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From the Department of Biology, University of Central Florida, Orlando, Florida. * Present address: Department of Pathology, M4233 Med Sci I, 1301 Catherine St, Ann Arbor, MI 48109-0602 (e-mail: monzyt{at}umich.edu)
| Correspondence to: Dr Catherine D. Thaler, Hopkins Marine Station, Stanford University, 120 Ocean View Blvd, Pacific Grove, CA 93950 (e-mail: cthaler{at}stanford.edu). |
| Received for publication January 22, 2004; accepted for publication March 15, 2004. |
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Abstract |
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Key words: Fertilization, gametes, membrane domain, protein mobility, sperm activation
During epididymal maturation, a number of cell surface changes take place, including increases in net negative surface charge (Bedford 1963; Yanagimachi et al, 1972; Lopez et al, 1989), changes in glycoprotein amount or exposure (Nicolson et al, 1977, 1979; Liu et al, 1991), adsorption of epididymal proteins to the sperm surface (Killian and Amann, 1973; Dravland and Joshi, 1981), as well as localization of proteins to restricted domains, as observed with PH-20 (Myles et al, 1987; Phelps et al, 1990). Proteins may be modified, as in the case of a sperm surface mannosidase (Tulsiani et al, 1995), or lost, as shown by the domain-selective loss of fucosyltransferase activity from the sperm tail but not the sperm head (Ram et al, 1989).
During capacitation, additional changes in composition and distribution of surface components occur. In guinea pig, plasma membrane proteins such as PH-20, PH-30 (Myles et al, 1987), and fertilin (Cowan et al, 2001) undergo redistributions during capacitation. In boar sperm, several antigens change localization when incubated in capacitating conditions (Saxena et al, 1986; Topfer-Peterson et al, 1990). Substantial changes in cholesterol and lipid distribution and mobility have also been reported (Wolfe et al, 1998; Flesch et al, 2001).
Recent work characterizing the molecular changes associated with capacitation in mouse sperm (Visconti et al, 1995, 1998) has shown that cholesterol efflux from the plasma membrane is required for the cyclic adenosine monophosphate–dependent tyrosine phosphorylations that are correlated with sperm capacitation (Visconti et al, 1999). Changes in cholesterol content, phospholipid dynamics, and protein mobility are intimately linked to capacitation (Jones et al, 1990; Topfer-Petersen et al, 1990; Lin and Kan, 1996; Martinez and Morros, 1996; Wolfe et al, 1998; Cowan et al, 2001; Flesch et al, 2001). In many cell types, cholesterol has been associated with specific plasma membrane domains called lipid rafts (Brown and London, 1998), which may be involved in assembly of signaling complexes (Hoessli et al, 2000; Miceli et al, 2001); and it is possible that capacitation provides the mechanism for similar changes in sperm, which also possess lipid raft domains (Travis et al, 2001; Trevino et al, 2001).
Lectins have been widely used to visualize cell surface glycoproteins because of their ability to recognize specific carbohydrate moieties (Elgavish and Shaanan, 1997). Lectins have revealed sperm surface changes during epididymal maturation in the sperm of several mammals including ram (Hammerstedt et al, 1982), rat (Olson and Danzo, 1981; Tulsiani et al, 1995), rabbit (Bedford, 1963; Nicolson et al, 1977), monkey (Navaneetham et al, 1996), bovine (Killian and Amann, 1973), boar (Calvo et al, 2000), and mouse (Liu et al, 1991). Lectins also reveal changes during capacitation of bovine (Medeiros and Parrish, 1996) and monkey sperm (Navaneetham et al, 1996), and exposure of new lectin-reactive sites during and after acrosomal exocytosis in human (Centola et al, 1990) and monkey (Navaneetham et al, 1996) sperm.
In the current study, we used lectins to assess changes in sperm head plasma membrane domains, before and after incubation of cauda epididymal mouse sperm under in vitro capacitating conditions. Several lectins showed significant redistributions correlated with incubation in capacitating conditions. The extensive reorganization of proteins on the sperm head during capacitation may be required for sperm to properly recognize and interact with the oocyte and to successfully complete fertilization.
