS)) to two volumes of cell suspension with a final molar label concentration of 1% of the membrane phospholipids. At distinct times, 70 µl aliquots were taken from the labeled cell suspension and mixed with 30 µl of 3% fatty acid-free BSA for 1 min on ice to extract the sl analog. Since BSA is only able to extract analogues from the outer membrane leaflet, a translocation of sl analogues to the cytoplasmic leaflet leads to a lower sl analog concentration in the BSA containing supernatant. After centrifugation (30 sec, 12 000 x g), 40 µl from the supernatant was mixed with 10 mM hexaferricyanide and stored for ESR analysis of the sl analog concentration. By measuring the ESR signal intensities of the cell pellets at time zero with and without BSA, we could demonstrate that nearly all analogues were incorporated into sperm membranes and extracted from membranes by BSA at this time point. In the case of sl-PS, kinetic of translocation was fitted to a single-exponential equation in order to estimate rate constants and the plateau of transbilayer movement (see [56]).

Cryopreservation

One volume of sperm suspension was mixed with 3 volumes of Cryofish (IMV, France, ref 014491) supplemented with 10% v/v avian egg yolk and 10% v/v Me2SO. Extended sperm was injected into 500 µl French straws (IMV, France, 3 straws per sperm sample) and immediately frozen in nitrogen vapors 3 cm above the liquid nitrogen surface. After 10 min equilibration, the straws were transferred into liquid nitrogen. Sperm was thawed by plunging the straws into 37°C water for 10 sec, the time needed for the samples to reach +4°C.

Fertilization

Fertilization was performed as described in [45]. Two fertilization batches were performed for each sample. Briefly, 0.1 x 109 fresh spermatozoa or 1 x 109 frozen-thawed spermatozoa were mixed with 200 eggs, and sperm activation was triggered by adding 10 ml of of the motility activating solution Actifish. After 3 wk development at 10°C in recycled water, eggs bearing eyed embryos were counted as fertilized and related to the total number of eggs used for fertilization. We showed previously [57–58] that no embryo mortality occurs between the first cell divisions and the eyed stage. The eyed stage, which is easier to assess, was therefore used as a reliable estimator of the early development rate and extrapolated to yield the fertilization rate.

Statistics

Statistical comparisons were done using the t-test for paired observations (SigmaStat32). Temperature- and time-dependent parameters were compared for different treatments using the Tukey-Test (SigmaStat32).

RESULTS

Optimization of Cholesterol Modification Protocols

To reduce the native cholesterol content, different incubation times in the presence of different quantities of MbCD were tested at 18°C. The reduction of the sperm cholesterol/phospholipid ratio (C/P) was dependent on MbCD quantity as well as time of incubation (Fig. 1). After only 15 min exposure of 109 sperm cells to 3 µmol MbCD, the sperm cholesterol content was significantly diminished from 299 ± 45 (control) to 211 ± 35 nmol/109 cells (P < 0.01, n = 6), whereas the phospholipid content did not change (618 ± 68 for control vs. 615 ± 30 nmol/109 cells for the reduction treatment). The C/P ratio decreased from 0.484 ± 0.05 to 0.345 ± 0.07 (P < 0.01). A further decrease of the cholesterol content to 160 ± 15 nmol/109 cells was obtained with 6 µmol MbCD/109 spermatozoa resulting in a C/P of 0.250 ± 0.03. A reduction of 30% to 50% in sperm cholesterol did not impair motility (Fig. 1). We found that lower MbCD ratios (0.5 and 1 µmol/109 cells) were ineffective in cholesterol reduction whereas a higher ratio (12 µmol/109 cells) markedly impaired sperm motility (37 ± 26%, C/P = 0.203 ± 0.035) after 30 min incubation.


Figure 1
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  		FIG. 1. Optimization of cholesterol reduction in trout spermatozoa. C/P: cholesterol/phospholipid ratio of spermatozoa expressed as a percentage of the values of control sperm (mean ± SD of 6 males). A significant difference (P < 0.05) between conditions is indicated by a different superscript.

