Simultaneous Study of Cholesterol and GM1 Ganglioside by Specific Probes: Lipid Distribution during Maturation, Capacitation and the Acrosome Reaction

Lipid distribution has been extensively studied in the plasma membrane of the male gamete due to its relevant role in sperm physiology. However, there are controversial reports about their distribution. Because of different reports about this topic, the aim of this work was define cholesterol and GM1 ganglioside location by simultaneous detection during sperm maturation, capacitation and the acrosome reaction in CF1 mouse sperm. We observed that throughout the epididymis, cholesterol detected by filipin III or complex and GM1 ganglioside by cholera toxin, colocalized in the acrosomal domain of the head and midpiece of the flagellum. During sperm capacitation, a decrease of cholesterol can be observed like diffuse pattern on the whole head, while GM1 ganglioside was restricted to the apical acrosome. Capacitated sperm incubated with acrosome reaction inducers showed a diffuse location on the head with both probes after acrosomal exocytosis. Also, both probes did not show different locations on the flagellum. These findings indicate that cholesterol and GM1 ganglioside have a dynamic behaviour during the life of the sperm, which could indicate that a continuous assembly or disassembly of the lipid raft could be involved in the responsiveness of the sperm to different environments. Thus, during sperm capacitation and acrosome reaction there is a redistribution of the lipids. However, there are not apparent changes during sperm maturation. Cholesterol efflux, a characteristic event of sperm capacitation, could be analyzed through diffuse pattern described by filipin, and GM1 ganglioside patterns observed by the cholera toxin could be interesting when it is needed to evaluate sperm capacitation and the acrosome reaction in fixed sperm.


Introduction
The plasma membrane is considered as a whole and it cannot assert that there is a random distribution of its components; in contrast, they are highly defined. Molecular association generates a heterogeneous plasma membrane, allowing them to establish scaffolding for a wide variety of cellular functions within different regions. These regions of membrane have been defined as microdomains or "rafts", and they are enriched in cholesterol (CHL), glycosphingolipids, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and diverse signal transduction proteins [1]. Their organisation restricts the access of proteins in such a way that only those attached to lipid raft reside in them [2]. The male gamete membrane is an interesting model in the study of specific lipids and proteins. The spermatozoa have lipids and proteins that stabilize the cell during its transit through both the male and female genital tracts. A number of these molecules become attached to the sperm surface during its transit through the epididymal duct, whereas other factors become associated to the sperm surface during ejaculation, when sperm cells are mixed with secretor fluids (seminal plasma) from diverse accessory sex glands [3]. Mammalian sperm undergo morphological, biochemical and functional changes during epididymal transit, known as sperm maturation [4]. However, these sperm are unable to fertilize an oocyte, and they must undergo sperm capacitation in the female reproductive tract [5,6]. To acquire fertilizing capacity, male gamete need to be in contact with molecules present in the female. This results in hyperactivated motility because of changes in metabolism and intracellular signaling cascades and the sperm capacitation also results in the readiness of sperm cells to bind to the cumulus layer and the zona pellucid [7][8][9]. Besides changes in the flagellum, sperm capacitation is also related to the ability of spermatozoa to undergo the acrosome reaction. Although the acrosome reaction is needed for fertilization and initially this process, it is considered part of the capacitation process, and it is a post-capacitation event. Acrosome reaction is required for fertilization and it is considered part of the sperm capacitation, and these processes are temporally coordinated/ activated by similar signaling pathways [10]. Lipid rafts are involved in physiological changes that occur during sperm maturation, capacitation and acrosome reaction, as relative amounts of these specific molecules, the re-positioning of antigens from one region to another and the exchange of proteins with epididymal fluid [9,[11][12][13]. For example, CHL efflux is known to be an early event closely associated with signaling cascades correlated to an increase in protein tyrosine phosphorylation (p-Y) during sperm capacitation [7,[14][15][16]. Although lipid rafts have been studied during sperm life, their organization and function remains a controversial topic. Several authors have reported different points of view regarding the existence of lipid rafts in nature, or if they represent an artifact induced by the applied methods for their study [17][18][19][20][21]. Fixed and live sperm has been demonstrated to have an acrosomal plasma membrane rich in CHL, while the postacrosomal plasma membrane is relatively poor and it appears to be conserved in mammals [22][23][24][25].

