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Biology of Reproduction 64, 69-79 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Incorporating Lipids into Boar Sperm Decreases Chilling Sensitivity but Not Capacitation Potential1

L. Heb, J.L. Baileyc, and M.M. Buhr2,,b

b Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 c Centre de recherche en biologie de la reproduction, Dept. des sciences animales, Université Laval, Québec, Québec, Canada G1K 7P4

ABSTRACT

Fresh boar sperm were incubated with small unilamellar liposomes composed of either the total lipids extracted from head plasma membranes (HPM) of fresh boar sperm or selected lipids (SL) of five defined phospholipids with specific acyl chains. To optimize fusion, liposomes with 2 mol% octadecyl rhodamine fluorophore in Beltsville Thawing Solution ± 1 mM CaCl2 were incubated at 35°C with 1 ;ts 107 or 108 spermatozoa/ml and monitored over 60 min, using flow cytometry and fluorescence microscopy. The HPM fused to both sperm concentrations faster than SL but was equivalent by 30 min (108 sperm/ml) or 60 min (107 sperm/ml; 57.5 ± 3% and 67.1 ± 8% sperm fused to HPM and SL, respectively) ± Ca2+. Neither HPM nor SL affected onset of capacitation or spontaneous or ionophore-induced acrosome reactions at 0 or 3 h (chlortetracycline and fluorescein isothiocyanate-Pisum sativum agglutinin; n = 3). During cooling and after cryopreservation (n = 4 ejaculates), SL but not HPM significantly improved sperm motility and viability (Sybr14/propidium iodide staining) ± 20% egg yolk, but egg yolk alone was more effective than SL alone. Liposomes of complex composition can fuse to boar sperm without harming in vitro capacitation or acrosome reaction and reduce sperm chilling sensitivity.

sperm, sperm capacitation/acrosome reaction

INTRODUCTION

Cryopreserving boar sperm results in an extreme reduction in fertilizing ability [1, 2], possibly due to damage to sperm membranes [3, 4]. Plasma and acrosomal membranes influence sperm shape and volume [5, 6], motility [7], energy production [8], permeability [9], capacitation and acrosome reaction (AR) [10, 11], and interaction with oocytes [12, 13]. Both the structural and functional integrity of the plasma membrane are, therefore, very important to sperm. Cryopreservation (dilution, cooling, freezing, and thawing) alters selective permeability of sperm membranes, reduces motility, decreases energy production, changes membrane lipid composition [14–16], and changes membrane dynamic behavior [15, 17, 18].

Membrane lipid composition is intimately involved in the degree and nature of the damage caused by cryopreservation [19]. Erythrocytes that are very sensitive to chilling lose their differentiated shape and deform as they are cooled below 22°C, which is also the temperature where the membranes undergo a lipid phase transition [18]. Sperm from different animal species with similar cold shock resistance have membranes with rather similar lipid composition, cholesterol:phospholipid ratio, and length and saturation of fatty acid chains [3, 14]. Sperm from different parts of the epididymis have different sensitivity to cold shock, which is correlated with the changes in lipid composition during sperm maturation from caput to cauda epididymis [20]. Cryopreservation of ram sperm increased the concentration of diphosphatidylglycerol [21] and phosphatidylserine (PS) [22] in the outer leaflet of the membrane, reversing the asymmetric distribution of these lipids in the membrane bilayer of fresh sperm. Some lipids are released from sperm membranes during cold shock [20, 23]. Egg yolk, a common protectant in cryopreservation media, is crude lipid that interacts with sperm plasma membrane [24–27], and addition of some lipids to the extender has been suggested to have beneficial effects on cold resistance [28].

Liposome-mediated transfer has been successfully used to incorporate molecules such as ATP [29] or DNA [30] into spermatozoa or to incorporate exogenous lipids into sperm membranes (bull [31], human [32], or boar [33]). Using exogenous lipid as a cryoprotectant for semen cryopreservation has not improved post-thaw sperm function [34–36], perhaps reflecting poor efficiency in lipid incorporation and/or an inappropriate mix of lipids. Composition of the lipid carrier vesicles [37] affects the efficiency of vesicle fusion. We hypothesized that a carefully selected combination of lipids incorporated into boar sperm using controlled, repeatable, and quantifiable procedures would improve their ability to survive cryopreservation.