To enhance the cholesterol content of trout spermatozoa, we used different quantities of cholesterol in cho-MbCD. The increase of C/P in spermatozoa after 15 min incubation at 18°C was dependent on the amount of applied cholesterol (Table 1). We checked that longer incubation time (30 and 60 min) did not improve sperm cholesterol enrichment. At a cholesterol concentration of 375 nmol in cho-MbCD applied to 109 cells, sperm cholesterol content increased by about 80% (from 324 ± 35 in the control to 569 ± 22 nmol/109 cells, P < 0.01, n = 6) without any change in phospholipid content (688 ± 89 for control vs. 696 ± 54 nmol/109 cells). The corresponding C/P increased from 0.477 ± 0.06 to 0.813 ± 0.12 (P < 0.05). Sperm motility was not affected by the treatments (Table 1). This cholesterol increase was not due to cho-MbCD sticking to the cells, since MbCD was not detected in the washed spermatozoa after an enzymatic glucid assay.


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  		TABLE 1. Optimization of cholesterol enhancement.

Sperm Resistance to Osmotic Stress

When trout spermatozoa were exposed to a hypo-osmotic shock in distilled water, they swelled according to the flexibility of their membrane until membrane disturbances allowed the dye PI to enter the cell (Fig. 2, inset). As shown in Figure 2, the half-time of PI entrance taken as a measure of membrane integrity increased linearly with the C/P (R = 0.87; P < 0.01; n = 54). Cholesterol-enriched trout sperm cells were more resistant to hypo-osmotic shock than those with less cholesterol.


Figure 2
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  		FIG. 2. Half-time of propidium iodide (PI) entrance in hypo-osmotically stressed trout spermatozoa in relation to their cholesterol/phospholipid ratio (C/P). Control (closed circles), cholesterol-reduced (open circles), and cholesterol-enriched (triangles) samples of 18 different fish are shown. Inset: representative kinetics of the PI staining of sperm cells with reduced (left curve), enriched (right curve), and nonmodified (middle curve) cholesterol content (a.u., arbitrary fluorescence unit).

Membrane Fluidity Assessed by DPH and Spin-Labeled Lipids

Fluorescent DPH incorporates into the hydrophobic core of plasma membranes sensing the fluidity/viscosity of the surrounding molecules. An increase of DPH anisotropy, calculated from fluorescence depolarization measurements, reflects a decreased mobility of membrane constituents. In cholesterol-depleted and control cells, i.e., up to a C/P of about 0.5, we observed a nearly linear dependence of DPH anisotropy with C/P (Fig. 3). However, a further increase in cholesterol content did not cause a lower fluidity of membranes measured in cholesterol-enriched sperm cells.


Figure 3
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  		FIG. 3. DPH-assessed fluidity of trout sperm plasma membrane after cholesterol modification. The fluorescence anisotropy (r) of DPH inversely reflecting fluidity was recorded at 20°C on nonmodified control (closed circles), cholesterol-reduced (open circles), and cholesterol-enriched (triangles) trout spermatozoa. N = 7 different males.

As another approach toward investigating membrane fluidity, we recorded ESR spectra of spin-labeled lipids, sl-chol, sl-PC, sl-SM.

From ESR spectra of sl-chol, the hyperfine splitting (Fig. 4 left, double arrow) was determined as a measure of the analog mobility reflecting the fluidity of membrane cholesterol. Compared to control and cholesterol-enriched trout spermatozoa, the decrease in membrane cholesterol concentration significantly increased the fluidity as seen from the diminished outer hyperfine splitting (P < 0.05). As expected, a rise in temperature also increased the membrane fluidity, as seen from the decreasing outer hyperfine splitting, in all treatments (Fig. 4, right). Regarding the high field peak (Fig. 4 left, single arrow), the spectrum of sl-chol in untreated sperm cells at 4°C clearly differs from the spectra of both cholesterol-depleted and cholesterol-enriched sperm cells.

