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From the * Gamete Signalling Laboratory, The
Babraham Institute, Cambridge, United Kingdom; the
Department of Biochemistry, Sofia University,
Sofia, Bulgaria; and the Institute of
Reproductive Medicine of the University, Münster, Germany.
| Correspondence to: Dr Roy Jones, Gamete Signalling Laboratory, The Babraham Institute, Cambridge CB3 0NQ, United Kingdom (e-mail: roy.jones{at}bbsrc.ac.uk ). |
| Received for publication September 20, 2001; accepted for publication November 27, 2001. |
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Abstract |
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Key words: Photobleaching analysis, lipid dynamics, fertilizing capacity, germ cells
In addition to compositional changes, repositioning of specific components of the sperm's plasma membrane during maturation has been suggested as one of the ways by which spermatozoa regulate their functionality in relation to the external environment. Notable examples of antigens that undergo spatial rearrangement are fertilin in the guinea pig (Hunnicutt et al, 1997) and the transmembrane glycoprotein CE9 in the rat (Petruszak et al, 1991). In both cases, migration is preceded by endoproteolysis and ectodomain shedding. How glycoproteins with classic transmembrane domains such as fertilin and CE9 are able to move against large concentration gradients and across putative intramembranous barriers such as the annulus is not known, but it may depend on some special property of the membrane or its underlying cytoskeleton. It is known that actin assembly/disassembly is especially sensitive to pH (Schafer and Cooper, 1995) and that migration of certain glycoproteins within the membrane (eg, guinea pig PH20 and its rat ortholog 2B1) is dependent on extracellular Ca2+ (Cowan et al, 1986; Jones et al, 1990). The pH, osmotic pressure, and ionic balance of the epididymal luminal fluid are regulated mainly by the principal cells lining the epididymal duct (Hinton and Palladino, 1995; Clulow et al, 1998; Brown and Breton, 2000; Gong et al, 2000), thereby providing a simple and direct mechanism for the epididymis to influence sperm plasma membrane organization and control intracellular signaling pathways. This is demonstrated clearly in the c-ros knockout mouse, where the initial segment fails to differentiate during neonatal development (Sonnenberg-Riethmacher et al, 1996), and hence, the appropriate signals between the epithelium and spermatozoa are missing. The result is formation of hairpin-shaped sperm after their release from the epididymis as a consequence of their inability to regulate volume (Yeung et al, 1999). The c-ros knockout mouse therefore provides a suitable model to investigate the effects of sperm-epithelium cross talk and cell swelling on membrane functionality.
To understand more about the response of the sperm's plasma membrane to the
constantly changing conditions within the epididymal lumen, we have applied
the technique of fluorescence recovery after photobleaching (FRAP) to measure
the rates of diffusion of fluorescent lipid analogs across this membrane
(Ladha et al, 1997; James et al, 1999;
Mackie et al, 2001). Lipids
comprise 55%-60% of a cell membrane, and because they are relatively small
molecules, they are 
50 times more abundant than proteins. Together with
their diverse and heterogeneous nature, they have a major influence on the
overall properties of a membrane. In this report, we have: 1) compared lipid
diffusion in the plasma membranes of mouse sperm collected from the caput and
cauda epididymides, 2) investigated their response to temperature, pH, and
osmotic pressure of the medium, and 3) examined if the c-ros knockout
model has any subtle defects in lipid diffusion in the membranes of sperm from
the cauda epididymidis.
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Materials and Methods |
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Collection and Labeling of Epididymal Spermatozoa With ODAF,
NBD-C6-PC, and NBD-Cholesterol Reporter Probes
Solutions of 5 mM ODAF in 100% ethanol were freshly prepared on the day of
the experiment (the concentration was estimated from its molar absorbance
coefficient [E497nm = 85 x 103 cm-1
M-1; Wolfe et al,
1998]). NBD-C6-PC was stored as a stock solution (10
mg/mL) in 100% ethanol at -20°C until required. Spermatozoa were collected
from the caput and cauda epididymides/vas deferens by carefully mincing the
tissue in Whittingham medium (Whittingham,
1971) supplemented with 20 mM HEPES; pH 7.5, 300 mOsm/kg unless
otherwise stated) and 0.3% (wt/vol) bovine serum albumin (BSA), followed by
gentle shaking for 30 minutes at room temperature. Sperm concentration was
estimated from hemocytometer counts and adjusted to between 0.5 and 1.0
x 107/mL. Fifty microliters of a sperm suspension was mixed
with 50 µL of 3.5 µM ODAF in Whittingham medium containing 2% (vol/vol)
ethanol and incubated in the dark for 10 minutes at room temperature
(20°C-22°C). Spermatozoa were washed twice with 1 mL glucose- and
BSA-free Whittingham medium at 350 x g for 5 minutes and
finally resuspended in 0.5 mL of the same medium.