Mouse sperm collection and samples preparation
Mice were sacrificed by cervical dislocation. Sperm from caput and cauda epididymis were obtained by cutting the epididymal tubules and swim out into 0.5 mL of HMB or HM media, for capacitating or noncapacitating conditions, respectively. Sperm suspension was diluted to a final concentration of 5-10 × 10 6 cells/ml in fresh medium and incubated for 90 min at 37°C in an atmosphere of 5% CO 2 in the air, maintaining a constant pH 7.4 [31].

Motility and sperm vitality
After incubation, sperm viability was evaluated by yellowish eosin staining (Eosin Y), following the method described in the World Health Organization Laboratory Manual for the Examination and Processing of Human semen [32]. Briefly, sperm from each incubation condition were mixed with equal amounts of 0.5% (w/v; final concentration) Eosin Y prepared in phosphate buffered saline (PBS: 0.01 mol l -1 phosphate buffer, with the addition of 0.137 mol l -1 NaCl and 0.0027 mol l -1 KCl). Sperm suspensions were observed under an optical microscope, and one hundred cells were counted, differentiating unstained (live) and stained (dead) sperm cells. This technique was also used to check the motility (progressive and in situ) expressed as percentage of motile sperm over 100 cells.

Tyrosine phosphorylation: sperm capacitation status
Tyrosine phosphorylation detected by immunoblot assay was used to establishing the sperm capacitation status [14,15]. Sperm aliquots under capacitating and non-capacitating conditions were concentrated by centrifugation at 800 g for 15 min at room temperature, washed twice in PBS containing 0.2 mmol l -1 Na 3 VO 4 (unspecific phosphatase inhibitor). These aliquots were resuspended in sampler buffer (25 mmol l -1 Tris, 0.5% SDS and 5% glycerol, pH 6.8) without β-mercaptoethanol [33], and boiling for 10 min. After centrifuging at 9,000 g for 15 min, the supernatant was recovered and frozen until use.
Total protein extracts were supplemented with 5% β-mercaptoethanol (v/v, final concentration) and boiled for 10 min. Proteins were subjected to SDS-PAGE, using 10% mini-gels and performed for 120 min at 100 mV in electrophoresis equipment Miniprotean II (Bio-Rad, Hercules, CA). Protein extracts loaded per lane were equivalent to 5 -10 × 10 6 sperm. Molecular weight markers (Bio-Rad, Hercules, CA) were run in parallel.
Immunodetection was performed at room temperature for 1 h with the monoclonal anti-phophotyrosine antibody (clone PY20, ICN Biomedicals) 1:5,000 in TTBS. Post-incubation membranes were washed three times in TTBS. Biotin-conjugated anti-mouse IgG (Sigma-Aldrich) was used as secondary antibody (1:1,250) and horseradish peroxidase-conjugated extravidine (Sigma) was added (1:750), both were placed in blocking buffer with a period of incubation (GM1) still remain unclear and its behavior has been largely discussed because of the different results reported in the literature, which depend on the method used for its study. Previous reports have observed GM1 acrosomal distribution in live sperm at physiological temperature; while the postacrosomal location was found in dead or weakly fixed cells [20].
Another study suggests that GM1 is located in postacrosomal domain both in non-capacitated and in capacitated sperm. These results indicate that GM1 is restricted to the head, but during the acrosome reaction changes its distribution to the midpiece of the flagellum [26]. Other authors reported that GM1 is found in the midpiece, but not in the sperm head. Besides, GM1 has also been shown to cross from the tail to the head in capacitated cells [27]. In rat sperm, GM1 was observed in the postacrosome domains and then moved to the acrosome during capacitation [28]. Finally, in human sperm, GM1 has been reported to present a uniform distribution and this does not change during sperm capacitation [29]. However, these assays were performed below the physiological temperature and/or various fixing methods have been employed, both conditions could the cause of different reports. Visualization of these membrane domains in living cells is very valuable, but their study is technically difficult. Moreover, the use of fixed cells is an important tool in male reproduction research and/or in predictive diagnosis. The aim of this work is to define the simultaneous location of CHL and GM1 by epifluorescence microscopy, using specific lipid probes in spermatozoa under a prefixative condition. A postfixative condition was also used as an alternative way to study in living cells. In the present study, lipid rafts undergo changes across epididymal transit (maturation), sperm capacitation and acrosome reaction. Cholesterol maintains its acrosomal location in immature cells, whereas during sperm capacitation there was loss. Ganglioside GM1 during epididymal transit has an acrosome distribution similar to the CHL. After sperm capacitation, apical acrosomal location predominates for GM1. However, during acrosome reaction its apical acrosomal distribution disappears as well as isothiocyano-fluoresceinated Pisum sativum (Edible pea) agglutinin (FITC-PSA) signal, indicating acrosome exocytosis. Fluorescence patterns obtained by both probes could be used when it is necessary to evaluate sperm capacitation and acrosome reaction. Therefore, specific experiments were conducted to analyze the lipid changes during sperm maturation, capacitation and acrosome reaction, to contributing to the knowledge of sperm physiology. of 1 h at room temperature. Excess of first and second antibodies was washed three times in TTBS. Detection was accomplished with an enhanced chemiluminescence system (ECL; Amersham Biosciences) and subsequent exposure to Eastman Kodak Co. XAR film for 5-30 s.