The correlation time ({tau}c) calculated from sl-PC and sl-SM spectra reflects the rotational mobility of these analogues. Raising the temperature, the rotational correlation time decreased for both analogues in the different conditions (Fig. 4, right). With the exception of 15°C, removal of cholesterol from the sperm membrane caused a significantly higher fluidity in sl-PC and sl-SM compared to control (P < 0.05). These differences are underscored by comparing the spectra (Fig. 4, left) in that the width of the low field peak of cholesterol-depleted cells is less than those of control and cholesterol-enriched cells. Increasing the cholesterol content of sperm membranes had no influence on {tau}c at higher temperatures (15°C both analogues, 8°C only sl-SM). At lower temperatures, enrichment of cholesterol caused a decrease in membrane fluidity; however, these treatment differences were only significant (P < 0.05) for sl-SM. Again, differences in mobility were also obvious from a comparison of the spectra. Comparing the mobility between sl-PC and sl-SM at identical conditions, we measured a smaller fluidity, i.e., higher {tau}c values, for the SM analog. This could be explained by a tighter packing of SM in membranes due to its saturated chains [59]. The correlation times measured for both analogues mainly reflect the characteristics of the outer membrane leaflet since most of the analogues were localized in the outer leaflet within the time frame that spectra were recorded (see Fig. 5).


Figure 5
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  		FIG. 5. Translocation kinetics of spin-labeled lipid analogues in trout sperm membrane after cholesterol modification. The transbilayer movement of sl-PS, sl-PC and sl-SM to the inner leaflet was measured in nonmodified (closed circles), cholesterol-reduced (open circles), and cholesterol-enriched (triangles) trout sperm cells at 15°C. Data represent the mean ± SD of 3 independent measurements.

On the whole, the results obtained with spin-labeled lipids confirm those of DPH in that a decrease of cholesterol content in sperm cells results in a significant increase in membrane fluidity, whereas enrichment with cholesterol has a lower impact or no impact at all on the membrane fluidity compared with control cells.

Phospholipid Transbilayer Movement Across the Membrane

As in eukaryotic cells including spermatozoa, the inward movement of PS to the cytoplasmic membrane leaflet in trout spermatozoa has been shown to be actively mediated by an aminophospholipid translocase [56]. In contrast, choline-containing phospholipids like PC and SM move across the membrane by slow passive diffusion. Likewise, we found a rapid translocation of sl-PS and a slow movement of sl-PC and sl-SM to the inner membrane leaflet of trout sperm cells at 15°C (Fig. 5). For sl-PS, the kinetics of phospholipid translocation were dependent on the cholesterol content of trout spermatozoa; the translocation was slower with increasing C/P ratios but finally reached the same plateau (Fig. 5).

For sl-PC, we found similar kinetics for control and cholesterol-enriched sperm cells, but cholesterol depletion led to significantly faster kinetics (P < 0.01). For sl-SM, the kinetics of control cells were somewhat slower than those of cholesterol-enriched and -reduced cells, although these treatment differences were not significant.

The degree of cholesterol reduction and enrichment as well as the motility of sperm cells (within the time scale of the translocation experiments) were routinely measured and gave results similar to those shown in Figure 1 and Table 1. Sixty minutes after labeling, motility could be induced in 86 ± 15%, 93 ± 3%, and 79 ± 14% of cells for control, cholesterol-depleted and cholesterol-enriched samples, respectively.