For labeling with NBD-C6-PC, caput and cauda sperm were released in BSA-free Whittingham medium, and then 50 µL of this sperm suspension was mixed with 50 µL of 12.5 µM NBD-C6-PC in Whittingham medium containing 2% (vol/vol) ethanol. Sperm suspensions were incubated for 30 minutes at 35°C in the dark and washed twice as described above. A similar protocol was used for labeling with NBD-cholesterol.
To investigate the effects of pH on lipid diffusion, spermatozoa were
released into Whittingham medium previously adjusted to pH 6.5, 7.5, or 8.5
with HCl (osmolality maintained between 290 and 305 mOsm/kg), followed by
staining and washing with NBD-C6-PC in the same medium throughout.
Similarly, the osmolality of Whittingham medium was adjusted to 200-300
or 400 mOsm/kg by altering the levels of NaCl while maintaining the pH at
7.5 with 20 mM HEPES buffer. Osmolality was measured by freezing point
depression using an Advanced Instruments Micro-Osmometer model 3M0 (Boston,
Mass).
Measurement of Lipid Diffusion
Detailed descriptions of the FRAP instrumentation and data analysis systems
have been published (Ladha et al,
1997; Wolfe et al,
1998). Prior to FRAP, spermatozoa were viewed in full-field
fluorescence, and only intact spermatozoa showing uniform fluorescence
throughout the head and tail (known as "live pattern" spermatozoa)
were selected for analysis (Ladha et al,
1997; Wolfe et al,
1998). ODAF was excited at 488 nm and NBD-C6-PC and
NBD-cholesterol at 457 nm using an argon laser. Two parameters relevant to
lipid diffusion—diffusion coefficient (D) and percentage of mobile
fraction (%R)—were recorded from 5 different regions of mouse
spermatozoa: equatorial segment, postacrosomal region, midpiece, cytoplasmic
droplet, and principal piece. Unless stated otherwise, measurements were made
at room temperature (20°C-22°C). All experiments were repeated at
least 3 times using sperm from a minimum of 3 different animals.
Statistics
Statistical analysis was performed using Microsoft Excel 97 SR-1 and
SigmaStat (Jandel, Erkrath, Germany); the Student's t test was used
for samples of unequal variance and for samples with normal distribution, and
the Rank Sum test was used for other distributions.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Although not quantified here, we have good reasons to believe that ODAF and NBD-C6-PC remain primarily in the outer leaflet of the membrane bilayer. When labeled caput or cauda sperm are incubated in Whittingham medium containing 5% fatty acid-free BSA, the intensity of background fluorescence increases rapidly, while that associated with the sperm declines until they are barely visible (James et al, unpublished data). This is consistent with back exchange of the fluorescent lipid from the sperm membrane by the BSA. If ODAF or NBD-C6-PC had been translocated to the inner leaflet, then, provided the plasma membrane is intact, back exchange would have been considerably reduced, and sperm would have remained fluorescent. Given the nature of the probes, this is not unexpected since phosphatidylcholine is found mainly in the outer leaflet of most cell membranes (Devaux, 1991).
