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Localization of Low-Density Detergent-Resistant Membrane Proteins in Intact and Acrosome-Reacted Mouse Sperm

Abstract Mammalian sperm become fertile after completing capacitation, a process associated with cholesterol loss and changes in the biophysical properties of the sperm membranes that prepares the sperm to undergo the acrosome reaction. Different laboratories have hypothesized that cholesterol efflux can influence the extent and/or movement of lipid raft microdomains. In a previous study, our laboratory investigated the identity of sperm proteins putatively associated with rafts. After extraction with Triton X-100 and ultracentrifugation in sucrose gradients, proteins distributing to the light buoyant-density fractions were cored from polyacrylamide gels and microsequenced. In this study, a subset of these proteins (TEX101, basigin, hexokinase 1, facilitated glucose transporter 3, IZUMO, and SPAM1) and other molecules known to be enriched in membrane rafts (caveolin 2, flotillin 1, flotillin 2, and the ganglioside GM3) were selected to investigate their localization in the sperm and their behavior during capacitation and the acrosome reaction. These molecules localize to multiple sperm domains, including the acrosomal cap (IZUMO, caveolin 2, and flotillin 2), equatorial segment (GM3), cytoplasmic droplet (TEX101), midpiece (basigin, facilitated glucose transporter 3, and flotillin 2), and principal piece (facilitated glucose transporter 3). Some of these markers modified their immunofluorescence pattern after sperm incubation under capacitating conditions, and these changes correlated with the occurrence of the acrosome reaction. While GM3 and caveolin 2 were not detected after the acrosome reaction, flotillin 2 was found in the equatorial segment of acrosome-reacted sperm, and IZUMO distributed along the sperm head, reaching the post- and para-acrosomal areas. Taking into consideration the requirement of the acrosome reaction for sperm to become fusogenic, these results suggest that membrane raft dynamics may have a role in sperm-egg membrane interaction.


INTRODUCTION
Mammalian sperm are highly specialized cells that undergo a complex series of differentiating processes to become fertile. These processes start in the male reproductive tract with epididymal maturation and continue in the female tract, where sperm undergo capacitation and become fully able to fertilize an oocyte [1,2]. Molecular events implicated in the initiation of capacitation include lipid rearrangements in the sperm plasma membrane that are coupled to changes in ion fluxes and to several signaling cascades. Among the changes in lipids, capacitation has long been thought to involve loss of cholesterol, leading to a different organization of the sperm plasma membrane.
The release of cholesterol appears to be necessary for the signaling events that accompany capacitation [3][4][5], including the preparation of the sperm to undergo an agonist-induced acrosome reaction. However, our understanding of how sterol efflux couples to the regulation of signal transduction pathways intrinsic to capacitation remains rudimentary. Years after the description of the plasma membrane fluid mosaic model [6], specialized platforms clustering signaling molecules were identified [7]. These structures are highly enriched in cholesterol and glycosphingolipids; this lipid composition provides their characteristic biochemical properties (insolubility in nonionic detergents such as Triton X-100 at 48C and light buoyant density after centrifugation in a sucrose gradient). Although several limitations should be taken into account [8], sucrose light buoyant detergent-resistant membranes (DRMs) have been widely used as a first approach to identify membrane rafts [9][10][11][12][13][14][15]. Current concepts attribute important signaling properties to the existence of membrane rafts acting to bring protein assemblies together [16][17][18]. In the case of mammalian sperm, it has been hypothesized that cholesterol release during capacitation modifies the function/location of proteins in sperm raft microdomains [19][20][21][22][23][24]. In an effort to elucidate the mechanisms by which the release of cholesterol couples to signaling events in the sperm, we previously conducted a proteomic analysis of the sperm light buoyant fractions and identified a number of proteins, including hexokinase 1 (HK1), testis serine proteases 1 and 2, TEX101, hyaluronidase (SPAM1), facilitated glucose transporter 3 (SLC2A3), lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin (BSG), and CRISP1 [20].
Sperm are highly polarized cells, and their membranes can be categorized into different compartments. In intact sperm, the plasma membrane on the head surrounds the anterior acrosome, the equatorial segment, and the postacrosomal region. In the tail, the membrane covers the anterior part of the flagellum (midpiece) containing the mitochondria and the posterior portion of the tail (principal piece) containing the fibrous sheath surrounding the outer dense fibers [25]. These sperm compartments should be taken into account when the role of DRM proteins is analyzed; therefore, in this study we used antibodies against a subset of these proteins to investigate their localization in mouse sperm and their behavior during capacitation. Using filipin staining, the cholesterol-rich regions have been mapped to the plasma membrane overlaying the acrosome [5,26,27]. However, our findings indicate that proteins present in the light buoyant fractions, as well as other molecules known to distribute dynamically in membrane rafts, localized in multiple sperm compartments, including the anterior head, equatorial segment, cytoplasmic droplet, midpiece, and principal piece. When the behavior of these molecules during capacitation was studied, no changes were found in the tail-resident proteins. On the other hand, while some of the molecules in the head were lost, others change their immunofluorescence pattern; these changes were observed only in sperm that had undergone acrosomal exocytosis. IZUMO, present exclusively in the acrosomal region in intact sperm, spread to the post-and para-acrosomal regions of the sperm head in acrosome-reacted cells, while flotillin 2 (FLOT2), originally in the dorsal acrosome and midpiece, appeared in the equatorial segment. Considering that sperm acquire fusogenic capacity after the acrosome reaction, these changes in DRM-associated proteins could indicate their involvement in sperm-egg interaction.