MATERIALS AND METHODS

Semen Collection

Boar semen was collected by the gloved-hand technique from mature Yorkshire boars. The spermatozoa-rich fraction was collected through gauze into a thermos warmed to 35°C. Sperm motility was examined immediately after collection, and only ejaculates with >70% motile spermatozoa were utilized. Spermatozoa concentration was determined with a calibrated spectrophotometer (Spectronic 20; Bausch & Lomb, Rochester, NY).

Preparation of Lipids

Head plasma membrane lipids Head plasma membrane (HPM) lipids were prepared from boar sperm as before [15, 38]. Briefly, fresh boar spermatozoa were washed with two silicon-based oils and Tris-sucrose buffers. Plasma membranes were removed by Parr cavitation and purified with differential centrifugation. Membrane lipids were obtained from the membrane pellet by three chloroform-methanol extractions (CM, 2:1, v:v). The extracts were combined and dried with a rotating evaporator. The lipids were resuspended in CM and any remaining proteins removed by adding 0.7% NaCl (1:5, v:v), vortexing, and centrifugation. The lower layer of solvent containing the lipids was evaporated again, resuspended in a small volume of CM, and stored in the dark under N2 at -70°C. After 8–10 ejaculates had been processed in this manner, all lipids were pooled, divided into 1.5-ml aliquots, and dried under a stream of N2 followed by 30 min under vacuum. Each aliquot was weighed and stored under N2 at -70°C until use. Relative proportions (mol%) of phosphatidylcholine (PC):phosphatidylethanolamine (PE):sphingomyelin (SPH):PS:phosphatidylinositol (PI):lysoPC were approximately 40:23:22:5:3:5, with a total saturated:unsaturated fatty acid ratio of approximately 81:17 [15].

Select lipids A proprietary mixture of 15 phospholipids, consisting of PC, PE, SPH, PS, and PI, each containing specific fatty acid chains were purchased from Sigma (Sigma-Aldrich, Mississauga, ON, Canada), Avanti (Alabaster, AL), and Matreya (Pleasant Gap, PA). The purity for each lipid was >98%. Approximate lipid ratios were PC:PE:SPH:PS:PI of 21:26:42:5:5, with a total saturated:unsaturated fatty acid ratio of approximately 85:15.

Preparation of Small Unilamellar Vesicles

Small unilamellar vesicles (liposomes) were prepared by the method of Huang [39] with some modifications. Briefly, lipids (HPM or select lipids [SL]) were dissolved in CM and then pipetted into screw-capped tubes. When the experiment was to test the efficiency of liposome incorporation into spermatozoa, octadecyl rhodamine B (R18; Sigma, St. Louis, MO) was added to a final concentration of 2% mol of lipids. The mixtures of lipids or lipids-R18 were dried under N2 (10 min) and then under vacuum desiccation (30 min), then rehydrated with Beltsville Thawing Solution (BTS, 0.2 M glucose, 0.02 M sodium citrate, 0.015 M NaHCO3, 3 mM EDTA, 0.01 M KCl, pH 7.3, 60°C), and vortexed. The tubes were filled with N2, capped tightly, and sonicated at room temperature for 30 min. After sonication, liposomes were stored in 2-ml aliquots under N2 at -70°C for no more than 1 wk. Before use, each aliquot was thawed at 25°C, its pH adjusted to 7.3, and resonicated for 30 min. For the liposome-R18, the solution was then run through a Sephadex G50 column (1 x 21 cm) to remove R18 not incorporated into the liposomes [40]. The final lipid concentration was 0.3465 µmol/ml.

Efficiency of Liposome Fusion with Spermatozoa

To optimize the percentage of sperm incorporating liposomes, two types of liposomes (HPM and SL, 0.3119 µmol/ml in the final reaction mixture), two Ca2+ concentrations (0 and 1 mM CaCl2 in H2O), and two sperm concentrations (107 and 108 spermatozoa/ml) were evaluated. Fresh undiluted spermatozoa were pooled from ejaculates from two boars in each trial as one replicate. Three replicates were assessed for fusion efficiency using flow cytometry, using a Coulter Epics Elite ESP (Coulter Corporation, Hialeah, FL) equipped with 640 DL and 610BP filters, with the emission photomultiplier tube wavelength settings of 610–640 nm. Samples were maintained at 34°C in a water bath. At 1, 10, 30, and 60 min of incubation, 100 µl were taken from each treatment tube, diluted to 1 x 106 spermatozoa/ml with BTS buffer, and injected into the flow cytometer. Three populations (liposomes, spermatozoa and liposomes with spermatozoa) were identified by size and fluorescence characteristics (Fig. 1). A total of 20 000 particles were counted per sample over approximately 2–3 min.



