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From the Baker Institute for Animal
Health, College of Veterinary Medicine, Cornell University, Ithaca, New York;
Department of Biology, Seton Hall University,
South Orange, New Jersey; and the Population
Council, The Rockefeller University, New York, New York.
| Correspondence to: Alexander J Travis, Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (e-mail: ajt32{at}cornell.edu). |
| Received for publication December 1, 2006; accepted for publication March 19, 2007. |
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Abstract |
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Key words: Rafts, ganglioside, cholesterol, cholera toxin, annulus
In vitro capacitation of sperm using such stimuli effects changes in membrane properties that have been reported to lead either directly or indirectly to several downstream events. These include plasma membrane hyperpolarization (Zeng et al, 1995; Arnoult et al, 1999), cyclic adenosine monophosphate-dependent protein kinase A activation and protein tyrosine phosphorylation (Visconti et al, 1995a; Visconti et al, 1995b), loss of plasma membrane bilayer phospholipid asymmetry and lipid order (Harrison et al, 1996; Gadella and Harrison, 2000; Flesch et al, 2001; Gadella and Harrison, 2002; Cross, 2003), phosphatidyl inositol signaling-mediated cytoskeletal remodeling (Brener et al, 2003; Breitbart et al, 2005), and calcium influx/release from internal stores (Ho and Suarez, 2001a, 2001b; Carlson et al, 2003; Herrick et al, 2005). However, it remains unclear how the changes at the level of the membrane and the downstream signaling events are transduced into hyperactivated motility in the tail and priming of the membranes of the sperm head for acrosomal exocytosis (AE). Although it has been shown that only a subpopulation of sperm responds to the stimuli for capacitation through protein tyrosine phosphorylation (Urner et al, 2001), there is lack of reliable and easy means to evaluate the capacitation status of sperm in response to a given stimulus/stimuli, be it within a population or a comparison between populations.
Currently, the most widely used assay for capacitation status involves patterns of fluorescence intensity in the sperm head using the fluorescent antibiotic chlortetracycline (Saling and Storey, 1979). More recently, studies on sperm membrane lipid organization have led to assays using merocyanine 540 to detect changes in packing order of lipids on the outer leaflet of the plasma membrane (Williamson et al, 1983) that are believed to change with capacitation (Rathi et al, 2001). In addition, annexin V has been used to bind and detect phosphatidyl serine on the outer leaflet of the plasma membrane indicating activation of phospholipid scramblase activity (Flesch et al, 2001). However, concerns about the effectiveness of merocyanine 540 in detecting capacitated vs abnormal/damaged sperm have been raised (Muratori et al, 2004), and phospholipid scramblase-mediated phosphatidyl serine exposure in capacitated sperm does not appear to be conserved in all species (Baumber and Meyers, 2006). Further complicating the study of changes to the state of the plasma membrane of the sperm head is the organization of this membrane into discrete micron-scale subdomains based on sterol and sphingolipid composition (Friend and Fawcett, 1974; Selvaraj et al, 2006). In the heads of fixed sperm from several species, the plasma membrane overlying the acrosome (APM) was found to be enriched in sterols and distinctly segregated from the postacrosomal plasma membrane (PAPM) that was found to be relatively sterol poor (Friend, 1982, 1989; Pelletier and Friend, 1983; Lin and Kan, 1996). In addition, within the APM are at least 2 distinct areas of membrane—one over the apical acrosome (AA) and a larger one over the equatorial segment (ES) (Friend, 1989; Lin and Kan, 1996; Selvaraj et al, 2006).
Membrane rafts are defined as small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Groups of small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (Pike, 2006). However, studies on membrane rafts have generated significant controversy regarding the existence and dynamics of these subdomains in cells—specifically, that artifacts appearing as rafts might be induced by the use of detergents or crosslinking reagents/fixatives in attempts to visualize these subdomains (Munro, 2003).