Antibodies
Specific antibodies directed against the different molecules analyzed in this study were obtained from several commercial and noncommercial sources as detailed herein. Monoclonal anti-TEX101 was generously provided by Dr. Yoshihiko Araki from Yamagata University (Yamagata, Japan). Dr. John Wilson from Michigan State University (East Lansing, MI) contributed the rabbit anti-HK1. Polyclonal anti-IZUMO was a gift from Dr. Masaru Okabe at Osaka University (Osaka, Japan). A specific polyclonal antiserum recognizing SPAM1 was obtained from Dr. Diana Myles and Dr. Paul Primakoff at the University of California, Davis. The antibody recognizing SLC2A3 was purchased from Alpha Diagnostic International. Dr. Kenji Kadomatsu from Nagoya University (Nagoya, Japan) generously provided the anti-BSG. Monoclonal antibodies reacting against FLOT1 and FLOT2 were purchased from BD Biosciences. Anti-CAV2 was obtained from Santa Cruz Biotechnology Inc. The monoclonal IgM that specifically recognizes the ganglioside GM3 was purchased from Seikagaku Corporation. Dr. Lonny Levin and Dr. Jochen Buck from the Department of Pharmacology, Weill Medical College of Cornell University (New York, NY) provided the antibody R2, which reacts against bicarbonate-dependent cyclase (ADCY10).

Sperm Preparation
Mouse sperm were collected from CD1 retired male breeders (Charles River), euthanized in accord with Institutional Animal Care and Use Committee guidelines and following experimental protocols previously approved by the Animal Care Committee of the University of Massachusetts. For the sperm to swim out, the cauda epididymis from each animal was placed in 0.5 ml of modified Whitten-Hepes medium (WH) containing 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5.5 mM glucose, 1 mM Pyruvic acid, 4.8 mM L(þ)-lactic acid hemicalcium salt in 20 mM Hepes, pH 7.3, at 378C. After 10 min, sperm suspension was diluted into capacitating media or collected and centrifuged at 500 3 g for 5 min at room temperature (RT). For capacitation, 50 ll of the original suspension was diluted into 450 ll of capacitating media (WH supplemented with 20 mM NaHCO 3 and 3 mg/ml of bovine serum albumin [BSA], A-0281; Sigma) and incubated for 60 min as previously described [28]. To induce the acrosome reaction, capacitated sperm were treated with 3 lM calcium ionophore A23187 for 30 min. Total sperm extracts were prepared as detailed in Materials and Methods, separated using 10% (A) or 15% (B) acrylamide gels and analyzed by Western blot using different antibodies directed against the corresponding proteins. Arrowheads represent bands highlighted in Figure 2.
FIG. 2. Distribution of proteins along the sucrose gradient. Sperm were extracted with 0.5% Triton X-100 and subjected to ultracentrifugation on a sucrose gradient as described in Materials and Methods. Nine fractions were collected from top to bottom and were analyzed by SDS-PAGE and Western blot using different antibodies. The molecular weights of the bands shown are in the right column. In the case of SPAM1 and ADCY10, bands shown are those marked with an arrowhead in Figure 1. A nonraft protein (ADCY10) was included as control.