Effects of Temperature on ODAF Diffusion in Cauda Sperm Plasma
Membranes
Temperature is well known to influence the phase disposition of lipids in
cell membranes (Lee and Chapman,
1987). This should be reflected in FRAP data, provided the probe
is in contact with the hydrophobic region of the bilayer. If the probe is
bound electrostatically or remains within the water microlayer on the membrane
surface, then diffusion will be very fast and relatively insensitive to
temperature. As shown in Figure
2, lowering the temperature to 6°C significantly reduced
D values on all regions of the sperm relative to those at 20°C. The
reduction was 3.9-fold on the equatorial segment, 3.5-fold on the
postacrosome, and 3.0-fold on the midpiece. Conversely, at 37°C, D values
increased 3.4-fold on the equatorial segment, 6.1-fold on the postacrosome,
and 4.3-fold on the midpiece relative to 20°C (differences significant,
P <.01). These results therefore indicate that ODAF diffusion in
the plasma membrane of live mouse cauda sperm behaves in a manner consistent
with the known response of lipids to shifts in temperature and confirm that
the probe is in contact with the hydrophobic region of the bilayer. Assuming a
linear response between 20°C and 37°C, the rates of change of D values
were 3.99/°C for the equatorial segment, 4.53/°C for the postacrosome,
and 1.35/°C for the midpiece.
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Comparison of ODAF Diffusion in Caput vs Cauda Sperm Plasma
Membranes
Having confirmed the validity of the reporter probes for measuring lipid
diffusion in the plasma membrane of mouse spermatozoa, immature sperm from the
caput epididymidis were compared with mature sperm from the cauda
epididymidis. As in all of the above experiments, only live cells showing
uniform staining over their entire length were analyzed. The effects of
epididymal maturation were confined mostly to the plasma membrane overlying
the head region (Figure 3a). In
the equatorial segment and postacrosomal regions, there was a 2.0- and
1.5-fold increase, respectively, in D values between caput and cauda
(differences significant, P <.01). No significant differences were
found on the midpiece or the membrane surrounding cytoplasmic droplets, but on
the principal piece, D values doubled (differences significant, P
<.01). Significant differences were also detected in %R in the equatorial
segment, postacrosomal region, and principal piece plasma membranes between
caput and cauda sperm (Figure
3b), although mean values were very close. Percentage recoveries
on the midpiece were consistently less than 50% on both caput and cauda
sperm.
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Effects of pH on Lipid Diffusion in Caput and Cauda Sperm Plasma
Membranes
Sperm are exposed to a mildly acidic pH (6.5) as they pass through the
epididymal duct that is created by proton pumps in the epithelial cells lining
the duct (Cooper, 1998). To
investigate whether external pH has any influence on lipid diffusion, caput
and cauda sperm were labeled with NBD-C6-PC (ODAF fluorescence is
pH sensitive and therefore unsuitable for these experiments) at pH 6.5, 7.5,
and 8.5, and diffusion coefficients on the equatorial segment and
postacrosomal regions were measured by FRAP analysis. As shown in
Figure 4a, on caput sperm,
there were small but significant differences in D values on the equatorial
segment between pH 7.5 vs 6.5 and pH 7.5 vs 8.5. The postacrosomal region also
showed a significant difference at pH 6.5. Lipid diffusion in cauda sperm,
however, was largely insensitive to pH changes within the ranges investigated
(Figure 4b), the only
significant difference measured being on the equatorial segment at pH 8.5 vs
7.5. It is also evident from Figure
4 that, irrespective of the pH, there was a significant increase
(P <.01) in D values for NBD-C6-PC between caput and
cauda sperm on both the equatorial segment and postacrosomal regions,
confirming the results obtained earlier with ODAF (see
Figure 3).
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ODAF Diffusion in Cauda Sperm Plasma Membranes From c-ros Knockout
Mice
Male c-ros (-/-) mice are infertile in natural matings due to the
appearance of hairpin-shaped sperm in the uterus that are unable to penetrate
the uterotubal junction (Yeung et al,
2000). The primary effect of the c-ros mutation is a
failure of the initial segment of the epididymis to differentiate correctly
during postnatal development. Since hairpin-shaped sperm from c-ros
(-/-) males cannot control cell swelling (Yeung et al,
1998,
1999), this failure could be
due to inadequate levels of intracellular osmolytes required for volume
regulation or to an altered state of channels in the plasma membrane that
control osmolyte efflux. Thus, we investigated if this was reflected in a
disturbance to lipid diffusion in their plasma membranes. For this purpose,
cauda sperm from c-ros (-/-) and (+/+) mice were labeled with ODAF
and subjected to FRAP analysis. As shown in the Table, there were no
significant differences between the 2 types of sperm for D values on any
region of the plasma membrane. Similarly, values for %R were not significantly
different (data not shown).
