Lipid dynamics in the plasma membrane of fresh and cryopreserved ...

Human Reproduction vol.14 no.7 pp.1827–1832, 1999

Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa

P.S.James1, C.A.Wolfe2, A.Mackie2, S.Ladha2, A.Prentice3 and R.Jones1,4 1Department of Signalling, The Babraham Institute, Cambridge CB2 4AT, 2Department of Food Biophysics, Institute of Food Research, Norwich NR4 7UA and 3Department of Obstetrics &

Gynaecology, Rosie Maternity Hospital, University of Cambridge, Cambridge CB2 2SW, UK 4To

whom correspondence should be addressed at: Gamete Signalling Laboratory, The Babraham Institute, Cambridge CB2 4AT, UK. E-mail: [email protected]

Preserving the integrity of the plasma membrane of spermatozoa is crucial for retention of their fertilizing capacity, especially after stressful procedures such as freezing and storage. In this investigation we have measured lipid diffusion in different regions of the plasma membrane of fresh and cryopreserved human spermatozoa using a sensitive, high resolution fluorescence photobleaching technique (FRAP) with 5-(N-octadecanyl)aminofluorescein as reporter probe. Results show that diffusion was significantly faster on the plasma membrane overlying the acrosome and decreased progressively in the postacrosome, midpiece and principal piece. The midpiece plasma contains a higher proportion of immobile lipids than other regions. In cryopreserved spermatozoa, lipid diffusion in the plasma membrane was significantly reduced on the acrosome, postacrosome and midpiece relative to fresh spermatozoa. Diffusion, however, could be restored to normal levels by washing spermatozoa in a medium containing 0.4% polyvinylpyrrolidine but not in medium alone or in medium containing 0.4% albumin. These results suggest that (i) lipid dynamics in the plasma membrane of human spermatozoa varies significantly between surface regions; (ii) in-plane diffusion is adversely affected by cryopreservation; and (iii) washing frozen spermatozoa in 0.4% polyvinylpyrrolidine restores membrane lipid fluidity to normal levels. The latter finding has important implications for improving the fertility of human spermatozoa following cryopreservation. Key words: cryodamage/diffusibility/membrane lipid domains/ photobleaching

Introduction A characteristic feature of differentiated cells is compartmentalization of their plasma membranes into separate regions that are commensurate with specialized function (reviewed by Rodriguez-Boulan and Nelson, 1989). The problem of how this lateral asymmetry is maintained over relatively large distances (5–10 µm) against the forces of random diffusion is © European Society of Human Reproduction and Embryology

fundamental to understanding many aspects of membrane functionality, especially the ability of some antigens to migrate between regions against large concentration gradients whilst others are restricted in their position. The mammalian spermatozoon is a good example of a morphologically polarized cell whose plasma membrane contains proteins and lipids asymmetrically distributed, both laterally and transversely (Friend, 1982). This asymmetry exists at both the micrometre and nanometre levels. In all spermatozoa, five macroregions, known as the acrosome, equatorial segment, postacrosome, midpiece and principal piece, can be readily distinguished from each other by morphological and cytochemical means (Holt, 1984). Within these macroregions, nanometre scale domains (especially those consisting of lipids) are thought to exist and to vary considerably in their dimensions and half-life. How this membrane polarity is established during spermiogenesis in the testis and maintained throughout the life of the spermatozoon is unclear, but possible reasons include interaction with the submembranous cytoskeleton, the presence of diffusion barriers within the bilayer, localized differences in lipid phase disposition caused by compositional heterogeneity as well as complex interactions with membrane proteins and calcium ions (Cowan et al., 1997; Ladha et al., 1997). Lipids make up ~60–65% of a plasma membrane and have a major influence on its properties. The lipid composition of whole spermatozoa (and to a limited extent purified plasma membranes derived from them) is well documented (Poulos et al., 1973; Lenzi et al., 1996), but much less is known about the spatial distribution, dynamics and physical state of individual lipids within the bilayer. This information is important to understand phenomena such as how localized membrane fusigenicity develops over the acrosome and equatorial segment prior to fertilization (Yanagimachi, 1994) and how some glycoprotein antigens can migrate between domains concomitant with endoproteolytic processing (Jones et al., 1997; Myles and Primakoff, 1997). In these respects, application of biophysical techniques to probe membrane structure has been informative. Using differential scanning calorimetry (DSC) (Holt and North, 1986; Canvin and Buhr, 1989; Wolf et al., 1990), at least two thermotropic phase transitions have been detected in ram and boar spermatozoa during cooling suggesting the coexistence of fluid and gel phase lipids. Drobnis et al., however, failed to detect any abrupt phase transitions between 4°C and 37°C in human spermatozoa by Fourier transform infrared spectroscopy (FTIR) and attributed this to the modulating influence of cholesterol (Drobnis et al., 1993). Later (Palleschi and Silvestroni, 1996), it was concluded from an analysis of generalized polarization spectra (GP) with Laurdan that lipids 1827