Isolation of Light Buoyant-Density DRM Fractions
Sperm suspensions were centrifuged at 500 3 g for 10 min and washed with WH medium. The pellet was resuspended in 400 ll of TEN buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM edetic acid, pH 7.3) supplemented with 0.5% Triton X-100 and a protease inhibitor cocktail (1 mM PMSF, 1 mM NaF, 2 mM sodium orthovanadate, 20 lg/ml of leupeptin, 15 lg/ml of pepstatin, 0.8 mM aprotinin, 10 mM benzimidine, 3 lg/ml of TLCK, 1 mM AEBSF, 40 lM bestatin, and 14 lM E-64). This suspension was Dounce homogenized and sonicated with five brief bursts of 1 sec each. Samples were kept on ice for 5 min and then rotated at 48C for 45 min. Lysates were adjusted to 40% sucrose with the addition of 400 ll of 80% sucrose in TEN buffer and placed in the bottom of a 2-ml Beckman centrifuge tube. This suspension was gently overlaid with 800 ll of 30% sucrose in TEN buffer, followed by 400 ll of 5% sucrose in TEN buffer. The samples were then centrifuged at 200 000 3 g for 18 h in a TLS 55 swinging bucket rotor in a Beckman Optima-TLX ultracentrifuge. After centrifugation, 200-ll fractions were carefully collected from the top to the bottom of the gradient. Fractions were prepared for SDS-PAGE by the addition of 0.2 volumes of 53 nonreducing Laemmli buffer, boiled for 5 min, and kept frozen until use.

SDS-PAGE and Western Blot
Total sperm extracts were obtained by cell suspension in nonreducing Laemmli buffer and by boiling for 5 min. Before running, samples were supplemented with 5% b-mercaptoethanol when required. The SDS-denaturing gels of different acrylamide concentrations (10% or 15%) were used depending on the protein under study. After electrophoresis, proteins were electroblotted to polyvinylidene fluoride membrane and blocked with 5% skimmed milk. All incubation and washing procedures were done with PBS supplemented with 0.1% Tween 20 (PBST). Membranes were blocked for 1 h at RT and then incubated with the different first antibodies overnight at 48C as previously described [29]. After washing three times for 5 min, the specific peroxidaseconjugated secondary antibody was added, and incubation was carried out for 1 h at RT. The membranes were washed with PBST, and immune complexes were located using ECL Plus and Kodak Biomax light films.

Immunocytochemistry
Sperm were fixed in suspension by the addition of fresh formaldehyde (prepared from paraformaldehyde [final concentration, 2%]) and incubation for 30 min at 48C. After washing two times with PBS, sperm were immobilized on slides and air dried. Cells were permeabilized with 0.1% Triton X-100 and 0.2% formaldehyde for 5 min at RT. After washing with PBS, blocking was done by 30min incubation with 1% BSA in PBS. Primary antibodies were diluted in PBS supplemented with 0.1% BSA and incubated overnight at 48C. After washing with PBS, cells were incubated with the respective Alexa Fluor-conjugated secondary antibody for 60 min at RT. When double staining was required, PNA coupled to Alexa Fluor 488 was included in the solution of the secondary antibody. After washing, cells were mounted using Slow Fade (Molecular Probes).

Validation of Antibodies and Confirmation of Sperm DRM-Resident Proteins by Western Blot
We previously identified a number of proteins using a combination of one-dimensional PAGE and tandem mass  [20]. To confirm their association with the light-density fractions, antibodies against these proteins were obtained from different commercial and noncommercial sources as enumerated in Materials and Methods. Each of these antibodies was validated by Western blot of total sperm extracts as shown in Figure 1A, and the antibodies recognized proteins of the predicted molecular weight. In addition to the proteins identified previously [20], we assayed for the presence of other proteins known to be enriched in membrane rafts in many cell types such as CAV2, FLOT1, and FLOT2. Antibodies against these proteins react with polypeptides of the expected molecular weight, indicating the presence of these proteins in mouse sperm (Fig. 1B). We also tested antibodies against ADCY10, a nonraft protein.
The validated antibodies were then used to probe Western blots of sucrose gradient fractions obtained as described in Materials and Methods (Fig. 2). Among the assayed proteins, it was possible to distinguish three different gradient distributions. Although all of them were found in the light fraction used previously for tandem mass spectrometry identification [20], TEX101, SPAM1, FLOT1, and FLOT2 were present exclusively in the light fractions; IZUMO was more widespread and was also found in intermediate-density fractions; and a third set of proteins comprising SLC2A3, BSG, HK1, and CAV2 was found in most fractions, including the heavy ones. ADCY10 distributed exclusively in the heavy fractions.