Cholesterol and GM1 ganglioside detection by filipin and cholera toxin
Lipids were located by specific probes: filipin complex or filipin III (FIL) for CHL and cholera toxin subunit beta AlexaFluor 594 conjugate (CTB) for GM1. The methodology is based on protocols selected from literature and preliminary experiments [27,35].
FIL stock solution (7.6 mmol l -1 ) and CTB stock solution (1.8 × 10 -3 mmol l -1 ) were prepared dissolving FIL in dimetyl-sulfoxide (DMSO) and CTB in PBS, respectively. Both solutions were stored in the dark at -20°C in aliquots for single use under a nitrogen atmosphere.
The study of both lipids was performed under prefixative and postfixative conditions.
In prefixative conditions, capacitated and non-capacitated sperm from cauda and caput epididymis were fixed for 30 min at room temperature with 4% (w/v) paraformaldehyde (PF) in PBS prepared at the time, pH 7.4. Then, spermatozoa were washed by centrifugation at 800 g for 15 min at room temperature to remove the excess of PF and then resuspended in PBS [25,[35][36][37][38][39].
Filipin (0.15 mmol l -1 in PBS) and CTB (1.8 × 10 -4 mmol l -1 in PBS) were applied sequentially to the spermatozoa for 60 and 30 min, respectively. This lipid detection protocol was carried out in the darkness at room temperature in a moisture chamber.
In postfixative condition, capacitated and non-capacitated sperm from cauda and caput epididymis were previously treated with specific probes, and then fixed for 30 min at room temperature with 4% (w/v) PF in PBS. The final concentration of each specific probe and incubation times were the same as those described above.
All samples were analyzed by phase contrast and fluorescence microscopy (see below). Samples without treatment with specific probes were tested to check the possibility of spurious signal.

Lipid specific probes and fluorescence patterns
Several authors have divided the sperm plasma membrane into five domains known as the acrosome, the equatorial segment, the postacrosome, the midpiece and the principal piece [22,[40][41][42]. In view of these plasma membrane domains, fluorescence patterns were defined for both specific probes independently of the incubation/ treatment conditions: diffuse pattern with weak signal on the whole head (Figure 1a
After incubation and centrifugation at 800 g for 15 min at room temperature, samples were fixed for 30 min at room temperature with 4% (w/v) PF in PBS, pH 7.4. Sperm were washed in PBS to remove the excess of PF and stored at 4°C until use.

Evaluation of acrosome reaction by coomassie blue staining
Coomassie Blue staining was performed to assess acrosome reaction status in capacitated cauda sperm [46]. Capacitated sperm under acrosome reaction induced were sequentially treated for 5 min with distilled water, methanol, and a second wash with distilled water. Samples were incubated with a 0.22% (w/v) Coomassie blue G-250 in methanol (50%) and glacial acetic acid (10%) at room temperature for 2 min, and mounted with 90% (v/v) glycerol in PBS. Three hundred cells were recorded considering the presence (no reacted) or absence (reacted) of characteristic dark blue acrosomal-cap.

Lipid rafts and acrosome reaction
In order to assess the relationship between the lipid rafts and acrosome reaction, capacitated sperm from cauda epididymis subjected to induction with PG and/or A23187 were treated with lipid specific probes and FITC-PSA.
Specific probes for CHL (FIL) and GM1 (CTB) were used as previously described. Thereafter, sperm suspensions were incubated with 50 ug/ml of FITC-PSA in PBS for 60 min at room temperature in a moisture chamber in darkness. Samples were examined by epifluorescence microscopy [47,48]. Non-capacitated sperm from cauda epididymis were treated too with lipid probes and FITC-PSA under equal conditions.