Resistance of Cholesterol-Modified Spermatozoa to Cryopreservation

Finally, we tested the influence of cholesterol manipulation on the quality of cryopreserved trout spermatozoa, namely, their motility and their ability to fertilize eggs (Fig. 6). As generally observed with cryopreserved rainbow trout spermatozoa, freezing of the cells reduced the motility to a value of 3–15%, and these low rates were independent of the cholesterol modification. A concomitant reduction in fertilization rate was observed, but this later parameter was dependent on the cholesterol treatment of sperm cells. Cholesterol reduction caused a greater decrease in sperm fertilization rates (P < 0.05) than cholesterol enhancement, which resulted in fertilization rates similar to control samples. Notably, those control sperm samples having the lowest post-thaw fertilization rates were also the ones with the lowest fertilization values after cholesterol enrichment and freezing. This shows that spermatozoa with a low fitness for cryopreservation could not be improved by increasing cholesterol. Separately from the experiment on cho-MbCD-mediated cholesterol enhancement, adding cho-MbCD directly to the cryopreservation medium did not yield better post-thawing fertilization rates.


Figure 6
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  		FIG. 6. Fertilization rate of fresh and cryopreserved trout spermatozoa after cholesterol modification. C/P: cholesterol/phospholipid ratio. The fertilization rate was measured for nonmodified control (control), cholesterol-reduced (– CHO), and cholesterol-enriched (+ CHO) trout sperm before (hollow squares) or after (filled squares) cryopreservation. Data represent the results of 9 male individuals.

DISCUSSION

In the present study we investigated the extent to which cholesterol content could be modified without affecting fresh cell functional parameters, i.e., motility and fertilization ability, in order to further analyze the impact of cholesterol modifications on biophysical parameters of the plasma membrane and on spermatozoa cryopreservation ability. Our data indicate that these ultimately specialized cells are highly adaptable to a wide range of membrane cholesterol concentrations. Up to 50% of the membrane cholesterol could be removed or the membrane cholesterol could be doubled without a noticeable change in motility and fertilization ability of fresh spermatozoa. In contrast to what has been observed for acrosome-bearing spermatozoa, where cholesterol manipulation may interfere with capacitation events [60–63], the fertilization ability of rainbow trout spermatozoa was fairly insensitive to a modification of their cholesterol content. This makes trout spermatozoa an excellent model for studying cholesterol-induced changes in plasma membranes and the consequences to their ability to resist freezing-thawing stresses.

Although sperm functions were maintained over a wide range of cholesterol changes, biophysical properties of the plasma membrane were very responsive to cholesterol manipulations. We showed that the resistance of trout spermatozoa to hypo-osmotic stress was positively correlated with the cholesterol content of their plasma membrane. The membrane resistance to hypo-osmotic stress is dependent on membrane permeability to water, a property essentially dependent on membrane structure. Cholesterol has important effects on the organization of phospholipids in the membrane, e.g., it increases the molecular order of the acyl chains of phospholipids, which leads to an increase in the bilayer thickness by modulating the packing of the lipids in the membrane [64–65]. Such a cholesterol-induced membrane condensation decreases the membrane permeability to water in model membranes [66–67] and in erythrocytes [68]. Li et al. [69] demonstrated by differential scanning calorimetry that water permeability during cooling was reduced in cholesterol-loaded bovine spermatozoa, but to our knowledge, our results are the first demonstrating a linear response of sperm cells in such a wide cholesterol range.

Membrane fluidity is also considered to be closely related to membrane condensation, which influences the permeability of the membrane to small polar molecules. A decreased fluidity reflects a tighter packing of membrane components that reduces passive molecule movement across the plasma membrane (reviewed in [66, 70, 71]). Our data in trout spermatozoa suggest that membrane fluidity and permeability diverge at high cholesterol content; in contrast to the continuous increase in membrane resistance to osmotic stress observed with increasing cholesterol content, DPH-assessed fluidity no longer decreased at a C/P of about 0.5. Likewise, a similar plateau of membrane fluidity has been observed in erythrocyte ghosts and in model membranes [11, 72, 73]. These authors have postulated (i) that an excess of cholesterol molecules sequesters into micro domains which exclude DPH, in accordance with a possible cholesterol sequestration in crystallites [47, 74] or (ii) that at high cholesterol concentration, the DPH motion is drastically reduced by the close neighborhood of cholesterol molecules, and any additional cholesterol would not further increase DPH anisotropy. Our results would also explain the lack of membrane fluidity change assessed with a TMA-DPH probe observed in bull spermatozoa upon addition of cholesterol [10]. It is likely that, as observed with trout spermatozoa in our experiments, bull sperm cells already exhibit a plateau of membrane fluidity.