To visualize potential membrane subdomains in live sperm in the absence of any fixative, we used the B subunit of cholera toxin (CTB) to bind the ganglioside GM1. We found that this sphingolipid does indeed segregate to the APM, as did sterols and the sterol-binding protein caveolin-1 in fixed sperm (Travis et al, 2001b). Other investigators have reported the use of fluorescent lipid probes to suggest that the APM subdomain behaves in a liquid-ordered fashion consistent with a raft (Sleight et al, 2005). The size and stability of the APM and PAPM subdomains in mammalian sperm are quite extreme in comparison with their counterparts in somatic cells, making it possible that the APM of live sperm represents a "super raft" of stably segregated smaller sub-subdomains. Fitting the proposed theory behind larger raft platforms, we found that the lipid segregation in sperm is maintained at least in part by disulfide-bonded proteins (Selvaraj et al, 2006). This is consistent, albeit at a larger scale, with a membrane compartmentation model of segregation (Kusumi et al, 2004). We found that this segregation to the APM was highly conserved across mammals, being present in murine, bovine, and human sperm, and that discrepancies in the literature between species were at least in part due to confounding effects of seminal plasma (Buttke et al, 2006). The organization of these membrane subdomains in sperm continues to be of great interest because of the pathways that potentially might be targeted to the APM super raft, which could function in capacitation, binding to the zona pellucida, and/or AE.
Although able to demonstrate distinct segregation of endogenous lipids in live sperm, the pitfalls inherent to localizing lipids in a biological system did reveal themselves in our studies. For example, we observed the interesting phenomenon that within seconds of a sperm's death (inferred by cessation of motility), CTB bound to GM1 helped induce a dramatic redistribution to the PAPM (Selvaraj et al, 2006). In the present study, we show a similar redistribution phenomenon seen in the sperm tail while exploring variations in sperm GM1 dynamics in response to stimuli for capacitation and AE. Our results not only demonstrate changes in individual cells but also shed light on the nature of functional subpopulations of sperm and the temporal dynamics of capacitation pathways.
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Methods |
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Preparation of Media
For murine sperm, a modified Whitten medium (MW; 22 mM HEPES, 1.2 mM
MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic
acid hemicalcium salt, pH 7.35 [Travis et
al, 2001a]) was used for all incubations. Glucose (5.5 mM),
NaHCO3 (10 mM), and 2-hydroxypropyl-ß-cyclodextrin (2-OHCD; 3
mM) were supplemented as needed. The 2-OHCD supports sperm capacitation and in
vitro fertilization (IVF) by functioning as a sterol acceptor and is preferred
over the more potent methyl-ß-cyclodextrin
(Visconti et al, 1999).
For diluting and transporting bovine semen, a HEPES-buffered Tyrode-albumin lactate pyruvate medium (TALP-H; 100 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 21.6 mM sodium lactate, 0.4 mM MgCl2, 40 mM HEPES, 0.4 mM ethylenediamine tetraacetic acid [EDTA], 10 mM NaHCO3, 2 mM CaCl2, 1 mM pyruvic acid, 1 mg/mL polyvinyl alcohol [PVA] [Parrish et al, 1988]) was used. For washing and incubation of bovine sperm, TALP medium (100 mM NaCl, 3.1 mM KCl, 0.3 mM NaH2PO4, 21.6 mM sodium lactate, 0.4 mM MgCl2, 10 mM HEPES, 0.4 mM EDTA, 25 mM NaHCO3, 2 mM CaCl2, 1 mM pyruvic acid, 1 mg/mL PVA [Parrish et al, 1988]) was used. Bovine serum albumin (BSA; 6 mg/mL) and heparin (20 µg/mL) were supplemented as needed to facilitate capacitation (Galantino-Homer et al, 1997).
Sperm Collection and Handling
Murine sperm were collected from the cauda epididymides of male CD-1 mice
by a swim-out procedure as described previously
(Travis et al, 2001b). All
steps of collection and washing were performed at 37°C using MW medium,
and large-orifice transfer pipettes or large-orifice pipette tips were used
for handling sperm to minimize membrane damage. Bull semen collected from
proven high-fertility bulls was immediately diluted (1:4) using TALP-H and
transported to the laboratory at 39°C. All steps of washing and sperm
handling were performed at 39°C as described previously
(Buttke et al, 2006). After the
initial washes but prior to experimental incubations, motility assessment was
carried out for both mouse and bull sperm, and samples showing less than 60%
motility for murine sperm and less than 80% motility for bovine sperm were not
used.