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  		FIG. 1. Flow cytograms. To monitor fusion, liposomes containing R18 fluorophore were incubated with boar sperm. For each replicate (n = 3), the flow cytometer detected labeled liposomes (b; region C), sperm alone (a; region B), and the fusion product (sperm with incorporated liposomes, c; region A)

Capacitation Assays

Capacitation and the AR were evaluated as described previously [41]. One ejaculate from each of three boars was diluted in BTS to a concentration of 40 x 106 sperm/ml and used within 30 min of collection. Diluted sperm were put on a discontinuous 35%/75% Percoll gradient and centrifuged (200 x g, 5 min and then 900 x g, 20 min; room temperature). Pellets were washed in PBS (350 x g, 10 min), resuspended in BTS to 108 sperm/ml, and 200 µl were added to 1800 µl of BTS alone or BTS containing SL or HPM liposomes, prepared as above. Sperm were incubated (30 min, 37°C), then pelleted (350 x g, 10 min, 37°C), and resuspended to a final concentration of 107 sperm/ml in capacitation medium [42]. Replicate aliquots of each treatment were incubated for 0 or 3 h (39°C, 5% CO2/95% air, humidified atmosphere). Sperm capacitation was determined by the ability of the sperm to undergo the AR in the presence of calcium ionophore A23187 (Molecular Probes Inc., Eugene, OR) and by the chlortetracycline (CTC) fluorescence assay [43, 44]. Two micromolar A23187 in dimethylsulfoxide (DMSO, or DMSO alone for negative controls) was mixed with sperm samples (20–40 x 106 sperm/ml) and incubated for 30 min at 39°C in a 5% CO2, humidified atmosphere in order to induce the AR. Post-ionophore (and post-DMSO) values were corrected for spontaneous AR (i.e., % AR prior to treatment). Assays were also conducted at 0 h on samples not incubated for the 30-min induction.

Acrosome-reacted sperm were detected with the fluorescein isothiocyanate-labeled Pisum sativum agglutinin (PSA-FITC [45]). In brief, 20–30 µl sperm suspension were smeared onto a slide, allowed to dry, then fixed and permeabilized in absolute ethanol for 10 min. After fixation, the smears were covered with 50 µl PSA-FITC (100 µg/ml) and placed in a moist environment in the dark at room temperature for 30 min. The slides were rinsed and then mounted with glycerolized water (90% water) and a coverslip. One to two hundred sperm were scored and classified as acrosome-intact or acrosome-reacted with a Nikon microscope equipped with fluorescence optics (excitation 450~490 nm: B2-A filter, 400x).

The CTC assay has been described for pig sperm by Wang et al. [44]. In brief, CTC (750 µM) and cysteine (5 mM) were dissolved in Tris-NaCl buffer (20 mM and 130 mM, respectively), and the pH was then adjusted to 7.8. At the time of the assay, 15 µl CTC solution was mixed with an equal volume of sperm, and 12.5% glutaraldehyde in 1 M Tris buffer (pH 7.8) was added at a final concentration of 0.1%. A coverslip was applied after gentle stirring. Two hundred sperm were scored by fluorescence microscopy (excitation 400~440 nm: BV2-A filter, 400x). Three fluorescence patterns were observed: pattern F sperm demonstrating bright uniform fluorescence over the head, representing sperm that are not yet capacitated; pattern B in which the anterior head is bright with a faintly fluorescent postacrosomal region, representing capacitated sperm; and pattern AR that have poorly fluorescent heads with a thin bright band along the equatorial segment, representing acrosome-reacted sperm.

Cryopreservation of Boar Semen

Boar sperm were frozen in 0.5-ml straws using the freezing method of Pursel and Johnson [46] as modified for straw freezing [47].

Semen handling Identical numbers of spermatozoa from two boars were pooled for each replicate (n = 4), and these two boars were selected as a balanced incomplete block from four boars. Linco-spectin (0.3 ml, 50 mg/ml) was added to 50 ml pooled semen that was then filtered through a double layer of Miracloth (CalBiochem, La Jolla, CA) and then diluted with BTS to 1 x 108 spermatozoa/ml.

Extenders Two kinds of cryopreservation extenders were used in this experiment. One was the original Beltsville F5 extender, including egg yolk (20%) and Orvus ES paste (BF5); another was Beltsville F5 extender with neither egg yolk nor Orvus ES paste (BF5nul). For each extender, two fractions were prepared, containing 0 or 6% glycerol.