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Methods |
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Sperm Preparation
ICR mice, 12–15 weeks of age (Harlan Sprague Dawley, San Diego,
Calif) were killed, and the epididymides recovered in M2 "salts"
(94.66 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM
MgSO4, 20.85 mM HEPES, 23.28 mM Na-lactate, 5.56 mM glucose, 0.33
mM Na-pyruvate, pH 7.4). Capacitated sperm were collected from the epididymis
in complete M2 (see below). Uncapacitated sperm were collected in
phosphate-buffered saline (PBS). Based on previous work
(Visconti et al, 1995;
Harrison et al, 1996), we
decided that a noncapacitating medium should be made without BSA,
Ca2+, or bicarbonate. We conducted an initial survey to optimize
staining procedures of live sperm, and, in these assays compared the use of
sperm that were collected in PBS or in M2 "salts." We found no
significant differences in staining patterns (four lectins tested: WGA, LEA,
PNA, and STA) among sperm that were collected in PBS versus those collected in
M2 "salts" (data not shown). Because we did not detect any
differences in staining, further experiments were conducted in PBS.
Specifically, live, uncapacitated sperm were collected by mincing one
epididymis in 2 mL PBS and swirling gently on a laboratory rotator for 10
minutes at room temperature. Capacitated sperm were prepared by mincing the
epididymis in 2 mL of complete M2 (M2 "salts" plus 4 mM
bicarbonate, 0.5 mM CaCl2, and 4% BSA), and incubating at 37°C
in 5% CO2 in a humidified atmosphere for 1 hour. Populations of
acrosome-reacted (AR) sperm were obtained by incubating capacitated sperm in
10 µM ionomycin for 1 hour. Acrosomal status of the population was
determined by Coomassie staining as previously described
(Thaler and Cardullo, 1995). Sperm samples from each of these preparations were stained with lectins as
described below.
Lectin Fluorescence and Quantitation
The lectins used in this study were Arachis hypogaea (PNA),
Anguilla anguilla (AAA), Lycopersicon esculentum (LEA),
Pisum sativum (PSA), Solanum tuberosum (STA),
Tetragonolobus purpureas (TPA), Ulex europaeus I (UEA-1),
and Triticum vulgare (WGA). Suspensions of live uncapacitated,
capacitated, or AR sperm were added directly to an equal volume of lectin in
blocking solution (20% FBS in PBS for uncapacitated or AR samples, and 20% FBS
in M2 for capacitated samples). The final concentrations of lectin were 100
µg/mL for LEA, PSA, and PNA; 200 µg/mL for WGA, AAA, STA, and TPA; and
400 µg/ml for UEA-1. The sperm were incubated for 20–25 minutes
either at room temperature (uncapacitated and AR sperm samples) or at 37°C
(capacitated sperm samples). Sperm were pelleted at 200 x g.
The supernatant was removed and sperm were resuspended in 2 µg/mL
avidinD-FITC in blocking solution. Sperm were incubated for 20 minutes and
pelleted again. The supernatant was removed, and 10 µL of buffer (PBS or
M2) was added to resuspend the sperm. Background staining was determined in
parallel samples by omitting the lectin during sample preparation. No
fluorescence was detected, except for a slight autofluorescence of the
midpiece. Sperm were placed on poly-L-lysine coated slides, mounted
in Gelmount, and viewed by epifluorescence microscopy.
Fluorescence images were recorded with a Zeiss Axiphot epifluorescence microscope (Carl Zeiss Inc, Thornwood, NY) using a Kodak MDS290 digital camera and image-capture software (Kodak, Rochester, NY) or a Nikon Eclipse E600 microscope (Nikon USA, Melville, NY) using SoftwoRx for image capture (Silicon Graphics Inc, Mountain View, Calif). Images were viewed in Adobe Photoshop (Adobe Systems Inc, San Jose, Calif), and individual sperm displaying a representative staining pattern were selected for presentation.
Quantitation of staining patterns was conducted by scoring a minimum of 100 sperm for each experimental condition (ie, each lectin and capacitation state). For AR sperm, 1 experiment was performed. For uncapacitated and capacitated sperm populations, 3 independent experiments for each lectin and capacitation state were conducted. Percentage scores were calculated to normalize the data. Significant differences were identified using a Student's t test (SigmaStat, SPSS Science, Chicago, Ill).