Besides the rather unspecific probe for membrane fluidity, DPH, we also characterized this parameter by more specific lipid analogues: spin-labeled cholesterol (a good analog for native cholesterol [75]) and spin-labeled phospholipids [76]. As expected, we observed an increase in fluidity with increasing temperature for all three analogues. As found with DPH, a fluidizing effect of cholesterol depletion was observed for sl-chol at all temperatures and for sl-SM and sl-PC at temperatures below 15°C. Also in accord with the DPH measurements, no decrease of membrane fluidity was observed after enhancement of sperm cholesterol, but rather the mobility parameters of spin-labeled lipids reflected an increased fluidity. The results obtained with sl-PC and sl-SM mainly reflect the situation of the outer membrane leaflet since these analogues were concentrated on this leaflet during the time scale of the experiment (Fig. 5). Besides, PC and SM are known to be main constituents of the outer leaflet of trout sperm membrane [56].

It is known that cholesterol exerts different effects on membrane domains depending on their lipid composition or the degree of saturation of the fatty acids. Cholesterol strongly associates with certain lipids such as sphingomyelin [77–78] and disrupts the highly ordered gel phase of brain sphingomyelin, leading to a more fluid membrane, whereas the fluidity of brain PC bilayers decreases [79]. We surmise that the modification of trout sperm cholesterol content interferes with the formation of specific membrane domains composed essentially of sphingomyelin and cholesterol. As with DPH, it may be assumed that the spin-labeled lipids do not incorporate equally into those cholesterol-rich versus cholesterol-poor domains and, therefore, do not necessarily sense a rise in viscosity upon the addition of cholesterol. Moreover, cholesterol-rich domains include lipid species like sphingomyelin whose rotational motion is even increased after cholesterol incorporation.

Our results indicate that the cholesterol modification of trout sperm cells affected the structural organization of the membrane. This is supported by the different ESR-spectra of sl-chol. In particular, comparison of the shape of the high-field peaks revealed the presence of an additional spectral component in cholesterol-depleted and cholesterol-enriched cells, i.e., some of the label molecules were in a new environment having a different mobility. This component could be extracted by spectra subtraction (+CHO vs. control and –CHO vs. control), but could not be quantified since the quantity of these molecules was low and/or the differences in mobility between the components were small (spectra not shown). The presence of the additional component could reflect changes in cholesterol-protein and/or cholesterol-lipid interactions upon alterations of the cholesterol content, and indicates that an artificial modification of the membrane cholesterol does not influence all membrane domains equally. In line with this, a divergent contribution of several coexisting domains involving membrane protein was recently observed in goat spermatozoa [80].

To analyze the influence of cholesterol on transbilayer membrane organization, we followed the transverse movement of spin-labeled lipids after their incorporation into the outer membrane layer. In agreement with Müller et al. [56], we found a slow inward movement of sl-SM and sl-PC to the inner membrane leaflet of trout spermatozoa, whereas sl-PS is rapidly transported from the outer to the inner monolayer due to the action of aminophospholipid translocase [76]. Upon increasing the membrane cholesterol content, the activity of aminophospholipid translocase decreased, whereas the equilibrium distribution of sl-PS was not influenced. In cholesterol-modified sperm cells, about 95% of sl-PS on the cytoplasmic membrane half indicates the maintenance of the well-known transverse phospholipid asymmetry, with PS being concentrated on the inner leaflet. A similar dependence of sl-PS transport from cholesterol concentration has been reported in human erythrocytes [54]. With regard to the passive diffusion of choline-containing lipids, the modification of cholesterol content (increase and decrease) resulted in a low (and statistically insignificant) increase of sl-SM inward movement and equilibrium concentration on the inner leaflet. For sl-PC, the removal of cholesterol caused a slight increase of the analog inward movement and inside concentration, as was shown by Morrot et al. [54] for human erythrocytes. These data show that the small cholesterol-dependent changes of phospholipid distribution did not essentially affect the transbilayer phospholipid asymmetry in trout sperm cells as one of the basic parameters characterizing membrane structure.