Treatments and cooling For each replicate, duplicate cork-capped test tubes (12 x 75 mm) were prepared for each of three treatments. Tubes with 3.6 ml of BTS containing either SL or HPM liposomes or BTS alone (control) were placed in a programmable bath (Biocool II; FTS System Inc., Stone Ridge, NY) and incubated at 34°C for 20 min. Each tube then received 400 µl of prediluted sperm (1 x 108 sperm/ml); the final concentrations of lipids and sperm were 0.3119 µmol/ml and 1 x 107 sperm/ml. These mixtures were then cooled to 24°C at 0.1°C/min and then centrifuged (800 x g, 10 min, 24°C). Sperm pellets were resuspended with nonglycerolated extender to 4 ml, with one tube from each treatment receiving BF5 and the other receiving BF5nul. These suspensions were cooled to 5°C (0.1°C/min) and the appropriate glycerolated fraction of the extender was added (5°C; 1:1 v:v; final glycerol concentration 3%) and gently mixed in.

Freezing and thawing All equipment and materials were at 5°C. Immediately after addition of the glycerolated extender, semen was loaded into 0.5-cc straws (cat. no. AA 101; IMV International Corp., Minneapolis, MN) and sealed with stainless steel sealing balls (Minitube of America, Madison, WI). Straws were placed in N2 vapors to cool at 30°C/min for 3 min, immediately plunged into liquid N2, and held for at least 3 days before quality tests. Straws were thawed (60°C, 5 sec, gentle agitation), emptied into 37°C BTS with caffeine (0.02 M), and held for 10 min.

Estimation of viability and motility Samples (500 µl for viability, 50 µl for motility) were taken from each treatment at the following stages of the cryopreservation process: 34°C within 1 min of adding liposomes; 24°C before centrifugation; 24°C after addition of extender; 5°C before and after addition of glycerolated extender; and after thawing. Viability was assessed by Sybr14 and propidium iodide (PI)(Live/Dead Sperm Viability Kit; Molecular Probes Inc., Eugene, OR), preparing two slides, and doing a blind count of a minimum 100 sperm per slide, scoring sperm as green, red, or dual stained using a fluorescence microscope (Leitz, Laborlux S, Wetzlar, Germany) at 400x magnification. Viability (% live sperm) was calculated as no. green/(no. green + no. red + no. dual) x 100, and the average was calculated from two slides for each sample. For motility determination, sperm were diluted to 0.5–1 ;ts 106 sperm/ml, two slides were made, and motility was examined under phase-contrast microscope (Zeiss, Jena, Germany) at 400x magnification. For each slide, 100 sperm were counted in total and categorized as moving forward (progressive motility), moving in place (motile), and not moving. The % progressively motile and total % motile (progressive + motile) were calculated for each slide, and the average from the two slides in each sample was calculated.

Data Analyses

Fusion efficiency The percentage of sperm that had incorporated lipids and the percentage of available liposomes that were incorporated by sperm were calculated from the number of particles in the appropriate areas of the cytogram (Fig. 1). Specifically, % of sperm fused to lipids = [particles in area A (fluorescent and sperm size)/particles in areas A + B (nonfluorescent, sperm size)] ;ts 100%; and % of available liposomes fused to sperm = [particles in area A/particles in areas A + C (fluorescent, liposome size)] x 100%.

Data were arcsine transformed and general linear model analysis (GLM; SAS/STAT, Cary, NC) tested the main effects and their interactions with the model that included replicate, calcium, liposome type (SL or HPM), and sperm concentration. Data from treatments that were not significantly different were pooled and a GLM analysis of the pooled data evaluated the factors affecting the efficiency of fusion between sperm and lipid liposomes. The t-tests with least square means tested specific differences between the two sperm concentrations within each lipid type at each time and between 0 and 1 mM Ca2+ within each lipid type and sperm concentration.

Viability and motility Viability and motility from cryopreservation were adjusted by adding 1 to all values to eliminate zeros and normalized by using arcsine transformation. A full model was used to test the overall main effect of replicate, egg yolk, liposome type, and temperature and their interactions in the whole process of cryopreservation. Aliquots destined to receive egg yolk or not were similar before extender addition (at 24°C), and therefore these data were pooled to determine liposome effect with a model including liposome type and replicate. After adding the extender, the main effects of egg yolk and the interaction of egg yolk and liposome at each temperature were tested using GLM analysis of variance and a model that included replicate, extender, and liposome. Egg yolk was highly significant, so data from sperm processed with and without egg yolk were tested separately. To monitor sperm performance over time, a model that included replicate and temperature tested data from each treatment. All conclusions were based on probabilities obtained with the type III sums of squares, with P < 0.05 considered significant.