Sperm Capacitation and Induction of AE
For murine sperm, incubation with different stimuli for capacitation was
carried out with 2 x 106 sperm in 300 µL of medium with
glucose under 1 of 4 conditions: (a) MW base medium, (b) MW supplemented with
10 mM NaHCO3, (c) MW supplemented with 3 mM 2-OHCD, and (d) MW with
both 10 mM NaHCO3 and 3 mM 2-OHCD for 45 minutes (or 60 minutes for
all conditions when inducing AE). The pH of medium for all incubation
conditions was adjusted to 7.35. The medium in incubation condition
("d") has been shown to be sufficient to support IVF
(Travis et al, 2004) and
capacitation-induced tyrosine phosphorylation
(Travis et al, 2001a) in
murine sperm. Progesterone was added to a final concentration of 20 µM to
induce AE in capacitated murine sperm
(Roldan et al, 1994;
Murase and Roldan, 1996;
Kobori et al, 2000) (A 2 mM
working stock was prepared in MW immediately before use from a 20 mM stock of
progesterone in dimethylsulfoxide [DMSO]; 0.2% vol/vol final DMSO
concentration.). The dead spaces of tubes used for all incubations were filled
with nitrogen to avoid the generation of bicarbonate anions in the aqueous
media in conditions "a" and "c." This had no effect on
protein tyrosine phosphorylation events associated with capacitation (data not
shown).
For bovine sperm, incubation with different stimuli for capacitation was carried out with 2 x 106 sperm in 300 µL of medium under 1 of the 4 conditions: (a) TALP base medium, (b) TALP supplemented with BSA (6 mg/mL), (c) TALP supplemented with heparin (20 µg/mL), (d) TALP with both BSA and heparin at the same concentrations for 90 minutes at 39°C. Lysophosphatidyl choline (100 µg/mL final concentration) was used for the induction of AE (Parrish et al, 1989). For live sperm of both species, the localization pattern of GM1 was visualized using CTB after incubation under one of the conditions described and/or after the induction of AE.
Fluorescence Localization of GM1 in Mature Sperm
All steps of localization experiments using either live or fixed sperm were
carried out under dim lighting at 37°C in a humidity chamber. In all
cases, the localization of GM1 was visualized with CTB. For
localization of GM1 in fixed samples, sperm were allowed to adhere
to coverslips for 20 minutes (at the end of incubations for capacitation
experiments) and then fixed for 10 minutes with either 0.004% paraformaldehyde
(PF) in phosphate buffered saline (PBS) for murine sperm or 1% PF with 12.5 mM
CaCl2 in PBS for bovine sperm. The sperm were then washed with PBS
and incubated for 10 minutes with CTB (5 µg/mL). The sperm were washed
again and mounted using a GVA mountant (Invitrogen). For all conditions in
experiments evaluating pattern change associated with AE, murine sperm were
fixed while in suspension by adding an equal volume of 0.008% PF, were
incubated with CTB as above, and aliquots then placed directly on slides for
microscopy.
Microscopy and Image Collection
Cells were viewed with a Nikon Eclipse TE 2000-U microscope (Nikon,
Melville, NY) equipped with a Photometrics Coolsnap HQ CCD camera (Roper
Scientific, Ottobrunn, Germany) and Openlab 3.1 (Improvision, Lexington, Mass)
automation and imaging software. Assignments of sperm to GM1
localization patterns were performed in a blind fashion regarding incubation
condition. To compare shifts in population tendencies, the numbers of sperm
having a given pattern were converted to percentages prior to statistical
evaluation. In all cases, 100 or more cells were counted for each test
condition, and every sperm in a given field was counted to avoid potential
bias. Sperm with morphologic abnormalities showing aberrant GM1
localization patterns (as described in
Buttke et al, 2006) were not
included in the count.
Scanning Electron Microscopy
SEM was performed using 2 techniques to visualize surface topography. In
the first method, sperm were fixed for 2 hours with 2.5% glutaraldehyde in 100
mM sodium cacodylate and 1% tannic acid at pH 7.4 in a culture tube. After
washing by centrifugation and resuspension, they were again fixed with 2%
osmium tetroxide and 2% sodium cacodylate at 4°C overnight. The cells were
washed and dehydrated in ethanol with a 20-minute incubation in 2% uranyl
acetate at 70% ethanol. Once in absolute ethanol, they were critical point
dried, coated, and viewed using a Hitachi S4500 scanning electron microscope
(Hitachi, Pleasanton, Calif). Digital micrographs were collected using a
Princeton Gamma Tech digital beam acquisition program (Imix, Princeton,
NJ).