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in the plasma membrane of human spermatozoa were in a single liquid crystalline phase throughout the cell. From a flow cytometric analysis (FACS) of lipid asymmetry in bull spermatozoa, Nolan et al. demonstrated differential flip-flop movement of phospholipids similar to that reported in somatic cells and found that it was inhibited by extracellular calcium and sulphydryl agents (Nolan et al., 1995). The major disadvantage of FTIR, DSC, GP and FACS techniques is that they utilize suspensions of whole cells or membrane vesicles derived from them and lack the necessary resolving power to interrogate discrete areas of the plasma membrane of a single cell. Currently, only photobleaching (FRAP) has this potential. Recently, we applied FRAP analysis to investigate lipid dynamics in different regions of live bull, boar, ram, and mouse sperm plasma membranes and found that diffusion of the lipid reporter probe ODAF [5-(N-octadecanoyl)aminofluorescein] was 3–5 times faster on the acrosome than on the midpiece (Ladha et al., 1997; Wolfe et al., 1998). In addition, there was differential temperature sensitivity between surface regions with the formation of large immobile phases following membrane perturbation and cell death, most noticeably over the midpiece. Guinea pig spermatozoa were unusual in that their plasma membranes were largely insensitive (or alternatively, very stable) to the above treatments (Wolfe et al., 1998). In this investigation, we have used FRAP to compare lipid diffusion in different regions of the plasma membrane of freshly ejaculated and cryopreserved human spermatozoa. Results show that in live spermatozoa ODAF diffusion is significantly faster (33) on the acrosome relative to the principal piece plasma membrane and that this difference disappears following cryopreservation. Some of the deleterious effects of freezing, however, can be mitigated by post-thaw treatment of spermatozoa in media containing macromolecules such as 0.4% polyvinylpyrrolidine, suggesting methods for improving recovery of fertility of human spermatozoa following long-term storage for artificial insemination by donor (AID) programmes. Materials and methods Chemicals All routine chemicals and solvents were of the highest purity available commercially and were obtained from Sigma (London, UK) or MerckBDH (Poole, Dorset, UK). ODAF was purchased from Molecular Probes (Eugene, OR, USA) and polyvinylpyrrolidine (PVP; MW 40 000) was from Sigma. Spermatozoa Semen was provided by patients (age range 25–45 years) attending the infertility clinic at Addenbrooke’s and Rosie Hospitals, Cambridge, and was allowed to liquefy at room temperature (23–25°C). A spermiogram was performed in accordance with World Health Organization (1992) guidelines and surplus semen (with the patient’s consent) used for experimental analysis. All ethical recommendations were strictly adhered to and rigorous precautions were taken for operator safety. Only semen specimens with good motility (.60%) and a high incidence of normal morphology (.60%) were used in the study. Straws containing frozen human semen from fertile donors