Each lectin used in this study generated three distinct staining patterns that were found in both uncapacitated and capacitated sperm populations. We scored the 3 staining patterns given by each lectin and calculated the percentage of sperm displaying each pattern. Student's t tests were performed on the percentage of sperm staining in the uncapacitated population versus the capacitated population for each of the 3 patterns seen with a given lectin. For example, the percentage of LEA posterior head (PH) staining in uncapacitated sperm was compared to the percentage of LEA PH staining in capacitated sperm. There was a significant difference (P = .003), with uncapacitated sperm favoring this staining pattern. The LEA posterior head and acrosomal crescent (PH/AC) pattern was compared, and there was a significant difference (P = .001), with capacitated sperm favoring this staining pattern. The third pattern observed for LEA staining, AC, did not differ between the uncapacitated and capacitated sperm populations. This analysis was repeated for each of the lectins used in this study.
The lectin staining pattern, which had significantly greater representation in the uncapacitated sperm population, was chosen as the "uncapacitated pattern," and the pattern that had significantly greater representation in the capacitated sperm population was chosen as the "capacitated pattern." In several cases, no significant differences were found (P > .05). For these lectins, the patterns with the highest representation in the uncapacitated or capacitated populations were chosen as the representative patterns, and the P values are listed in Table 1.
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Results |
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In order to understand how the patterns identified in Figure 1 correlated with capacitation, we conducted additional assays, quantified the lectin staining patterns, and determined the statistical significance of these pattern changes for each of the 8 lectins. Based on our observations of the staining patterns, we distinguished 4 subdomains of the mouse sperm head: anterior head (AH), PH, AC, and postacrosomal ring (PAR). Additionally, some lectins stained the whole head (WH), in some cases in combination with more intense staining in a subdomain.
The sperm head staining patterns for both uncapacitated and capacitated sperm populations are illustrated in Figure 2. The pictorial representations of the sperm head show the staining patterns observed, and the bars show the fraction of uncapacitated (black bar) or capacitated (gray bar) sperm displaying each pattern. Seven of 8 lectins tested (LEA, PSA, PNA, AAA, UEA-1, WGA, and STA) showed a reciprocal correlation between staining pattern and capacitation status. Only 1 lectin tested, TPA, showed no apparent shift in staining pattern. Table 1 summarizes the trends in lectin staining. The lectins are presented according to the "strength" of the trend, as determined by P value from the Student's t test between uncapacitated and capacitated patterns. Several lectins exhibited a significant difference between the two staining patterns. For example, LEA stained the PH of uncapacitated sperm and the AC/PH of capacitated sperm. The reciprocal shift in staining pattern suggests that the redistribution is correlated with the process of capacitation and may reflect membrane-associated changes that occur during this process. Not all of the changes in lectin staining pattern were statistically significant. However, it is clear from the data described below that the shift in staining patterns was not the result of spontaneous acrosome reactions.
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The data in Figure 2 and
Table 1 focus on the major
staining patterns of the sperm populations; however, we observed,
consistently, a third minor staining pattern (
15%–30%) that was not
correlated with incubation conditions (ie, capacitating or noncapacitating
medium). The minor pattern was identical to the AR pattern, with the exception
of TPA. It was interesting that the AR pattern for TPA, PH, was not observed
in the staining patterns of uncapacitated or capacitated sperm samples. AR
patterns were identified by lectin staining of sperm that had been incubated
with ionomycin and were >95% AR as judged by Coomassie staining (data not
shown). The AR patterns are represented by the third, minor sperm pattern
shown in Figure 2, where the
sperm head is marked AR. The lectin staining patterns for AR sperm are
summarized in Table 2. The
fraction of sperm staining with the proposed AR pattern correlated well with
the assessment of acrosomal status by Coomassie staining.
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Discussion |
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Only some of the lectins in the study showed a significant shift in staining pattern after a 60-minute incubation under capacitating conditions. The 60-minute time point was chosen in order to minimize spontaneous acrosome reactions, which commonly accumulate during in vitro manipulations of mouse sperm. Earlier work has shown that a molecular indicator of capacitation, tyrosine phosphorylation, is significantly greater than controls by 60 minutes, and maximal by 90 minutes (Visconti et al, 1995). Tyrosine phosphorylation was correlated with significant increases in other measures of capacitation such as chlortetracycline (CTC) staining pattern and zona pellucida–induced AR (Visconti et al, 1995). Although we did not conduct an independent assessment of capacitation such as CTC staining, we reasoned that, given the previously observed trends (Visconti et al, 1995), a 60-minute incubation would enable us to visualize capacitation-related changes but still provide a high proportion of acrosome-intact sperm. It is possible that those lectins that showed a substantial, but not significant, trend toward redistribution represent cell surface proteins that undergo changes in domain localization late in the capacitation process, in contrast to the proteins that were detected by the lectin staining as significant changes by 60 minutes, and may represent proteins that have completed redistribution early in capacitation. Lipid distributions undergo changes quite rapidly (ie, within 1.5 minutes) during capacitation (Harrison et al, 1996), but the timing of protein changes is less well-characterized.