In the second method, sperm were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate (pH 7.4) overnight at 4°C. A 20 µL aliquot of sperm suspension was then washed in 100 mM sodium cacodylate buffer by centrifugation and resuspension. Aliquots were placed on poly-L-lysine–coated 12-mm round coverslips and allowed to incubate for 45 minutes at 4°C. The coverslips were rinsed by gentle dipping 3 times in cacodylate buffer and then dehydrated in ethanol followed by 2 changes in 100% ethanol for 20 minutes each. Coverslips were critical point dried, sputter-coated with gold/palladium (Au/Pd) and imaged on a Hitachi S4700 cold field-emission scanning electron microscope operating at 10-kV accelerating voltage and 7-µA emission current. Digital images were captured at 2500 x 1900 pixel resolution.
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Results |
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For murine sperm we used 0.004% PF in PBS, which has been reported to be sufficient to immobilize sperm but not permeabilize their membranes (Harrison and Vickers, 1990). After fixation, noncapacitated murine sperm showed GM1 over the PAPM as previously observed (Selvaraj et al, 2006) but, in the presence of capacitating stimuli, new patterns of GM1 distribution were seen (Figure 1A). These patterns were highly reproducible in terms of specific patterns occurring in response to specific stimuli. Under all conditions, labeling over the APM similar to that seen in live sperm was only seen in rare cells after weak fixation. In the presence of NaHCO3, a significant percentage of cells (36.1% ± 2.5%) showed an incomplete redistribution to the PAPM with residual GM1 labeling over the AA (the AA/PA pattern; Figure 1B) when compared with noncapacitated sperm. In the presence of 2-OHCD, a significant percentage of cells (42.0% ± 3.4%) had GM1 diffusely distributed over the entire APM in addition to the PAPM (the D pattern; Figure 1B) when compared with noncapacitated sperm. The presence of both NaHCO3 and 2-OHCD caused no additional increase in the percentages of sperm showing either the AA/PA or the D patterns (Figure 1B).
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Also unlike murine sperm, a very small percentage of bovine sperm showed the PAPM pattern and a large percentage of sperm showed the APM pattern under all conditions (Figure 2B). Incubation with heparin was associated with a significant increase in a diffuse localization, pattern D (70.9% ± 3/7%; Figure 2B). In the presence of BSA, there were no significant changes in patterns vs incubating sperm under noncapacitating conditions, but when both BSA and heparin were present in the medium, there was a significant increase in a pattern showing localization in both the AA and ES (AA/ES pattern; 39.1% ± 9.6%; Figure 2B). These patterns in bovine sperm were similar but did not correspond directly to the patterns seen in murine sperm. However, the GM1 redistribution in response to weak fixation (0.004% PF) in noncapacitated sperm from both species was from the sterol-rich APM to the sterol-poor PAPM (Buttke et al, 2006; Selvaraj et al, 2006).
GM1 Localization in Acrosome-Reacted Sperm
Distribution of GM1 in live epididymal murine and bovine sperm
remained unchanged even after incubation with stimuli for capacitation.
However, induction of AE using progesterone in capacitated murine sperm and
lysophosphatidyl choline in capacitated bovine sperm showed an increase in a
pattern distinct from those seen after exposure to stimuli for capacitation
followed by fixation as described above. After capacitation, a subset of
murine and bovine sperm induced to undergo AE demonstrated a pattern
characterized by a hollow AA suggestive of loss of both acrosomal contents and
membranes from the AA (the AR pattern;
Figure 3A and B). In both
species, this pattern was characterized by the AA showing somewhat ragged
borders consistent with the nature of vesiculations associated with this
exocytotic process. In bovine but not murine sperm, this GM1
pattern in the AA was associated with a weak PAPM signal. In murine sperm,
there was a significant increase in the AR pattern after the induction of AE
(21.8% ± 1.3%; Figure
3C) when compared with sperm incubated under noncapacitating and
capacitating conditions. This increase in AR pattern was associated with a
significant decrease in the D pattern after the induction of AE when compared
with sperm incubated under capacitating conditions (D pattern in capacitated
sperm [48.9% ± 2.8%] and after AE [32.5% ± 1.6%]).