DRM-Resident Proteins Compartmentalized to Different Sperm Regions
A unique feature of sperm cells is the subdivision of the plasma membrane into well-defined regional domains with different compositions and functions [25]. In addition to the domains described in the Introduction, the cytosolic droplet that is present in a fraction of cauda epididymal mouse sperm constitutes another level of complexity. To analyze the specific distribution of the sperm DRM proteins in the different membrane regions, the already-validated antibodies were used to localize the respective proteins by immunofluorescence. As shown in Figure 3, DRM proteins were found in the anterior head (IZUMO, CAV2, and FLOT2), midpiece (SLCA3, FLOT2, BSG, and SLC2A3), and principal piece (SLC2A3). TEX101, a protein that localized exclusively to the light buoyant sucrose gradient fractions, was present only in the cytoplasmic droplet.
In addition to the proteins enriched in membrane rafts, DRMs are enriched in gangliosides. Recently, fluorescently labeled cholera toxin has been used to address the presence of GM1 in sperm from different mammalian species [19,21,23,[30][31][32][33]. Although GM1 is the best studied in sperm, other gangliosides are known to be enriched in membrane rafts [34], and differential ganglioside distribution among these membrane microdomains has been reported [35][36][37]. In particular, GM3 appears to be the major ganglioside in the male reproductive system [38], and several studies [39][40][41] demonstrate its abundance in bovine, ovine, and human sperm. However, localization of GM3 on sperm has not yet been analyzed, to our knowledge. To investigate the location of GM3 in mouse sperm, a specific anti-GM3 antibody was tested by immunofluorescence. This monoclonal antibody has been validated by Kotani et al. [42,43] and was used in several studies [35,[44][45][46][47][48]. Anti-GM3 fluorescent signal was found to be restricted to the equatorial segment (Fig. 4), whereas PNA signal exclusively stained the anterior acrosomal region.  4. Detection of ganglioside GM3 in mouse sperm. Mouse sperm fixed and permeabilized as already described were incubated overnight with a monoclonal anti-GM3 antibody, followed by Alexa Fluor 555-conjugated secondary antibody (GM3). Cells were also stained with Alexa Fluor 488-conjugated PNA. No primary antibody controls were used (lower panels). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.

Sperm Acrosome Reaction Correlates with Changes in DRM Protein Immunolocalization
As previously stated, cholesterol content is relevant for the function of raft proteins and for sperm capacitation. To analyze if there was any relationship between these two events, we studied DRM proteins after incubating sperm under capacitating conditions. Most of the proteins did not change their respective immunofluorescence pattern upon incubation (data not shown). However, some proteins (IZUMO, CAV2, and FLOT2) and the ganglioside GM3 showed two different staining patterns in the capacitated population as shown in Figure 5. In the case of CAV2 and GM3, some sperm displayed no signal; in contrast, FLOT2 and IZUMO showed a different staining pattern. Although these changes were observed in the capacitated population, in all cases they correlated with the loss of PNA staining, suggesting that the differential immunofluorescence patterns were due to the acrosome reaction and were not directly related to capacitation.
To further confirm this observation, the immunolocalization of FLOT2 and IZUMO was studied after inducing the acrosome reaction with the calcium ionophore A23187. As shown in Figure 6, sperm that have lost PNA staining showed a new IZUMO localization. Although both FLOT2 and IZUMO displayed a different staining pattern in acrosome-reacted cells versus intact sperm, they did not colocalize (data not shown). While FLOT2 was restricted to the equatorial segment, IZUMO distributed into adjacent regions.

DISCUSSION
Capacitation has been associated with changes in the lipid composition of the sperm plasma membrane; in particular, cholesterol efflux induces signaling events in the sperm, including the capacitation-associated increase in tyrosine phosphorylation and the preparation of the sperm to undergo the acrosome reaction [1,5]. Previous work from our group has used a proteomic analysis of low-density DRM-resident proteins in an effort to investigate whether cholesterol efflux regulates membrane rafts during sperm capacitation [20]. The use of detergent to relate protein association to rafts is the most widely used method to analyze the composition of membrane rafts and to identify putative resident proteins [9][10][11][12][13][14]. However, this procedure has been questioned on the basis that detergent can induce the formation of membrane domains and fails to provide physiologically relevant information [8,14]. Despite these criticisms, detergent separation can still be considered a first approach to determining components of membrane rafts. In particular, although proteins not belonging to rafts could be found in the low-density DRM fractions, it is FIG. 5. Differential immunofluorescence pattern of GM3, CAV2, IZUMO, and FLOT2 in capacitated sperm. Mouse sperm incubated under capacitating conditions were analyzed by immunofluorescence as described in Materials and Methods using anti-GM3, anti-CAV2, anti-IZUMO, and anti-FLOT2, followed by the respective Alexa Fluor 555-conjugated secondary antibody. Cells were also stained with Alexa Fluor 488-conjugated PNA to relate the staining patterns to the acrosomal status. Sperm that underwent the acrosome reaction correspond to those showing the new staining patterns (arrowheads). Experiments were repeated at least three times; representative sperm are shown. DIC, differential interference contrast. Original magnification 360.