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were obtained from Bourn Hall Clinic, Cambridge. Semen was diluted 1:1 with a cryoprotective medium (15% glycerol, 20% egg yolk, the remainder consisting of sodium citrate, glucose, fructose and salts; Dale and Elder, 1997) and frozen as described (Dale and Elder, 1997). Fresh spermatozoa were cold shocked by lowering their temperature rapidly from 37 to 0°C on melting ice, rewarmed to 37°C and the cycle repeated another 2 times. Labelling of spermatozoa with ODAF reporter probe With fresh spermatozoa, 10 µl of whole semen was diluted with 40 µl of Mann’s Ringer phosphate pH 7.4 containing 5.5 mmol/l glucose (MRP; Mann, 1964) and mixed with 50 µl of MRP containing 12.5 µmol/l ODAF in 2% ethanol. Suspensions were incubated in the dark for 15 min at room temperature (~23°C) and washed twice by centrifugation (500 g for 5 min) in 1 ml of MRP. Loose sperm pellets were resuspended to 100 µl in MRP containing 0.1% sodium azide and aliquots drawn up into capillary microslides (CamLab Ltd, Cambridge, UK) for viewing and FRAP analysis. The microslides are sealed at both ends to prevent evaporation. To investigate the effects of macromolecules on ODAF diffusion, 0.4% bovine serum albumin (BSA) or 0.4% PVP were included in the MRP during the washing and labelling steps described above. For labelling frozen spermatozoa, straws were thawed in a water bath at 37°C for 10 min and 50 µl of extruded semen washed twice by dilution into 500 µl of MRP or MRP 1 0.4% BSA or MRP 1 0.4% PVP followed by staining with ODAF as described above. Attempts at labelling thawed spermatozoa without washing were unsuccessful, presumably because the combination of seminal plasma, glycerol and egg yolk, glucose, and citrate in the extending medium inhibited uptake of the probe. Human seminal plasma in particular contains large numbers of prostasomes and extraneous membrane vesicles that would absorb ODAF very rapidly, thereby reducing its availability to spermatozoa. FRAP analysis Detailed descriptions of the FRAP instrumentation and data analysis systems have been published elsewhere (Ladha et al., 1997; Wolfe et al., 1998). Briefly, FRAP provides two indices of lipid behaviour within a membrane:(i) the rate of diffusion of the reporter probe (known as the diffusion coefficient, D); and (ii) the extent of fluorescence recovery after photobleaching (known as the percentage recovery, %R) which is a reflection of the proportion of the molecules that are freely diffusing within the membrane. The major membrane domains subjected to FRAP analysis were acrosome, postacrosome, midpiece and principal piece. Temperature was maintained at 24°C on a microscope warm-stage. Microscopy Spermatozoa stained with ODAF were photographed with Kodak Ektar 1000 film using a Zeiss Axiophot photomicroscope fitted with a 50 W mercury vapour lamp and an excitation filter at 485 nm and emission filter at 530 nm. Statistical analysis Results were analysed for statistical significance by one-factor ANOVA and Microsoft (Redmond, WA, USA) Excel 97.SR-1 ‘t-test’ assuming samples of unequal variances.

Results Human spermatozoa stained with 6.2 µmol/l ODAF in the presence of MRP containing 10% seminal plasma and 1% ethanol showed a variety of labelling patterns. Those spermato-

Lipid diffusion in human sperm plasma membranes

Figure 1. Fresh human spermatozoa stained with 5-(N-octadecanoyl)aminofluorescein (ODAF) to illustrate the different fluorescence patterns observed. (a) Uniform staining over the head and tail regions typical of live spermatozoa. (b) Strong staining on the midpiece. (c) Strong staining on the acrosome and proximal cytoplasmic droplet. (d) Left hand side spermatozoon shows stronger staining on the equatorial segment and neck regions. Bar 5 5 µm.