RESULTS

Fusion

Both types of liposomes actually fused to sperm in a rapid and stable fashion. This was true fusion not dye transfer or adherence [33], as measured qualitatively by spectrofluorometry (resonance energy transfer of the probes N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadeanoyl-sn-glycero-3-phospho-ethanolamine, triethylammonium salt and rhodamine, and release of self-quenching with R18; Buhr, unpublished results) and confirmed by flow cytometry (Fig. 1) and fluorescence microscopy (Fig. 2). Flow cytometry was used to measure fusion efficiency because of the quantitative nature of the data thus obtained.



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  		FIG. 2. Photomicrographs of boar sperm exposed to SL liposomes for 10 min. The SL liposomes containing 2 mol% of the fluorophore R18 were incubated with fresh boar sperm for 10 min and photographed under fluorescence (a) and by phase contrast microscopy (b). Original magnification x400.

Fusion Efficiency

Calcium (1 mM) did not increase fusion efficiency (P > 0.05) at any time for either HPM or SL liposomes, or for either sperm concentration. Data obtained with 0 and 1 mM Ca2+ were therefore pooled for determination of the effect of lipids, sperm concentration, and incubation time.

The HPM lipids fused to sperm faster than SL at both sperm concentrations (Fig. 3). With 107 sperm/ml, the percentage of sperm incorporating HPM lipids declined from 1 to 10 min (P = 0.0014), while the percentage of sperm incorporating SL increased from 1 to 10 min (P < 0.05); both then maintained this degree of incorporation to the end of incubation. For both lipids, more sperm at 107 than 108 sperm/ml had lipids fused (P < 0.05). The percentage of sperm taking up HPM and SL lipids was the same by 30 min (108 sperm/ml) or 60 min (107 sperm/ml).



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  		FIG. 3. Percentage of sperm incorporating lipids over time (mean ± SEM). Boar sperm (pooled data, n = 3 ejaculates) at 107 or 108 sperm/ml were incubated at 35°C with R18-labeled liposomes made with HPM or SL, and fusion was measured by flow cytometry. *Within a sperm concentration, the percentage of sperm with incorporated SL lipids differed from the percentage with HPM at this incubation time (P < 0.05). a, b, Within a sperm concentration and liposome type, times with no letters in common differ (P < 0.05)

The amounts of available liposomes that were taken up by sperm varied between HPM and SL over time and between sperm concentrations (Fig. 4). As expected, 108 sperm/ml took up more SL than 107 cells at all times, and more HPM at 1 and 10 min (P < 0.05). Uptake by 107 sperm/ml of SL was stable and of HPM increased over 60 min. By 60 min, sperm at 107 sperm/ml had used approximately 40% of available SL; approximately 60% of available liposomes were used by other treatments.



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  		FIG. 4. Amount of liposomes fused to sperm over time (mean ± SEM). Boar sperm (pooled data, n = 3 ejaculates) at 107 or 108 sperm/ml were incubated at 35°C with R18-labeled liposomes made with HPM or SL, and fusion was measured by flow cytometry. a–c, Within an incubation time, treatments with no letters in common differ (P < 0.05). W–Z, within a sperm concentration and lipid, times with no letters in common differ (P < 0.05)

Viability and Motility During Cryopreservation

The overall effects of lipid, egg yolk, and temperature on viability, progressive motility, and total motility were highly significant (P < 0.01). There was no interaction between lipid and egg yolk (P > 0.05).

Cooling and extension At 34°C, after sperm had incubated with BTS ± liposomes for 1 min, viability was significantly lower in SL than in HPM or BTS (Table 1, P < 0.05). The percentage of sperm that were progressively motile was higher in SL than in either HPM or BTS (P < 0.05), but the total percentage of motile sperm was the same for all treatments. After controlled cooling to 24°C, SL-treated sperm had better viability and motility than the HPM or control (BTS) sperm (Table 1).