Microscopy and image collection and processing
All experiments were analyzed by phase contrast and fluorescence microscopy using an Olympus FV10-ASW confocal microscope (New York, USA). For each treatment, cells were counted and categorized into different fluorescence patterns (see results).

Statistical analysis
All experiments were repeated at least three times in each incubation condition. Measurements were performed in triplicate and results were expressed as means ± S.E.M. Statistical differences between treatment groups were evaluated by two-way analysis of variance (ANOVA), using Bonferroni´s post-test. Differences were considered statistically significant at p<0.05 and p<0.01. Statistical analysis was performed using the GraphPad Prism 4 software (http//www.graphpad.com/ prism/Prism.htm, San Diego, CA, USA).

Vitality and motility control
For the present work, CF1 mice were selected due to their stability on spermatic parameters. Eosin test revealed high sperm viability (70.1% ± 9.7) [30]. After incubation and centrifugation treatments, vitality was again evaluated (64.6% ± 8.3). No statistic differences between both assays (p>0.05) were observed. Microscopic evaluation of sperm motility showed high and rapid progressive motility (~60%) prior to each experimental condition (data not shown). Samples that showed a low percentage of progressive motile sperm or eosin test below the values stated were discarded.

Sperm capacitation: tyrosine phosphorylation
In order to establish capacitation status, tyrosine phosphorylated proteins were examined in spermatozoa from each incubation condition. Tyrosine phosphorylation was revealed by western blot on total sperm protein extracts. After 1 h of incubation under capacitating conditions, only sperm from cauda epididymis presented phosphotyrosine immunoreactivity. In contrast, caput and cauda epididymal spermatozoa, incubated in non-capacitating conditions, did not show phosphotyrosine activity. A similar result was observed in samples from caput sperm even when they were incubated in the capacitating medium. The protein bands obtained are those expected for this specie (Figure 2).

Acrosome reaction and coomassie blue staining
Sperm capacitation status can be established through induced acrosome reaction, because non capacitated sperm and immature sperm (caput sperm) not support acrosome exocytosis. Thus, the acrosome reaction in capacitated spermatozoa was determined by Coomassie blue staining method. Samples from capacitated cauda spermatozoa without induction treatment (basal group) were statistically significantly lower than those treated with PG (p<0.05) and A23187 (p<0.01). Dimetyl-sulfoxide control did not show any difference compared with basal group (p>0.05) ( Table 1). These results were similar to previous reports [49].

Lipid specific probes and fluorescence patterns
For study to lipid rafts, CTB and FIL were employed to label the GM1 and CHL, respectively. Considering the reference patterns described previously, spermatozoa incubated with CTB presented diffuse (Figure 3Aa Fluorescence in the midpiece of the flagellum and the cytoplasmic droplets were characteristically stronger than the fluorescence in the principal piece. Interestingly, this phenomenon was observed with both specific probes, and they showed the same patterns independently of experimental conditions (data not shown).
Samples without specific probes treatment (autofluorescence) showed a very weak signal ( Figure 3C).

Cholesterol and GM1 ganglioside distribution in prefixed spermatozoa
The location of the CHL in caput sperm presented predominantly an acrosomal pattern (Ac) under non-capacitating conditions ( Figure  4A, white bars) and capacitating conditions ( Figure 4A, black bars).
These results indicate a loss of CHL during capacitation in cauda sperm as it was reported by others authors [14,16,20] while CHL in caput sperm (immature) not presented modifications under any condition.
The study of GM1 was more complex than the CHL because several fluorescence patterns were observed by epifluorencence microscopy. Sperm from caput ( Figure 4C) and cauda ( Figure 4D) epididymis subjected to non-capacitating conditions (white bars) and treated with CTB showed predominantly acrosomal patterns. These results could indicate that GM1 did not modify its distribution during epididymal transit.
Interesting acrosomal pattern (Ac) did not change in the immature sperm although under the capacitating conditions ( Figure 4C, compare white and black bars). However cauda sperm presented predominantly acrosomal-cap pattern (ACap) after the in vitro capacitation ( Figure  4D, black bars).
These results taken together suggest that GM1 was restricted to a narrow location over the acrosome (acrosomal-cap pattern) after in vitro sperm capacitation in prefixed sperm.