zoa that remained motile were uniformly labelled over the whole head and tail (Figure 1a) and correspond to the livepattern classification described for bull, ram, boar and mouse spermatozoa (Ladha et al., 1997; Wolfe et al., 1998). Other patterns observed include uniform staining on the head with stronger fluorescence over the midpiece (Figure 1b), strong fluorescence over the acrosome and cytoplasmic droplet/midpiece (Figure 1c) and strong fluorescence on the equatorial segment, postacrosome and midpiece (Figure 1d). The latter three types of spermatozoa approximate to the dead-pattern staining described in animal spermatozoa (Wolfe et al., 1998). Given the facility of two epi-illumination attachments on the viewing microscope, it was possible to classify spermatozoa according to their staining pattern before photobleaching. For the purpose of this investigation, only data on those spermatozoa that showed normal morphology and uniform staining with ODAF (Figure 1a) are presented. Diffusion coefficients for fresh spermatozoa that had been labelled with ODAF in the presence of 10% seminal plasma (Figure 2a) were highest over the acrosome (33.7 6 3.4310–9 cm2/s) decreasing significantly towards the postacrosome (22.4 6 2.5310–9 cm2/s; P , 0.05), midpiece (14.7 6 2.1310–9 cm2/s; P , 0.01) and principal piece (10.5 6 1.3310–9 cm2/s; P , 0.01). A similar pattern was obtained following washing of spermatozoa in MRP 1 0.4% BSA or MRP 1 0.4% PVP. In MRP alone without any added macromolecule, D values on the postacrosome and principal piece were significantly lower than those on the acrosome (P , 0.05) but on the midpiece they were more variable than those measured in the presence of seminal plasma proteins or 0.4% BSA or 0.4% PVP (differences not significant; P . 0.05). Percentage recoveries for ODAF on the acrosome, postacrosome and principal piece were ~70%, irrespective of the medium in which they were suspended (Figure 3a). Once

Figure 2. Comparison of diffusion coefficients (D) for ODAF in fresh (five samples) and frozen (two samples) spermatozoa washed in different media as shown. Ac 5 acrosome. PA 5 postacrosome. MP 5 midpiece. PP 5 principal piece. Values given are means 6 SEM. The number of spermatozoa analysed in each treatment is shown in brackets. Significance values are as follows. Significantly different from acrosome, *P , 0.05 or **P , 0.01. Significantly different from comparable figure for fresh spermatozoa, †P , 0.01. Significantly different from comparable region on frozen spermatozoa suspended in MRP alone, δP , 0.05.

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Figure 3. Percentage recoveries (%R) for ODAF in spermatozoa treated as described in Figure 2. Abbreviations as in Figure 2.

again, the exception was the midpiece where %R varied between 48 and 51%. By contrast, spermatozoa that had been cryopreserved followed by washing and labelling with ODAF in the presence of MRP or in MRP 1 0.4% BSA showed much reduced D values in all regional domains (Figure 2b). Washing previously frozen spermatozoa in MRP 1 0.4% PVP, however, had a restorative effect to the extent that D values were similar to those for fresh spermatozoa on the acrosome, postacrosome and midpiece regions (differences not significant (P . 0.05). Paradoxically, diffusion coefficients on the principal piece increased significantly relative to the value observed for fresh spermatozoa (P , 0.05). Despite low D values, %R for ODAF on frozen spermatozoa was similar to those found on fresh spermatozoa (Figure 3b). That is, they were ~70% on the acrosome, postacrosome and principal piece regions whereas on the midpiece %R was ~55% following washing and suspension in the presence of 0.4% BSA or 0.4% PVP. To investigate whether permeablizing the plasma membrane by a sudden reduction in temperature had any effect on ODAF diffusion, fresh spermatozoa were suspended in MRP 1 10% seminal plasma and subjected to three cycles of cold shock from 37°C to 0°C. Results showed that D values were significantly lower (P , 0.05) on the acrosome, postacrosome and midpiece (Figure 4a) amounting to ~66, 50 and 48% respectively of control values. No significant effect of cold shock on D was detected in the principal piece. As in the case of fresh and frozen spermatozoa, the %R 1830