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  		TABLE 1. Immediate impact of lipid on the viability, progressive motility, and total motility of boar sperm at 34°C (mean ± SEM, n = 4 ejaculates).*

Presence of egg yolk in the extender added at 24°C had an overall positive effect (Fig. 5, P = 0.0001), with viability, total, and progressive motility significantly (P < 0.05) higher than sperm in extender lacking egg yolk, regardless of liposome treatment (Fig. 5). The SL significantly improved viability and motility in the presence or absence of egg yolk. Simply adding egg yolk-containing extender increased progressive but not total motility in all treatments (P = 0.0001) and increased apparent viability in SL (P = 0.0263) and BTS (P = 0.0001) compared to values at 24°C immediately before adding extender. In contrast, adding extender without egg yolk significantly decreased total motility in all treatments (P < 0.002) and apparent viability in SL and HPM (P < 0.05).



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  		FIG. 5. Impact of lipids on sperm viability (a) and motility (b and c, progressive and total motility; mean ± SEM, n = 4) immediately after addition of extender ± 20% egg yolk and surfactant (EY) at 24°C. a, b, Within EY or no EY, values with no superscripts in common differ (P < 0.05). *Differs from corresponding treatment with EY. Statistical analysis performed on transformed data

Sperm cooled to 5°C in egg yolk had better overall function (P = 0.0001; data not shown). Sperm in SL plus egg yolk had better total and progressive motility than sperm in BTS alone (P < 0.05), while apparent viability was similar among the three treatments. In the absence of egg yolk, viability and total motility were higher (P < 0.05) in SL than in either HPM or BTS (P < 0.05). Neither these values nor relationships changed immediately after the addition of glycerolated extender, except for a decrease in the viability of sperm in BTS without egg yolk (P = 0.0193).

Post-thaw All functional measures declined after freezing and thawing. Cryopreservation with egg yolk significantly improved (P < 0.0001) viability and total and progressive motility (Fig. 6). The SL in egg yolk resulted in significantly (P < 0.05) better viability and motility than HPM, with values for the BTS industry-standard being intermediate. In the absence of egg yolk, SL significantly (P < 0.05) improved viability and progressive and total motility.



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  		FIG. 6. Impact of lipids on sperm viability (a) and motility (b and c, progressive and total motility; mean ± SEM, n = 4) after thawing in 0.02 M caffeine in BTS. a, b, Within EY or no EY, values with no superscripts in common differ (P < 0.05). *Differ from corresponding treatment with EY. Statistical analysis performed on transformed data

Capacitation and the AR

To determine if adding more native or non-native lipids (HPM, SL) to the sperm would affect the onset of capacitation and the AR, fresh boar sperm had liposomes fused to them and then were incubated for 3 h in capacitating conditions. Acrosomal status was then evaluated both by the CTC stain for capacitation and the FITC-PSA stain for acrosomal integrity. As expected, the percentage of sperm capacitated or undergoing spontaneous or induced ARs was significantly greater after 3 h of incubation in capacitating media than at Time 0, and calcium ionophore significantly increased the percentage of sperm undergoing the AR at both 0 and 3 h. Neither type of liposome affected either the timing or the percentage of sperm undergoing either capacitation or spontaneous or ionophore-induced ARs (Fig. 7).



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  		FIG. 7. Capacitation and AR in boar sperm with and without SL and HPM liposomes assessed by CTC and FITC-PSA. Boar sperm (n = 3 ejaculates) were incubated for 30 min in buffer alone (BTS) or liposomes made from HPM or SL. Sperm were washed, resuspended in capacitating media, and incubated for 3 h in capacitating media. At 0 and 3 h, sperm were assessed for capacitation status (pattern B, CTC stain; PAT B) and their ability to acrosome react by the CTC stain (AR CTC), and by FITC-PSA in the absence (AR FITC) or presence of the calcium ionophore A23187 (AR-A23 FITC). Values at 3 h are greater than 0 h; within a time, values with A23187 always exceed those without

DISCUSSION

Select lipids significantly improved the viability and motility of boar spermatozoa during cooling and after cryopreservation with or without egg yolk, confirming that incorporation of specific lipids into sperm membranes can improve the ability of boar sperm to resist chilling injury. Cryopreservation (dilution, cooling, freezing, and thawing) of boar sperm causes structural and functional damage in sperm head membranes, altering both the chemical composition and the dynamic behavior of the membrane lipids. Membranes from fresh boar sperm have multiple membrane domains [38, 48, 49] whose basic fluidity is altered by cryopreservation of the intact sperm [47, 50]. Even the lipids alone, whether isolated from the HPM of fresh or frozen-thawed boar sperm, form domains with unique fluidities, but the HPM lipids from cryopreserved sperm have a different composition and dynamic behavior [15, 51]. Such damage to membrane lipids may be in addition to, or involved in, damage to motility apparatus [52] and cryopreservation-induced premature capacitation, such as is seen in bull sperm [41]. Any or all of these types of damage could contribute to the reduced fertilizing ability of frozen-thawed boar sperm [1]. Membrane lipid composition could well influence the cold-shock sensitivity of sperm [14]; certainly the reversibility of cryopreservation-induced damage in somatic cells varies with the composition of the cell membrane [19]. The composition of the SL was designed to rectify lipid changes identified as resulting from cryopreservation of boar sperm.