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Effects of Osmotic Pressure on ODAF Diffusion in Cauda Sperm Plasma
Membranes
In view of the lack of any measurable effect of the c-ros knockout
model on lipid diffusion in the plasma membrane of cauda sperm, we next
investigated the effects of varying the osmotic pressure of the suspending
medium. Epididymal fluid from normal mice is hyperosmotic (430 mOsm/kg in
the cauda epididymidis: Yeung et al,
1999). In hypo-osmotic conditions, however, sperm swell, and their
tails become coiled or hairpin shaped. This should decrease lipid packing in
the plasma membrane (at least over the tail) and may influence lipid
diffusion. This possibility was investigated by suspending normal ODAF-labeled
cauda sperm in media of different osmolalities (range, 202-389 mOsm/kg). No
significant differences in D values (Table) or %R (data not shown), however,
were observed on any region of sperm exposed to extremes of osmotic
pressure.
Diffusion of NBD-Cholesterol in Caput vs Cauda Sperm Plasma
Membranes
It has been estimated that 70% of the cholesterol in a cell is present
in the plasma membrane, the remainder being found in the Golgi and associated
endoplasmic reticulum (Lange,
1991,
1992). Very little cholesterol
is present in mitochondrial membranes or the nuclear envelope. Filipin has
been used extensively to visualize cholesterol distribution in cells, as it is
strongly autofluorescent under ultraviolet light. However, it readily
permeabilizes cells and hence will gain access to cholesterol in intracellular
membranes, lipid droplets, etc. To determine the diffusion of cholesterol in
the plasma membrane of mouse sperm, therefore, we introduced NBD-cholesterol
exogenously rather than using filipin as a probe. FRAP analysis of caput and
cauda sperm showed that, unlike ODAF and NBD-C6-PC, diffusion of
NBD-cholesterol in all regions of the plasma membrane did not vary
significantly during maturation (Figure
5). It was noticeable, however, that in both caput and cauda
sperm, D values for NBD-cholesterol on the sperm tail were 10 times lower than
on the sperm head.
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Discussion |
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Although D values for NBD-C6-PC on the mouse sperm head are slightly higher than for ODAF (Figure 1), both probes reveal the same differential pattern between regions. That is, they show higher rates of diffusion on the equatorial segment than on the postacrosome, which, in turn, shows higher rates than on the midpiece/principal piece. With the exception of guinea pig sperm (Wolfe et al, 1998), this is a consistent finding in mouse sperm (Wolf et al, 1986) and other mammalian species and emphasizes the physiological as well as the morphological compartmentalization of the sperm plasma membrane. It is not known how this lateral heterogeneity in the plasma membrane is initiated during spermiogenesis in the testis and subsequently maintained during maturation in the ep ididymis and capacitation in the female tract, but longstanding possibilities include intramembranous barriers, regional differences in lipid phase disposition, and influence of the cytoskeleton (reviewed by Ladha, 1998). A role for the posterior ring and annulus as barriers to lipid diffusion has recently been questioned (Mackie et al, 2001). Instead, the present results suggest that in the epididymis, changes to the plasma membrane overlying the sperm head take place independently of those on the tail. At present, it is not known whether this is a consequence of maturation-associated changes in phospholipid composition (Jones, 1998) or whether it is a secondary effect caused by uptake of epididymal secretory proteins, many of which bind to specific regions of the plasma membrane (Cooper, 1998). The addition of a new cohort of glycoproteins to the sperm surface would not be inconsequential, as the so-called surface "glycocalyx" on cells is known to have a strong influence on lateral diffusion of membrane components (Zhang et al, 1991). The approximately twofold increase in ODAF diffusion on the equatorial segment and postacrosome between the caput and cauda mouse sperm is less than that reported for the ram (approximately a fourfold difference; James et al, 1999) but similar to that for the dog, goat, and marmoset (YC, PSJ, RJ, unpublished data).