To estimate sperm cryotolerance, we chose to assess both sperm motility and development rates after fertilization, as they are ultimate tests which take into account the whole cellular functionality. Indeed, changes in plasma membrane properties were likely to have cascading consequences on other cellular components. Membrane defects will alter: motility initiation, because of the role of membrane signaling in the process [39]; motility maintenance, because of the loss of intracellular ATP [4]; and membrane fusion and possibly the ability of the sperm nucleus to produce the first embryonic cell after fertilization [57]. A consistent improvement of plasma membrane resistance to freeze-thaw stress was therefore expected to ultimately affect fertilization and early development rates. Despite the fact that cholesterol manipulation modified biophysical plasma membrane properties of trout spermatozoa to such a great extent as demonstrated in our study, it was striking that no systematic relation with sperm cryotolerance was observed. In view of the discrepancy between the linear changes induced in membrane permeability and the lack of linear response of spermatozoa resistance to freezing-thawing as cholesterol increased, this biophysical parameter may be dismissed as an important factor for sperm cryotolerance in our model. Not only did the highest cholesterol contents fail to improve sperm cryotolerance, but membrane fluidity failed to decrease further upon cholesterol enrichment. On the contrary, the low cholesterol contents resulted in increased membrane fluidity together with decreased cryotolerance. These superimposed patterns between fluidity and the quality of freeze-thawed sperm makes fluidity a more likely candidate as a determinant of sperm cryotolerance. However, our data demonstrate that the functional consequences of changes in membrane biophysical properties with respect to sperm cryotolerance are not straightforward and that some extrapolations should be carefully considered before they can apply to those highly specialized cells that are spermatozoa. Cholesterol, additionally incorporated into the sperm membrane, was not able to protect those cellular structures that are involved in fertilization, but the removal of cholesterol even increased their impairment upon freeze-thawing. Future studies will have to consider the lateral structures of the sperm membrane whose importance has been indicated in this study.

In conclusion, we showed that the exchangeable pool of cholesterol in the trout sperm plasma membrane was very significant, and that cholesterol modifications strongly modified plasma membrane properties of fresh spermatozoa. Those modifications were tolerated in fresh semen and had no influence on fertility. However, after freeze-thawing, the fertilizing potential was reduced in cholesterol-depleted sperm cells, and could not be improved in cholesterol-enriched samples compared to that of control cells. Artificial modification of some biophysical properties in trout sperm plasma membrane before freeze-thawing was obviously not able to improve the fitness of intrinsic membrane structures to better survive cryopreservation.

ACKNOWLEDGMENTS

The authors thank Jean-Luc Roger for his major contribution to the experimental work.

FOOTNOTES

1Collaboration between authors was supported by EU fellowship PROCOPE. Back

Correspondence: 2Catherine Labbe, INRA SCRIBE, Campus de Beaulieu, 35000 Rennes, France; e-mail: catherine.labbe{at}rennes.inra.fr

Karin Müller, Leibniz-Institut für Zoo-und Wildtierforschung im Forschungsverbund Berlin e.V., Alfred-Kowalke-Strasse 17, 10315 Berlin, Germany; e-mail: mueller{at}izw-berlin.de Back

Received: 16 July 2007.

First decision: 6 August 2007.

Accepted: 9 October 2007.

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