Figure 4. Effects of cold shock on diffusion coefficients (D) and percentage recoveries (%R) of ODAF in human sperm plasma membranes. Abbreviations as in Figure 2.

values for cold shocked spermatozoa were not significantly different to controls, values ranging from ~70% on the acrosome and postacrosome to ~60% on the principal piece (Figure 4b). The exception once again was on the midpiece, where the %R was consistently lower than in the aforementioned regions (40–56%). Discussion This work has shown that in live human spermatozoa, lipid diffusion (as reflected in the dynamics of recovery of the lipid reporter probe ODAF during FRAP) is fastest on the plasma membrane overlying the acrosome and decreases progressively towards the postacrosome, midpiece and principal piece of the tail. Following cryopreservation, however, diffusion coefficients for ODAF are significantly reduced in all regions relative to fresh spermatozoa. Washing frozen– thawed spermatozoa with a medium containing 0.4% PVP has a significant restorative effect, most noticeably on the head. These observations have practical implications for improving the efficiency of AID programmes with frozen human semen. Our finding that there are differential rates of lipid diffusion in the plasma membrane of live human spermatozoa is in keeping with previous observations on mouse, bull, boar and ram spermatozoa (Ladha et al., 1997; Wolfe et al., 1998; James et al., 1999). The exceptions appear to be guinea pig spermatozoa, in which there were no measurable differences between surface regions (Wolfe et al., 1998), and hamster spermatozoa, where diffusion of DiIC14 was faster on the

Lipid diffusion in human sperm plasma membranes

postacrosome than the acrosome (Smith et al., 1998). In the latter case, D values were 10 times slower than we have recorded with ODAF, possibly because of the different nature of the probes. Differential diffusion between regions is initiated during the acrosomal stage of spermiogenesis (Wolf et al., 1986; Cowan et al. 1997) and is fully established on testicular spermatozoa (James et al., 1999). How these differences are maintained in a plasma membrane that appears morphologically continuous over the whole cell is not known but a possible reason is the presence of putative intramembranous barriers in the form of the posterior ring and annulus. These specializations are thought to be analogous to tight junctions between epithelial cells although there is no direct evidence that they can prevent lateral diffusion of either lipids or proteins in spermatozoa. It has been argued (Cowan et al., 1987, 1997) that barriers to diffusion must be present to explain the containment of PH20 glycoprotein to the posterior head of guinea pig spermatozoa and that a breakdown in the barrier has to occur during the acrosome reaction to enable directional migration to take place. Similar explanations have been invoked for migration of rat sperm glycoprotein 2B1 (the rat homologue of PH20) during capacitation (Jones et al., 1990) and CE9 antigen during epididymal maturation (Nehme et al., 1993). Compositional and/or organizational heterogeneity are also important factors. Transverse asymmetry of phospholipids in sperm membranes has been described on several occasions (Hinkovska-Galcheva and Srivastava, 1993; Nolan et al., 1995) but lateral heterogeneity between surface regions is less well documented. Cholesterol-rich and cholesterol-poor regions have been visualized within the head of hamster, ram and boar spermatozoa with filipin (Suzuki, 1990; James et al., 1999) and since cholesterol has complex effects on the behaviour of unsaturated lipids (in which spermatozoa are especially rich; Poulos et al., 1973; Jones et al., 1979; Zalata et al., 1998), it has the potential to initiate separation of fluid and gel phase lipids. DSC measurements of ram sperm membranes support this hypothesis (Holt and North, 1986; Wolf et al., 1990), as do recent findings on artificial lipid bilayers (Tocanne et al., 1994). Phase diagrams of phospholipid:cholesterol binary mixtures indicate that a critical level of sterol is ~20 mol% and that for a given temperature, abrupt changes in D occur on either side of this value (Mauritsen and Jorgensen, 1995). Thus, in theory localized differences in cholesterol concentrations in sperm membranes could lead to thermodynamically driven inplane phase separations. Although the present results do not disagree with earlier data (Palleschi and Silvestroni, 1996) that the lipids in human sperm plasma membranes are predominantly fluid phase, they indicate that at the higher resolution afforded by FRAP analysis significant amounts of immobile phase lipids are present and that the proportion is greatest overlying the midpiece. This is a consistent feature of mammalian spermatozoa, suggesting an influence of the underlying mitochondria (Wolfe et al., 1998). Large immobile phases are also characteristic of intracellular organelles, especially in plants (Metcalf et al., 1986). Other reasons for immobile lipids are interaction with the cytoskeleton, cross linking of negatively charged phospholipids by Ca21, peroxidation by oxygen free radicals and formation of