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GM1 Localization in the Midpiece and Principal Piece
GM1 was also seen in the flagella of both murine and bovine
sperm. In live sperm, the CTB signal was faint and appeared diffuse (data not
shown). After fixation with 0.004% PF for murine sperm and 1% PF for bovine
sperm, the localization of GM1 in the midpiece did not conform to a
specific pattern. In murine sperm, it appeared either distributed evenly
across the entire midpiece or was somewhat more prominent in the distal half
to third of the midpiece (Fig
1A), and in bovine sperm the midpiece labeled more uniformly
(Figure 2A). In murine sperm,
GM1 became greatly enriched in the plasma membrane at the region of
the annulus, where the mitochondrial sheath of the midpiece abuts the fibrous
sheath of the principal piece (Friend and
Fawcett, 1974) (Figures
4 and
1A). In the principal piece,
GM1 fluorescence was seen as a fine line coursing caudally from the
annulus (Figure 4). To our
knowledge, there is only 1 linear feature that runs the length of the plasma
membrane in the principal piece. This is a distinct membrane subdomain known
as the flagellar zipper that has been identified primarily through the use of
freeze fracture techniques in several species
(Friend and Fawcett, 1974;
Lin and Kan, 1996). We now
show by SEM that the flagellar zipper is a morphologically distinct membrane
subdomain within the flagellar plasma membrane
(Figure 5A). In an SEM
micrograph of a demembranated sperm (Figure
5B), structures underlying or comprising internal components of
the annulus and the flagellar zipper can be seen.
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Discussion |
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Although GM1 redistribution upon cell death appears to be induced by CTB, variations in the pattern of GM1 revealed several important points of information about the nature of membrane changes during capacitation, both within single cells and at the level of sperm populations. Supporting the hypothesis that different populations of sperm exist within a single ejaculate or collection, only a subset of both murine and bovine sperm (approximately 40%) responded fully to stimuli for capacitation. This figure corresponded to the approximate percentage of sperm showing protein tyrosine phosphorylation in response to incubation under capacitating conditions (Urner et al, 2001).
Interestingly, in murine sperm we observed that both 2-OHCD and NaHCO3 could independently alter membrane properties, showing that unlike either existing model for the chronology of response to stimuli for capacitation, both stimuli could induce membrane changes as an initial event. Here the D pattern was indicative of sperm that responded to sterol efflux (2-OHCD), and the AA/PA pattern indicated sperm that responded to NaHCO3. If the 2 patterns were induced together in the same sperm, they would overlap to provide a D pattern. Because there was no net increase in the percentage of sperm showing the D pattern when both stimuli were included in the medium, this result indicates that the sperm that responded to the presence of NaHCO3 were the same population as those that responded to sterol efflux.
These results suggested that the D pattern represented capacitated sperm. Capacitation can be assessed by means of different end points and is most rigorously defined by the ability to fertilize an egg. However, use of that end point would not allow visualization of the membrane GM1 pattern. Therefore, we used the acquisition of the ability to undergo AE as a marker for capacitation. We saw an increase in the AR pattern after AE and an almost identically sized decrease in the D pattern in this treatment. These results showed that it was indeed the population of sperm having the D pattern that responded to progesterone, which would be consistent with the D pattern being a marker for capacitated sperm. This finding confirmed our hypothesis that CTB labeling of GM1 could function as an indicator of membrane changes associated with capacitation, being able to identify populations of sperm responding to specific capacitating stimuli.
In bovine sperm, there were no significant changes in patterns compared with noncapacitated sperm even after exposure to BSA, which is commonly used to mediate sterol efflux. However, the presence of heparin alone did induce an increase in the D pattern in bovine sperm, suggesting that this stimulus could independently exert effects on bovine sperm membranes. The AA/ES pattern emerged only when both heparin and BSA were present in the medium, revealing that bovine sperm require both stimuli to show the full extent of membrane changes suggested by these GM1 localization patterns. Appropriately, similar patterns were not observed with bovine epididymal sperm (data not shown), suggesting that epididymal sperm of this species did not effectively respond to capacitating stimuli. This finding supports existing literature showing that epididymal bovine sperm incubated under capacitating conditions fail to undergo zona pellucida protein-mediated AE (Florman and First, 1988).