Fusion

A critical first step to altering membrane lipid composition was successful incorporation of lipids into the membranes of a high percentage of the sperm. The liposome fusion methods described here caused over 60% of the boar sperm to incorporate exogenous lipids in a readily repeatable fashion, with no adverse effects on sperm viability. This greatly exceeds the incorporation rates of 0 and 2% for live sperm and 18% for dead sperm in other trials [31, 36], but similar to the 80% fusion rate of proteoliposomes with erythrocytes [37]. Fusion was carefully monitored (Buhr, unpublished results) using resonance energy transfer [53] and by flow cytometry [54, 55]. Flow cytometric measures were made on populations of fresh spermatozoa in the presence or absence of fluorescently labeled lipids, with the flow cytometer gated to determine the amount of fluorescence transferred to the sperm population. The high fusion efficiency was achieved by optimizing liposome size, temperature, pH, and lipid composition, all of which influence the efficiency with which lipids fuse to cells [32, 56]. The diameter and homogeneous size of the small unilamellar vesicles permit close approach to cell membranes [56, 57], and this early adhesion-type reaction can be seen on one sperm in Figure 2. Liposomes that adhered, did not fuse, and subsequently drifted free are the most likely cause of the apparent decrease in the percentage of liposomes used from 0 to 10 min. Liposome size was somewhat variable and increased over time, as small vesicles coalesced. Extension of the sonication time reduces the initial variation in size and increases the percentage of sperm taking up the lipids (data not shown). Fluorescence microscopy clearly shows that the lipids incorporate in a diffuse pattern throughout the head and midpiece and even in the principal piece. Because our ultimate purpose is to extend the fertilizing life span of boar sperm, fusion conditions were chosen to be physiological for boar sperm (35°C; BTS buffer; pH 7.4). Although calcium promotes fusion in some systems [58, 59], it did not enhance fusion of HPM or SL liposomes and was omitted in the cryopreservation experiment to reduce the possibility of Ca2+-induced destabilization of sperm membranes [60] or premature capacitation [12, 41]. Predictably, the percentage of sperm taking up liposomes increased when fewer sperm or more lipids were present. A preliminary study found even higher incorporation with 106 than with 107 sperm/ml (data not shown), but this was too high a dilution for practical use in swine artificial insemination, and so further experiments used the 107 concentration.

The complex composition of both the HPM and SL lipids is likely largely responsible for the high degree of fusion achieved, with the different composition of HPM and SL probably driving their different incorporation dynamics. Liposomes from rat liver fused more readily to human sperm than did liposomes from human prostasomes, apparently due to the rat's much lower SPH and higher PE content [32]. Fusion clearly is an interaction between the liposome and target cell, because liposomes of PS and cholesterol fuse poorly or not at all to bull sperm [31, 36], although PS readily inserts in membranes from erythrocytes [61] and fibroblasts [62].

The HPM lipids acted as a control for the SL. It was assumed that HPM lipids, being native to the head plasma membrane of boar sperm, should be nontoxic, readily fusible, and they are known to consist of a complex mixture of phospholipids and fatty acids [15]. The SL contained specified proportions of PC, PE, SPH, PS, and PI, each with specified varieties of fatty acid chains. These lipids were deliberately selected to both promote fusion [56] and correct cryopreservation-induced damage. Relative to the HPM, SL had high concentrations of SPH, low concentrations of PC, PI, and PE, and fewer unsaturated fatty acids, particularly in the PE and PS fractions. The HPM lipids contained lysoPC and had more PE with long and unsaturated fatty acid chains [15]. The PS, PI, and PE present in both HPM and SL liposomes would exist as fusogenic anions or zwitterions at the working pH [63, 64]. Some types of PE, such as dioleyl-PE, act as fusogenic factors [65] and have been used as helper lipids to mediate gene transfer [66]. The HPM liposomes fused more quickly than SL, perhaps due to their lysoPC or higher content of PE with long unsaturated acyl chains [15], which would promote conversion to the highly fusogenic HII or inverted micelle, structure [67].