DIFFERENTIAL DISTRIBUTION OF DRM PROTEINS IN SPERM
generally believed that most proteins associated with membrane rafts will behave as detergent resistant [8]. In addition, an essential property of sperm cells is their highly compartmentalized structure [25]. Taking this into consideration, an alternative experimental approach is required to determine the original location of DRM proteins among the different sperm compartments.
In the present work, we chose a series of DRM-resident proteins to investigate their localization in sperm. These proteins were not restricted to one sperm region but were distributed among different sperm compartments; our findings regarding localization of these proteins are illustrated in Figure  7. Some of these molecules (e.g., CAV2, GM3, IZUMO, and FLOT2) change their localization during the acrosome reaction. While CAV2 and GM3 appear to be lost, IZUMO and FLOT2 show a different pattern of localization in a fraction of the capacitated sperm population. The new patterns were found only in acrosome-reacted sperm, suggesting that the changes were due not to capacitation but to the acrosome reaction.
Sphingolipids are derivatives of the long-chain amino alcohols sphingosine and dihydrosphingosine. The sphingoid long-chain base is linked to a fatty acid molecule via an amide bond-forming ceramide. The addition of carbohydrates to this ceramide backbone leads to the formation of a great diversity of glycosphingolipids, including the gangliosides GM1 and GM3 that differ only in their oligosaccharide chains. Like cholesterol, sphingolipids are found to be enriched in membrane rafts; this behavior is due to the presence of two long saturated alkyl chains that can be organized and condensed by sterols to form the liquid-ordered phase of membrane rafts [49]. In sperm, other investigations on nonprotein raft markers have focused on GM1. Using cholera toxin, this ganglioside was shown to localize to the plasma membrane overlaying the acrosome in living sperm [32]. On the other hand, using biochemical methods, it has been demonstrated by biochemical methods that GM3 is the most abundant ganglioside in the male reproductive tract and in sperm [39][40][41]; however, localization of GM3 in these cells was undetermined. In this study, we used previously validated antibodies against GM3 [42,43] to determine the immunofluorescence pattern of this ganglioside in sperm and found it to be restricted to the equatorial segment.
Considering the method used in our original study [20], the source of the proteins located in the light-density fractions could belong to more than one membrane. In addition to the aforementioned sperm compartments (e.g., head and tail), the subcellular structure of the sperm encompasses different membranes, including the plasma membrane, outer acrosomal and inner acrosomal membranes, and nuclear and mitochondrial membranes. Although results obtained from the proteomic analysis suggested the absence of mitochondria and nuclear membranes in the sperm light-density fractions, DRM proteins coming from the inner and outer acrosomal membranes were also found in these fractions. The results of this study are in agreement with this possibility and with the assorted origin of plasma membranes contributing to the light-density fractions. In the tail, localization of BSG, SLC2A3, and FLOT2 suggests that DRM can be found along the different sections comprising the sperm flagella. The results in this study provide some other insightful clues for the sperm head. IZUMO cannot be detected on live sperm and is only exposed when plasma membrane integrity is disrupted by freezing, permeabilization, or the acrosome reaction [50,51]; accordingly, it is hypothesized that this protein localizes to the inner acrosomal membrane. In this respect, the association of IZUMO with the light-density fraction provides evidence that the inner acrosomal membrane contributes to the total light buoyant DRM fractions. Even an ephemeral structure like the cytoplasmic droplet seems to preserve DRMs, as suggested by the highly restricted location of TEX101 in the light-density fractions and by the results of the immunofluorescence studies. These results confirm studies [37,49] supporting the heterogeneity of DRMs with regard to their protein and lipid composition.
Pursuing our analysis on the putative involvement of DRMresident proteins in sperm function, the location of selected proteins associated with the light-density fractions was reanalyzed in sperm incubated under capacitating conditions.   7. Schematic representation of the distribution of DRM proteins in sperm compartments depicting the results obtained using the antibodies directed against the different DRM-resident molecules analyzed in the study; HK1 localization as described previously [49] is also shown. As already noted in the text, IZUMO and FLOT2 demonstrate a different immunofluorescence pattern after the acrosome reaction (AR).

