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
P.S.James et al.
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
1828
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.
1829
P.S.James et al.
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
1831
P.S.James et al.
members of staff at the Rosie and Addenbrooke’s Hospitals infertility
clinic and Bourn Hall Clinic for their co-operation.
References
Behrman, S.I. and Ackerman, D.R. (1967) Freeze preservation of human
semen. Am. J. Obstet. Gynecol., 103, 654–658.
Canvin, A.T. and Buhr, M.M. (1989) Effect of temperature on the fluidity of
boar sperm membranes. J. Reprod. Fertil., 85, 533–540.
Cowan, A.E., Myles, D.G. and Koppel, D.E. (1987) Lateral diffusion of the
PH-20 protein on guinea pig sperm: evidence that barriers to diffusion
maintain plasma membrane domains in mammalian sperm. J. Cell. Biol.,
104, 917–923.
Cowan, A.E., Nakhimovsky, L., Myles, D.G. and Koppel, D.E. (1997) Barriers
to diffusion of plasma membrane proteins form early during guinea pig
spermiogenesis. Biophys. J., 73, 507–516.
Dale, B. and Elder, K. (1997) Cryopreservation. In In Vitro Fertilization.
Cambridge University Press, Cambridge, pp 144–147.
Drobnis, E.Z., Crowie, L.M., Berger, T. et al. (1993) Cold shock damage is
due to lipid phase transitions in cell membranes: a demonstration using
sperm as a model. J. Exp. Zool., 265, 432–437.
Friend, D.S. (1982) Plasma membrane diversity in a highly polarized cell.
J. Cell. Biol., 93, 243–249.
Hinkovska-Galcheva, V. and Srivastava, P.N. (1993) Phospholipids of rabbit
and bull sperm membranes: structural order parameter and steady-state
fluorescence anistropy of membranes and membrane leaflets. Mol. Reprod.
Dev., 35, 209–217.
Holt, W.V. (1984) Membrane heterogeneity in the mammalian spermatozoon.
Int. Rev. Cytol., 87, 159–194.
Holt, W.V. and North, R.D. (1986) Thermotropic phase transitions in the
plasma membrane of ram spermatozoa. J. Reprod. Fertil., 78, 447–457.
James, P.S., Wolfe, C.A., Ladha, S. and Jones, R. (1999) Lipid diffusion in
the plasma membrane of ram and boar spermatozoa during maturation in
the epididymis measured by fluorescence recovery after photobleaching.
Mol. Reprod. Dev., 52, 207–215.
Jones, R., Mann, T. and Sherins, R. (1979) Peroxidative breakdown of
phospholipids in human spermatozoa, spermicidal properties of fatty acid
peroxides, and protective action of seminal plasma. Fertil. Steril., 31,
531–537.
Jones, R., Shalgi, R., Hoyland, J. and Phillips, D.M. (1990) Topographical
rearrangement of a plasma membrane antigen during capacitation of rat
spermatozoa in vitro. Dev. Biol., 139, 349–362.
Jones, R., Ma, A., Hou, S.-T. et al. (1996) Synthesis and endoproteolytic
processing of the migrating antigen 2B1 on rat spermatozoa. J. Cell. Sci.,
109, 2561–2570.
Ladha, S., James, P.S., Clark, D.C. et al. (1997) Lateral mobility of plasma
membrane lipids in bull spermatozoa: heterogeneity between surface
domains and rigidification following cell death. J. Cell. Sci. 110, 1041–1050.
Lenzi, A., Picardo, M., Gandini, F. and Dondero, F. (1996) Lipids of the
sperm plasma membrane: from polyunsaturated fatty acids considered as
markers of sperm function to possible scavenger therapy. Hum. Reprod.
Update, 2, 246–256.
Mann, T. (1964) In The Biochemistry of Semen and The Male Reproductive
Tract. London, Methuen, p. 347.
Marmar, J.L. (1998) The emergence of specialized procedures for the
acquisition, processing, and cryopreservation of epididymal and testicular
sperm in connection with intracytoplasmic sperm injection. J. Androl., 19,
517–526.
Mauritsen, O.G. and Jørgenssen, K. (1995) Micro-, mono-, and micro-scale
heterogeneity of lipid bilayers and its influence on macroscopic membrane
properties. Mol. Membr. Biol., 12, 15–20.
McLaughlin, E.A., Ford, W.C.L. and Hull, M.G.R. (1992) Motility
characteristics and membrane integrity of cryopreserved human
spermatozoa. J. Reprod. Fertil., 95, 527–534.