Cholesterol and GM1 ganglioside distribution in postfixed spermatozoa
Postfixed spermatozoa from caput epididymis under non-     Figures 5C and 5D, white and black bars). Thus, under this postfixation protocol, GM1 presents postacrosomal/acrosomal cap and acrosomal cap pattern only in capacitated cauda sperm. These results suggest that GM1 has different location when the labelling method is performed after fixation (postfixed spermatozoa).

Lipids and acrosome reaction in prefixed capacitated spermatozoa
Mammalian male gamete undergoes acrosome reaction when sperm capacitation occurs. Besides, only sperm from cauda epididymis are susceptible to in vitro sperm capacitation. The apical acrosome is lost during acrosome reaction and this phenomenon could introduce modifications in the fluorescence signal.
In this study, prefixed sperm were incubated sequentially with lipid probes and FITC-PSA. Non-capacitated caput (data not shown) and non capacitated cauda sperm showed a predominantly acrosomal pattern by FIL and CTB probes ( Figure 6, row A, picture b and c). Isothiocyano-fluoresceinated Pisum sativum (Edible pea) agglutinin shows an acrosomal-cap pattern indicating that the sperm does not undergo acrosome reaction (Fig. 6, row A, picture d). Interestingly, the confocal image from the same cell shows a good co-location ( Figure 6, row A, picture e).
Capacitated cauda spermatozoa showed a decrease in the CHL fluorescent signal described here as the diffuse pattern (Compare Figure  6 row A picture b with row B picture g) and there is a redistribution of the GM1 fluorescence signal to the apical acrosome ( Figure 6, row   B picture h). As it was stated previously, CHL was loss and GM1 redistributed to apical acrosomal region (acrosomal-cap pattern). Both fluorescence signals did not co-located in capacitated cauda sperm. In this case, the FITC-PSA signal has acrosomal-cap pattern ( Figure 6, row B, picture i) indicating acrosomal integrity (unreacted), which colocalized with the GM1 (Figure 6, row B, picture j).
After PG or A23187 treatment, diffuse pattern observed by FIL probe did not change ( Figure 6, row C, picture l), while acrosomalcap pattern of GM1 observed in the capacitated cauda spermatozoa changed to diffuse pattern in the whole head ( Figure 6, row C, picture m). This diffuse pattern of GM1 was only observed in capacitated sperm that underwent acrosome reaction confirmed by the loss of the FITC-PSA fluorescence signal ( Figure 6, row C, picture n). During acrosome reaction the three signals did not co-localized on the head ( Figure 6, row C, picture o). Table 2. Table 2 includes relative counts (percentage) of GM1 distribution described by CTB probe in prefixed cauda sperm under non capacitating condition, sperm capacitation and acrosome reaction.
These results indicate that lipid rafts in spermatozoa undergo changes across epididymal transit (maturation), sperm capacitation and acrosome reaction. Cholesterol maintains its acrosomal location (acrosomal pattern) in immature cells, whereas during sperm capacitation there was loss (diffuse pattern). Ganglioside GM1 during epididymal transit has acrosomal distribution as the CHL. After sperm capacitation, acrosomal cap pattern predominates for GM1. During acrosome reaction GM1 acrosomal cap pattern disappears as well as FITC-PSA signal, indicating acrosome exocytosis.