One lectin, TPA, did not show changes in staining pattern relative to capacitation status, and this suggests that only a subset of plasma membrane proteins is involved in remodeling the sperm surface during the capacitation process. Additionally, some of the changes in staining patterns, particularly those lectins in which a region of staining is lost in the capacitated population (eg, PSA and WGA), could be the result of removal of surface-adhered material during capacitation as suggested by previous researchers (Nicolson et al, 1977; Dravland and Joshi, 1981).
It is interesting to note that the "uncapacitated" sperm populations were heterogeneous with respect to distribution of lectin binding sites and a minor fraction of the population exhibited staining patterns associated with capacitated sperm. Other studies, using markers other than lectins, have noted previously that some heterogeneity exists among freshly isolated sperm populations (Ward and Storey, 1984; Harrison, 1997; Flesch et al, 2001). The functional significance of this heterogeneity is not clear, but it may indicate that not all capacitation-associated changes are strictly dependent on the inclusion of bicarbonate, BSA, and Ca2+ in the capacitating medium, as is tyrosine phosphorylation (Visconti et al, 1995). Instead, some changes may be able to occur spontaneously at a slow rate. In future work, it will be important to determine which components of the capacitating medium are required to support the cell surface changes identified in the present study.
In many cell types, changes in cholesterol content of the plasma membrane have been correlated with changes in protein-protein interactions among plasma membrane–associated proteins. These associations may include not only interactions between integral membrane proteins, but also interactions among proteins that may be scaffolded at the plasma membrane (Hoessli et al, 2000) by their ability to interact with components of particular lipid microdomains. Cholesterol content is a major factor influencing the formation of lipid microdomains, including detergent-resistant membrane domains, caveolae, and lipid rafts.
One of the hallmarks of sperm capacitation is the loss of cholesterol from the plasma membrane. Changes in cholesterol content may enable sperm to assemble lipid rafts or to alter the proteins that associate with the raft domains of the plasma membrane. Many studies have indicated that signaling components associate with lipid rafts and that their activity may be modulated by this association (reviewed in Hoessli et al, 2000). Lipid rafts have been isolated from sperm (Travis et al, 2001; Trevino et al, 2001) and both signaling components (eg, trp Ca2+ channels; Trevino et al, 2001), and exocytotic machinery (eg, syntaxin-2; Travis et al, 2001) appear to be colocalized with these domains.
Recent work with mouse sperm has demonstrated that efflux of cholesterol from the sperm plasma membrane during capacitation is both necessary and sufficient to stimulate tyrosine phosphorylation of several sperm proteins (Visconti et al, 1999). The changes that occur during capacitation are not entirely understood; however, capacitation appears to alter the zona pellucida responsiveness of mouse sperm: uncapacitated mouse sperm can bind both soluble and intact zona glycoproteins but do not display significant stimulation of acrosomal exocytosis, whereas capacitated sperm respond to zona binding by undergoing the acrosome reaction (Visconti et al, 1998).
These observations suggest that one of the necessary changes that occurs during capacitation may be the redistribution of plasma membrane components to enable the interaction and activation of signaling components that modulate sperm function during gamete interactions. The redistribution of lectin-reactive components in the mouse sperm head plasma membrane shown here is consistent with this hypothesis. Further work is needed to identify specific players for which redistribution is required to make sperm fertilization competent. Additionally, it will be important to correlate in vitro data with in vivo data, such as the lectin staining patterns shown here, in order to understand sperm membrane structure and to identify a fully capacitated state.
In summary, lectins provide an excellent tool for studying protein redistributions and have enabled us to observe changes in plasma membrane components as sperm undergo capacitation. The lectins and their representative staining patterns provide a tool that will be useful for identifying the status of sperm populations and can be used to characterize acrosomal status as well as capacitation status of mouse sperm.
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Acknowledgments |
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Footnotes |
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