Metcalfe, T.N., Wang, J.L. and Schinder, M. (1986) Lateral diffusion of
phospholipids in the plasma membrane of soybean protoplasts: evidence
for membrane lipid domains. Proc. Natl Acad. Sci. USA, 88, 95–99.
Myles, D.G. and Primakoff, P. (1997) Why did the sperm cross the cumulus?
To get the oocyte. Functions of the sperm surface proteins PH-20 and
fertilin in arriving at, and fusing with, the egg. Biol. Reprod., 56, 320–327.
Nehme, C.L., Cesario, M.M., Myles, D.G. et al. (1993) Breaching the diffusion
barrier that compartmentalizes the transmembrane glycoprotein CE9 to the
posterior tail plasma membrane domain of the rat spermatozoan. J. Cell.
Biol., 120, 687–694.
1832
Nolan, J.P., Magorgee, S.F., Posner, R.G. and Hammerstedt, R.H. (1995) Flow
cytometric analysis of transmembrane phospholipid movement in bull
sperm. Biochemistry, 34, 3907–3915.
Palleschi, S. and Silvestroni, L. (1996) Laurdan fluorescence spectroscopy
reveals a single liquid-crystalline phase and lack of thermotropic phase
transitions in the plasma membrane of living human sperm. Biochem.
Biophys. Acta, 279, 197–202.
Perez-Sanchez, F., Cooper, T.G., Yeung, C.H. and Neischlag, E. (1994)
Improvement in quality of cryopreserved human spermatozoa by swim-up
before freezing. Int. J. Androl., 17, 115–120.
Poulos, A., Darin-Bennett, A. and White, I.G. (1973) The phospholipid-bound
fatty acids and aldehydes of mammalian spermatozoa. Comp. Biochem.
Physiol. B., 46, 541–549.
Prins, G.S. and Weidel. L. (1986) A comprehensive study of buffer systems
as cryoprotectants for human spermatozoa. Fertil. Steril., 46, 147–149.
Rodriquez-Boulan, E. and Nelson, J.W. (1989) Morphogenesis of the polarized
epithelial cell phenotype. Science, 245, 718–725.
Royere, D., Barthélémy, C., Hamamah, S. and Lausac, J. (1996)
Cryopreservation of spermatozoa: a 1996 review. Hum. Reprod. Update, 2,
553–559.
Smith, T.T., McKinnon-Thompson, C.A. and Wolf, D.E. (1998) Changes in
lipid diffusibility in the hamster sperm head plasma membrane during
capacitation in vivo and in vitro. Mol. Reprod. Dev., 50, 86–92.
Suzuki, F. (1990) Morphological aspects of sperm maturation: modification
of the sperm plasma membrane during epididymal transport. In Banister,
B.D., Cummins, J. and Roldan, E.R.S. (eds), Fertilization in Mammals.
Serono Symposia, Norwell, pp. 65–75.
Tocanne, J.F., Cézanne, L.D. and Lopez, A. (1994) Lateral diffusion of lipids
in model and natural membranes. Prog. Lipid. Res., 33, 203–237.
Watson, P.F. (1990) Artificial insemination and the preservation of semen. In
Lamming, G.E. (ed.), Marshall’s Physiology of Reproduction. Churchill
Livingstone, Edinburgh, vol. 2, pp. 747–869.
Wolf, D.E., Scott, B.K. and Millette, C.F. (1986) The development of
regionalized lipid diffusibility in the germ cell plasma membrane during
spermatogenesis in the mouse. J. Cell Biol., 103, 1745–1750.
Wolf, D.E., Maynard, V.M., McKinnon, C.A. and Melchior, D.L. (1990) Lipid
domains in the ram sperm plasma membrane demonstrated by differential
scanning calorimetry. Proc. Natl Acad. Sci. USA, 87, 6893–6896.
Wolfe, C.A., James, P.S., Mackie, A.R. et al. (1998) Regionalized lipid
diffusion in the plasma membrane of mammalian spermatozoa. Biol.
Reprod., 59, 1506–1514.
World Health Organization (1992) WHO Laboratory Manual for the
Examination of Human Semen and Sperm–Cervical Mucus Interaction.
Cambridge University Press, Cambridge.
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill,
J.D. (eds), The Physiology of Reproduction, 2nd edn. Raven Press, New
York, pp. 189–317.
Zalata, A.A., Christophe, A.B., Depurydt, C.E. et al. (1998) The fatty acid
composition of phospholipids of spermatozoa from infertile patients. Mol.
Hum. Reprod., 4, 111–118.
Received on April 4, 1999; accepted on April 19, 1999