Discussion
A comparative study of CHL and GM1 distribution was performed to reflect their arrangement in the sperm plasma membrane. The simultaneous study of two components of the lipid domains (CHL and GM1) by specific probes (FIL and CTB) during maturation, sperm capacitation and acrosome reaction was analyzed here. Thus, the colocation of lipids permits us to introduce the idea of the disruption of rafts during sperm capacitation and acrosome reaction, in contrast with previous reports, in which the location of lipids was studied independently and this behaviour was not detected.
Furthermore, important assays were employed to establish the sperm capacitation status; the detection of p-Y and the detection of acrosomal reaction by Coomassie blue staining. Complementary to these methods, FITC-PSA labeling was used to study behavior both lipids during acrosome reaction in capacitated spermatozoa. The combination of these methods in the same population of cells allows us to detect whether changes are due to physiological conditions, or dead sperm and/or methodological artifacts.
In this paper mainly observed a strong fluorescence signal in the head acrosome domain (acrosomal pattern) by FIL (CHL) and CTB (GM1) in non capacitated sperm from caput and cauda epididymis under both fixing conditions. Thus, through standard fixation methods, CHL and GM1 do not show a visual redistribution during the epididymal transit. However, after in vitro sperm capacitation or, more dramatically, the induced acrosome reaction, both lipids showed intense changes in their specific locations.
In capacitated cauda spermatozoa, CHL showed weak fluorescence in the whole head with FIL probes (diffuse pattern), indicating its loss from the plasma membrane. Interestingly, CHL did not show significant differences of fluorescence pattern with FIL between postfixed and prefixed sperm from caput and cauda epididymis under incubation conditions.
On the other hand, GM1 could be more sensitive to fixation methods, due to the different results observed in this paper and by other authors. In prefixed spermatozoa, GM1 showed changes in fluorescence pattern, from acrosomal to acrosomal-cap pattern between non capacitated and capacitated cauda cells; while the equatorial segment remained devoid of signal fluorescence for this glycosphyngolipid with CTB probes. However, postfixed cauda sperm labeled by CTB probe presented an acrosomal-cap/postacrosomal pattern and did not show significant differences between non capacitated and capacitated sperm.
In prefixative and postfixative conditions, sperm cells described as other were not a frequent pattern observed and they could be transitional states that have not completed the maturation/capacitation process. Besides, flagella midpiece showed an important fluorescence signal with both lipid probes, and these were maintained in all experimental conditions. Based on these observations, CHL and GM1 co-localized in epididymal sperm indicated a significant amount of both components in the acrosome domain, which were maintained during maturation. These results indicate that lipid rafts are defined from the spermatogenesis and they experiment important physiological changes during sperm capacitation and the acrosome reaction.
In the sperm, these high levels of CHL could be important to maintain a non capacitating condition in the epididymis through the balance between activation and the suppression of membrane signalling processes [27,50]. Sperm capacitation can be induced in vitro in sperm from cauda epididymis. However, sperm capacitation cannot be induced in caput cells, which may be caused by an inhibition of protein kinases and/or by the predominant CHL acrosomal in immature sperm [42,50].
Cholesterol plays a major role in the assembly of signaling protein complexes, allowing the activation of signal transduction. During sperm capacitation, CHL efflux triggers p-Y of capacitation-specific proteins [14]. In the present work, the decrease of CHL during sperm capacitation can be observed as a diffuse pattern detected by FIL probe. This evidence of CHL efflux is also reported by other authors using cryofracture and fluorescence methods [25,51,52].
However, GM1 behavior has been largely discussed because of the different results reported in the literature. For example, in non capacitated sperm from boar and mouse, GM1 was observed principally over the tail, and following the CHL efflux, it diffused progressively toward the sperm head with the exception of the equatorial segment [27]. In contrast, Kawano [21] reported a postacrosomal pattern in non capacitated mouse sperm. In the uterine cavity, GM1 shifted from postacrosomal region to the equatorial segment, and this location disappeared in the oviduct, site where the acrosome reaction occurs. The reason for these results could be because the sperm were incubated with CTB before treatment with PF.
Also in mouse was suggested that GM1 localizes to the postacrosome domain and this localization does not change during sperm capacitation [26]. Some of these differences observed may be due to that these studies were performed at 16°C, which is lower than the physiological temperature.
Jones et al. [13] observed that GM1 was first accumulated on apical acrosome region and then diffused to the equatorial segment in live boar sperm. The meaning of this movement should be elucidated further; because studies in live cells are those that present the largest number of differences in localization and behavior about lipid rafts.
Consequently, these different findings reported about GM1 depend on the experimental conditions: live sperm or under fixative methods.
In the present investigation, experimental assays were performed in vitro under conditions equivalents to physiologic environment; in order to reduce the presence of technical artifacts.