There have been several studies demonstrating changes in membrane distribution or mobility of membrane raft-associated proteins and/or lipids in the sperm head with capacitation (Cowan et al, 2001; Roberts et al, 2003; Cross, 2004; Shadan et al, 2004; Belmonte et al, 2005; van Gestel et al, 2005). Some of these studies have also examined GM1 localization in sperm from different species, with varying results. In the mouse, it was suggested that GM1 localizes to the PAPM and that this localization does not change with capacitation (Trevino et al, 2001); another study localized GM1 to the midpiece and moving to the head during capacitation (Shadan et al, 2004). Both these studies were done at 16°C, and phase transitions between this and physiologic temperatures (Wolf et al, 1990) might account for some disparity with our results. In rat sperm, it was suggested that GM1 localizes to the PAPM and then moves to the APM during capacitation (Roberts et al, 2003). In both this and the study in murine sperm showing no movement, it is clear that the initial localization to the PAPM was an effect of fixation condition (Selvaraj et al, 2006). In human sperm, it was reported that GM1 has a diffuse localization pattern and then assembles in the APM (Cross, 2004). Also as discussed, this was likely due to exposure of sperm to seminal plasma (Buttke et al, 2006).
Possible Explanation for the Redistribution Phenomenon
Based on our observations and current models of sperm membrane properties,
we have arrived at one possibility that could explain why CTB induced
GM1 redistribution from the APM to the PAPM. We suggest that once
crosslinked by CTB, GM1 moves from sterol-rich, liquid-ordered
membrane regions to sterol-poor, less-ordered areas on the sperm. In both live
and fixed sperm, it has been suggested that in noncapacitated sperm, the
sterol-rich APM is a liquid-ordered subdomain whereas the sterol-poor PAPM is
liquid disordered (Sleight et al,
2005).
Therefore, in sperm incubated under noncapacitating conditions, GM1 redistributed to the PAPM, being excluded from or forced out of the sterol-rich, more-ordered APM. Upon incubation of murine sperm with NaHCO3 in the presence of calcium (stimulators of sperm phospholipid scramblases [Gadella and Harrison, 2000]), some GM1 redistributed to the PAPM, but some also remained in the AA. The aminophospholipid transporter (SAPLT) with homology to a flippase localizes to this region (Wang et al, 2004). Studies comparing sperm from wild-type vs SAPLT-null mice suggest that phospholipid scramblase activity in this region depends on the activity of the flippase as well as the presence of NaHCO3 (Wang et al, 2004). Therefore, the presence of NaHCO3 could be inducing liquid disorder at the region of the AA giving rise to the AA/PA pattern. Sterol efflux from murine sperm has been shown to occur throughout the APM (Visconti et al, 1999), increasing lipid disorder throughout this subdomain (Cross, 2003; Sleight et al, 2005). Accordingly, there was a diffuse pattern of GM1 localization throughout the head (APM and PAPM) of sperm incubated with 2-OHCD. The presence of both NaHCO3 and 2-OHCD induced no additional increase in the percentage of sperm, suggesting again that these populations of sperm were the same.
Also supporting the notion that crosslinked GM1 redistributed to membrane subdomains of reduced-order/lower-sterol abundance was the redistribution seen in the flagellum of murine sperm upon death or fixation with 0.004% PF. Originally diffuse throughout the flagellum, GM1 became concentrated at the annulus and the flagellar zipper of the principal piece, structures shown by freeze fracture to be devoid of sterols (Pelletier and Friend, 1983; Lin and Kan, 1996).
Our data on the stimulus-specific patterns of changes in GM1 localization suggest strongly that temporally sperm can respond to NaHCO3 or mediators of sterol efflux independently of one another. This finding provides a refinement to existing models of capacitation. Yet, in addition to these points of basic science, our findings also suggest a clinical application for GM1 as a marker for detecting capacitation-associated membrane changes in murine and bovine sperm. Changes in localization patterns of GM1 in response to specific stimuli for capacitation could provide a diagnostic tool for predicting a male's reproductive fitness based on the proportion of sperm that are capable of responding to such stimuli. This information could be used in agricultural industries to make broad classifications regarding a male's fertility or could be used to help guide clinicians when choosing between techniques such as in vitro fertilization or intracytoplasmic sperm injection. Furthermore, because this potential assay is based upon functional membrane responses, it might also be useful when evaluating or comparing media or conditions used to handle sperm in vitro or to cryopreserve them.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this article and share
coauthorship. 
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