non-lamellar hexagonal-II phase structures (discussed in detail by Ladha et al., 1997). Whatever their aetiology, immobile lipids become a feature of sperm membranes following permeabilization and vary in extent between regions. In the extreme situation represented by dead bull spermatozoa they are so extensive that recovery is ,20% in all regions and diffusion cannot be measured accurately (Ladha et al., 1997). The present experiments showed that cold-shocking human spermatozoa significantly reduced D values on the head and midpiece but, unlike bull spermatozoa, this did not change the pattern of %R. Nonetheless, the consistency of only 40–50% recovery on the midpiece is noteworthy and is highly suggestive of specialized plasma membrane composition/structure in this region. Human spermatozoa are more resistant to cryodamage than animal spermatozoa (Watson, 1990; Royere et al., 1996) and recently a variety of extenders and freezing protocols have been developed specifically for the preservation of human testicular and epididymal biopsies for artificial conception by AID and ICSI (Marmar, 1998). Despite these methodological improvements, frozen human semen has significantly lower fertility than fresh spermatozoa (Behrman and Ackerman, 1967; Prins and Weidel, 1986; McLaughlin et al., 1992; PerezSanchez et al., 1994). The present results demonstrate that even when human spermatozoa survive freezing apparently undamaged, lipid diffusion in their plasma membranes is significantly compromised, especially over the acrosomal domain. Such changes, albeit subtle, would have important iterative effects on the recovery of fertilizing capacity as they would affect phenomena such as spatially dependent signalling pathways required for acrosomal exocytosis, development of membrane fusigenicity and antigen migration (Yanagimachi, 1994). It is not clear at present whether the cryoprotectants per se are having a direct effect or whether the changes are caused by low temperature, or both. Whatever the reasons, they are not irreversible since washing spermatozoa in a medium containing 0.4% PVP restored lipid diffusibility over the whole membrane to near normal levels. PVP has found a range of uses as a non-penetrating cryprotectant, for preserving the activity of purified enzymes and for preventing cells sticking non-specifically to plasticware (Watson, 1990). Its effects in the present context are probably indirect, e.g. such as displacing loosely-bound proteins (most likely seminal plasma proteins) from the surface membrane thereby altering its properties. However, the observation that washing frozenthawed spermatozoa in MRP containing 0.4% BSA did not have a significant effect on ODAF diffusion suggests that PVP is influencing the plasma membrane in ways that may not be entirely non-specific. It will be of interest in future studies to investigate the effects of other macromolecules on lipid diffusion in stressed sperm membranes. An obvious prediction from the present work is that washing human spermatozoa in PVP-containing media following cryopreservation should have a beneficial effect on their fertility. Acknowledgements This work was supported by the BBSRC and by a joint collaborative grant between IFR Norwich and The Babraham Institute. We thank

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members of staff at the Rosie and Addenbrooke’s Hospitals infertility clinic and Bourn Hall Clinic for their co-operation.

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