The responsiveness of the mouse sperm plasma membrane to changes in
temperature between 20°C and 37°C is of particular interest in
relation to sperm survival in the cauda epididymidis. It is known that
mammalian spermatozoa can survive for several weeks in the cauda/vas deferens
regions before they lose their viability and that this may be related to the
lower temperature in the scrotum (Foldesy
and Bedford, 1982). Bedford
(1991) has proposed that the
requirement for a lower temperature for sperm survival has been a major
selective force in the evolution of the scrotum. Assuming a temperature
differential of 2°C adjacent to the cauda epididymidis
(Brooks, 1973), then, following
deposition in the female reproductive tract, spermatozoa will be exposed to
37°C-38°C. This sudden 5°C-6°C increase in temperature would
effectively double lipid diffusion rates on the mouse sperm head. Essentially
similar effects have been found with bull sperm
(Ladha et al, 1997). Migration
of many glycoproteins between surface domains during maturation, capacitation,
and acrosome reaction is temperature sensitive (eg, PH20:
Myles and Primakoff, 1984; 2B1: Jones et al, 1990; CE9:
Cesario et al, 1995), so the
reduced rates of membrane lipid diffusion in the cauda epididymidis may be one
of the contributing factors that sustains sperm in a quiescent state before
ejaculation.
Cholesterol is known to be heterogeneously distributed in cell membranes being segregated into cholesterol-rich and cholesterol-poor domains (Schroeder et al, 1991). The former domains, known as "rafts," also contain concentrations of sphingomyelin, gangliosides, and a variety of GPI-anchored proteins that are involved in signal transduction (Simons and Toomre, 2000). Removal of cholesterol from these rafts leads to dispersal of their components and, in the case of mammalian sperm, initiation of intracellular phosphorylation pathways normally associated with capacitation (Visconti et al, 1999). Since capacitative changes to spermatozoa in the epididymis have to be held in check, cholesterol-binding proteins in epididymal fluid (in the mouse membrane protein [ME1]; Nakamura et al, 2000) may function as a cholesterol reservoir to maintain high levels in the plasma membrane, thereby preventing spontaneous acrosome reactions. However, the steady-state equilibrium for cholesterol exchange between the sperm plasma membrane and binding proteins in epididymal fluid is not known. Together with the poor temperature response and consistently low %R of the midpiece, the 10-fold lower diffusion rate for NBD-cholesterol in this region suggests a different lipid and protein environment, although the influence of the underlying mitochondria and cytoskeleton cannot be excluded. Filipin-accessible sterol first appears in the mouse sperm membrane in the corpus epididymidis, and the number of complexes per square micrometer increases progressively toward the cauda and vas deferens (Toshimori et al, 1985; Suzuki, 1988). It is tempting to speculate that varying cholesterol levels in maturing sperm membranes represent a subtle mechanism for regulating phenomena such as antigen migration and signal transduction in relation to the changing external fluid. As gene expression is highest in the mouse caput and corpus epididymides, it may be predicted from the underdeveloped caput of the c-ros (-/-) males that less ME1 would be secreted with appropriate consequences for membrane remodeling of the contained sperm. However, the lipid diffusion properties of (+/+) and (-/-) sperm membranes indicated no major differences in biophysical properties between the 2 genotypes. Since loss of tail angulation after detergent treatment of (-/-) sperm demonstrates that the hairpin-shaped flagellum is restrained within a confining membrane (Yeung et al, 1999), a comparison of hypoosmotically treated (+/+) spermatozoa was made. It has been shown that, under these conditions, sperm swell, implying a decrease in lipid "packing" in the plasma membrane (Petrunkina et al, 2001; Yeung and Cooper, 2001). If this was the case, then it had no measurable effect on lipid dynamics as measured by FRAP analysis.
In conclusion, our results show that lipid diffusion in the plasma membrane overlying the sperm head is faster than on the tail and that this difference increases during epididymal maturation. The plasma membrane on the midpiece contains an unusually high proportion of immobile lipids and is less temperature sensitive than that on the sperm head, especially between 20°C and 37°C. Variations in external pH and osmotic pressure of the medium have relatively little effect on this diffusion. Artificially reducing the concentration of cholesterol in the sperm head plasma membrane during epididymal maturation has the potential to induce premature acrosome reactions, either during storage in the cauda or after ejaculation, suggesting new avenues for contraceptive development in the male.
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Footnotes |
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); and MP, midpiece (
). *Significantly lower than at
20°C, P <.01. **Significantly higher than at
20°C, P <.01. Values are means plus or minus standard error of
the mean of 30-35 spermatozoa from 3 experiments.