Effect of Lipid on Boar Sperm Viability and Motility During Cryopreservation

The SL markedly (P > 0.0001) improved viability (Sybr14 and PI dual staining) and motility at all temperatures from 24 to 5°C, suggesting SL lipids ameliorate the effects of chilling. Post-thaw, protective abilities ranked SL + BTS/egg-yolk >= BTS/egg-yolk > BTS/egg-yolk + HPM >> SL > HPM > no lipid.

Using exogenous lipid as a cryoprotectant in semen cryopreservation has had little previous success [34–36, 68]. While SL lipids caused a very small, but significant, loss of sperm viability immediately after addition, they were beneficial at all subsequent times. The HPM lipids did protect viability compared to the lipid-free controls, suggesting that an overall increase in total lipid and concomitant decrease in the high protein:lipid ratio of boar sperm [14] protects sperm function. However, the extent of protection was less than that of SL lipids and HPM lipids did not alleviate cryopreservation damage. Clearly the simple addition of mixed lipids does not reduce chilling sensitivity, confirming that specific lipids have specific actions. This is consistent with previous reports that PS-cholesterol liposomes had a cryoprotective effect on bull sperm [69] and stallion sperm [36], but PS-PC liposomes did not.

The superior protection afforded by SL may be due to its component lipids interacting with specific membrane proteins, perhaps via annular lipids, or they may cause a more general shift in membrane fluidity [17, 38] that supports membrane-dependent actions. The SL improved egg yolk's protective actions, but egg yolk was clearly an overwhelmingly effective cryoprotectant. The significant difference between SL alone and BTS-egg yolk alone could be due to the different amount, or different structure, of lipids in those two groups. The SL concentration (0.3119 µmol/ml, about 200 µg/ml) is far less than egg yolk (20% of extender). The SL contain most of the same types of phospholipids as the extender [15] but in different proportions, and the liposomes are small unilamellar vesicles, rather than the presumptive micelles of the egg yolk extender. The SL also supplied a high proportion of SPH that was negligible in extender [15].

Capacitation and AR

Capacitation and the AR were monitored in fresh sperm over time using the CTC stain and FITC-PSA [42]. The AR was induced by ionophore A23187; lysoPC was not used because it causes the immediate death of sperm [4], and its lipid nature could compromise any conclusions. If exogenous lipids are to be useful in extending the viable lifespan of sperm, they must neither prevent nor prematurely induce fertilization events, and neither the SL nor HPM lipids affected either the percentage of sperm or the timing of onset of capacitation or the AR. Membrane lipids are undoubtedly involved in many sperm functions including capacitation and the AR. Sperm membrane lipids are changed during epididymal maturation [70, 71] and during ARs in vitro [10, 72] and in vivo [73, 74]. The involvement of the polyphosphatidyl inositol pathway in capacitation and the AR [75–77] clearly implicates membrane phospholipids directly in the AR. The postcryopreservation loss of SPH and increase in 20:4 in boar sperm head membranes may indicate early or abnormal ARs, because induced AR in epididymal sperm reduce SPH [72] and 20:4 can initiate an AR either directly [78] or via the polyphosphoinositide cycle as seen in fragile aged sperm [79]. Because neither SL nor HPM liposomes altered the induction of capacitation or the AR, a decrease in the membrane protein:lipid ratio to the extent induced by this fusion must be insufficient to affect these events. Therefore, HPM lipids would not be expected to induce any changes, because their component lipids derive from the HPM of freshly ejaculated sperm and should not imbalance any important lipid:lipid ratios. It is gratifying that the SL, being selected to redress cryopreservation damage that may include a premature AR, permitted capacitation and the AR to proceed naturally.

Conclusion

Liposomes of complex composition fuse to boar sperm at 35°C at a neutral pH and without any nonlipid fusogenic factors. Sperm concentration, lipid type, and incubation time affect fusion efficiency. The SL lipids have beneficial effects on boar sperm viability during slow cooling (0.1°C/min) and improve viability and motility in frozen-thawed sperm without any effect on the ability of fresh sperm to capacitate or acrosome react. Fusion of SL and other lipid mixtures to boar sperm will help elucidate the role of membrane lipids in sperm function.

FOOTNOTES

First decision: 20 June 2000.

1 This research was supported by Natural Sciences and Engineering Research Council of Canada, and the Ontario Ministry of Agriculture, Food and Rural Affairs. Back

2 Correspondence. FAX: 519 767 0573; mbuhr{at}uoguelph.ca Back

Accepted: August 15, 2000.

Received: June 8, 2000.

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