902
Although no relevant changes were noticed with capacitation, modifications in the immunofluorescence pattern of some proteins were observed in acrosome-reacted sperm. The acrosome reaction involves an extensive reorganization of the membranes located in the anterior head of the sperm cell. Multiple fusion points between the plasma and outer acrosomal membranes induce a fenestration process during which the components originally located in the apical portion of the sperm are lost [1]. After fusion, the inner acrosomal and plasma membranes become continuous, assembling a new sperm surface resulting from the mixing of their respective components. Caveolin 2, originally found in the anterior head of the sperm, could not be detected after the acrosome reaction, suggesting that this antigen is likely located at the plasma membrane covering the acrosomal cap and is consequently lost during exocytosis. In the case of GM3, although no signal was found on reacted sperm, the original localization of this ganglioside did not predict this behavior because the equatorial segment is excluded from the vesiculization process that takes place during the acrosome reaction [1]. Although the possible loss of GM3 cannot be ruled out, considering that anti-GM3 recognizes the glycoside portion of the GM3 molecule [42], the lack of signal of acrosome-reacted sperm could also be due to the removal of the respective epitope by acrosomal glycosidases released during exocytosis [1].
In contrast to CAV2 and GM3, the other two proteins analyzed, IZUMO and FLOT2, can still be detected by immunofluorescence in acrosome-reacted sperm, although they showed a different localization compared with the one originally displayed in nonreacted cells. In intact sperm, IZUMO was restricted to the dorsal portion of the sperm head; after the acrosome reaction, it distributed to a new region opposite to the anterior acrosome, including part of the postacrosomal region. Different IZUMO staining patterns have been previously observed [50], but their connection with antigen relocation due to the acrosome reaction has not been analyzed in detail. Concerning FLOT2, the staining formerly observed in the midpiece did not change with the acrosome reaction, but the signal located in the apical portion of the head extended along the acrosomal domain, revealing the presence of FLOT2 in the equatorial segment in cells that underwent the acrosome reaction. In addition to FLOT2, several proteins that have been reported to relocate after the acrosome reaction such as SPAM1, CRISP1, and IZUMO are present in the light buoyant DRM fractions [51][52][53]. This common feature suggests that changes in localization could be related to these membrane substructures rather than to a specific aspect of the individual proteins.
Although both IZUMO and FLOT2 change their location after the acrosome reaction, they do not seem to colocalize. While FLOT2 is restricted to the equatorial segment, IZUMO can move farther, crossing to the ventral para-acrosomal sperm domains. These compartments are separated by a specialized structure that seems to be defined during epididymal maturation and involves tyrosine phosporylation [54]. Other proteins reported to redistribute after the acrosome reaction are localized to the equatorial segment and are not able to pass the postacrosomal boundary [52,53]. This suggests that protein redistribution after the acrosome reaction is a regulated process that may comprise a particular mechanism for IZUMO. It is a matter to be resolved why only IZUMO among all the proteins that move after the acrosome reaction is able to cross the postacrosomal boundary and whether this has a functional significance considering its relevance in sperm-egg fusion [55].
The membrane reorganization taking place during the acrosome reaction involves an additional change of high functional significance: the sperm becomes capable of fusing with the egg. Although IZUMO has been shown to be necessary for sperm-egg fusion [55], the molecular mechanisms conferring fusogenic ability to sperm are not well understood. Membrane microdomains and their resident molecules have been implicated in processes requiring cell-cell fusion such as invasion [56,57]. With respect to the molecules depicting a different inmmunofluorescence pattern after the acrosome reaction, while FLOT2 has been positively correlated with metastatic processes [58], GM3 has been negatively associated with cell motility and invasiveness [59]. In the sperm, we can speculate that the presence of GM3 is associated with a nonfunctional equatorial segment and that GM3 modification or loss may be needed for the sperm to acquire the ability to fuse with the egg. Taking into consideration that IZUMO has a direct role in sperm-egg fusion as suggested by the null mutant phenotype [55], the putative participation of those regions where IZUMO localized after the acrosome reaction in sperm-egg fusion should be reanalyzed.