For sperm capacitation, several molecules have been used because of their ability to extract CHL from the plasma membrane. For example, methyl-β-cyclodextrin (MBCD) enhances capacitation and induces phosphorylation. However, lipid diffusion rates and sperm capacitation are affected when MBCD-mediated CHL efflux is excessive [27].
Bovine serum albumin is other molecule used in in vitro sperm capacitation. Albumin is present in the female tract and is responsible for the removal of CHL from the sperm plasma membrane during sperm capacitation [53][54][55][56][57]. Furthermore, calcium and NaHCO 3 are also important female tract components. Visconti et al. [10] reported that BSA, Ca 2+ , and NaHCO 3 in the medium are promoters of p-Y of multiple sperm proteins. For this reason, in the present study, cells were incubated in albumin (in vitro sperm capacitation by BSA), due to its physiological role as an essential molecule in sperm capacitation.
Also the fixative methods are critical for the analysis of lipid rafts. Selvaraj et al. [18] reported that live murine sperm incubated under non-capacitated condition, GM1 was found exclusively in the acrosome. Moreover, it was demonstrated that upon the cessation of motility or cell death in unfixed or lightly fixed sperm, GM1 moved from the acrosomal to the postacrosomal domain. This result suggests that a weak fixation is sufficient to immobilize sperm, but it is not adequate to immobilize GM1. The use of 4% PF plus 0.1% glutaraldehyde is adequate to immobilize lipids in the membrane sperm regardless of the incubation conditions [52][53][54][55][56][57]. In the present paper, similar results were obtained with 4% PF without the addition of glutaraldehyde.
A postfixative condition was employed as an alternative to study living sperm, allowing using specific fluorescence probes in unfixed cells. In this way the artifacts produced by the fixation technique could be avoided. However, this alternative postfixative condition may not be completely appropriate to study lipid changes in live sperm, possibly due to an alteration in the membrane composition.
In this investigation, successive centrifugation treatments did not produce a significant difference in the vitality in prefixed spermatozoa. However cells inevitably suffered some degree of stress when cells were previously labeled with specific probes and then fixed by PF. The degree of stress can be easily observed as postacrosomal and acrosomal-cap/ postacrosomal patterns, predominant for GM1under postfixatived condition. These patterns were described above as dead cells.
As previously mentioned, lipids of membrane do not undergo changes through epididymal transit. One likely reason for the lipid behavior could be their interaction with other molecules present in the plasma membrane. The molecular barriers formed by transmembrane proteins between the equatorial segment with apical acrosomal region and postacrosomal domain have an important role in the lipid balance [58]. Single molecules are free to exchange across this barrier, but larger complexes are unable to do so [59]. The signalling complex might show polarized migration under physiological conditions from regions where they are inactive to areas where they become fully functional. This barrier is stabilized by the cytoskeleton, which is sensitive to intracellular changes that could affect the migration of polarized molecular complexes [59].
On the other hand, many studies have documented an interaction between a lipid raft-specific protein such as caveolin 1 (Cav1) and various signaling molecules present in membrane microdomains [60]. During sperm capacitation, Cav1 changes its membrane distribution from the acrosome domain to the apical acrosome region [61]. A similar GM1 description by CTB probe was observed in prefixed capacitated sperm in the present investigation. GM1 ganglioside and Cav1, two components of the membrane microdomains, might be associated with the actin cytoskeleton during sperm capacitation and participate in acrosome reaction [62]. In the present work, the relationship between GM1 and acrosome reaction was studied by FITC-PSA labelling in prefixed capacitated sperm. In vivo, the zona pellucida stimulates the spermatozoa to undergo acrosome reaction. Calcium ionophore A23187 and PG are used to induct an in vitro acrosome reaction. Non-capacitated cauda sperm showed acrosomal-cap pattern with FITC-PSA, while capacitated cells incubated with several acrosome reaction inducers showed a characteristic diffuse pattern.
Reacted sperm showed a diffuse pattern with the three specific probes, indicating CHL efflux by FIL, early redistribution to acrosomalcap domain and subsequent loss of GM1 signal by CTB, and acrosome reaction by FITC-PSA. These findings might indicate that there is a dynamic behavior in lipid rafts, which could be employed to the correlation between morphology and function. Moreover, in this investigation, it is observed that the method applied is critical when interpreting the results of studies about lipid rafts and their changes during the epididymal transit (sperm maturation), sperm capacitation and acrosome reaction. Spermatozoa under postfixative conditions presented different results. These differences were most noticeable for GM1.
Results exposed in this investigation showed a redistribution that could be correlated with the physiological changes of other raft components like Cav1, as well as an association with actin cytoskeleton.

Conclusion
This paper is a clear demonstration that sperm capacitation and acrosome reaction produce a redistribution of the lipids. However, there were no changes in lipid rafts during sperm maturation, possibly to avoid early sperm capacitation. Cholesterol efflux, a characteristic event of sperm capacitation, could be analyzed through the diffuse pattern described by FIL probes; and GM1 fluorescence patterns observed by the CTB probe could be interesting when it is needed to evaluate sperm capacitation and the acrosome reaction under prefixed conditions.