Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.


Introduction
After leaving the testis, mammalian spermatozoa from many species are morphologically differentiated but have acquired neither progressive motility nor the ability to fertilize a metaphase II-arrested egg. During epididymal transit, sperm acquire the ability to move progressively; however, they are still fertilization incompetent. Fertilization capacity is gained after residing the sperm in female reproductive tract for a finite period of time. The physiological changes that confer on the sperm the ability to fertilize are collectively called capacitation [1]. Capacitation includes several cellular changes in the sperm particularly in the distribution and composition of certain glycoproteins, protein tyrosine phosphorylation, intracellular Ca 2+ and cAMP concentrations, as well as motility pattern [2,3]. This phenomenon is an absolute prerequisite that spermatozoa must undergo in order to interact efficiently with the zona pellucida and to accomplish one of the last steps leading to fertilization, namely the acrosome reaction (AR) [4].
The acrosome is an exocytotic vesicle overlying the anterior region of the sperm head and contains a variety of proteins, including several protease zymogens, protease inhibitors, zona pellucida (ZP) binding proteins, and other ligand-binding proteins [5]. Only capacitated sperm cells are able to undergo the zonatriggered AR and this process characteristically involves multipoint fusions of the sperm head plasma membrane (PM) with the outer acrosome membrane (OAM) [6,7]. This leads to the release of various hydrolytic enzymes principally, the trypsin like acrosin [2] and in the elimination of various surface antigens that are normally exposed on the acrosomal cap of spermatozoa and allows zona pellucida penetration [4]. Only acrosome reacted sperm can penetrate the ZP and fuse with egg plasma membrane [2].
Though sperm membrane modification occurs throughout the male reproductive tract, caput and corpus epididymis are involved in the acquisition of sperm fertilizing ability whereas the cauda segment are specialized in sperm storage [8]. During epididymal maturation, sperm membrane lipids undergo distinct physical and chemical alteration [9]. Changes in the distribution of sperm membrane protein occurring during this process reflect biochemical alterations of both membrane lipids and proteins. The sperm plasma membrane has both membrane integrated and surface adsorbed proteins when spermatozoa leave the testis. Some of these surface proteins change their location from one membrane domain to another during sperm maturation. Other sperm surface proteins are altered, masked or replaced by new proteins of epididymal origin [10]. Identification of epididymal sperm proteins involved in acquisition of sperm fertilizing ability has been investigated in many laboratories [11]. Previously, a low molecular weight 14 kDa protein from goat spermatozoa, named as p14, has been characterized and reported from our laboratory [12]. In the present study we have investigated the localization of p14 on caprine spermatozoa along the epididymal duct and established whether epididymal transit, capacitation or the acrosome reaction affect the distribution of this particular protein.
The role of this protein, if any, in sperm motility and acrosome reaction has also been investigated.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, Registration Number: 95/99/CPCSEA).

Collection of caprine epididymis
Fresh epididymides of adult goats were obtained from the local slaughterhouse (Haji slaughter house, CK Block, Sector 1, Salt Lake, Kolkata 700064, India) immediately after sacrifice and brought to the laboratory on ice. Sacrifice of the animals was done following local municipality's guidelines. Spermatozoa were extracted from the tissue within 2 hrs of slaughtering of the animals.

Preparation of spermatozoa and epididymal plasma
Spermatozoa were obtained from goat cauda (or other regions as and when required) epididymis using standard procedure. Highly motile spermatozoa were extracted at room temperature from the goat epididymis in a modified Ringer's solution (RPS medium: 119 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 10 mM glucose, 16.3 mM potassium phosphate, 50 Units/ml penicillin, pH 6.9). Number of spermatozoa in the sample was estimated with a haemocytometer. Freshly extracted sperm preparations contained about 10-20610 7 cells/ml. For the preparation of goat cauda epididymal plasma (EP), freshly extracted sperm preparation was centrifuged at 8006g for 10 mins. The supernatant was spun again at 120006g to obtain cell-free EP. The concentration of EP was expressed as its protein content [13].

Induction of capacitation and acrosome reaction
Highly motile swim-up spermatozoa were extracted at room temperature from the cauda epididymis in Tyrodes medium (100 mM NaCl, 3.1 mM KCl, 1.5 mM MgCl 2 , 0.29 mM potassium dihydrogen phosphate, 21.6 mM sodium lactate,1 mM sodium pyruvate, 20 mM HEPES, 50 mg/ml Kanamycin, pH 7.4). Control samples (non capacitated, NC) were taken immediately following swim-up procedure for processing by immunoblotting and immunofluorescence.
Acrosome reaction was induced by the addition of 3 mM calcium ionophore, A23187 in 0.3% DMSO to the capacitated sperm (2610 8 cells/ml) and incubated further at 39uC for 1 hr (AR samples) [17]. Control tubes were run in presence of DMSO but without any ionophore, which was found to have no effect. To study the effect of anti-p14 antibody on acrosome reaction, cells were incubated with the same for 1 hr at room temperature before induction of acrosome reaction. Acrosomal status was then assessed using Rose Bengal staining method.

Extraction of proteins
To prepare whole-cell extract aliquots of 0.5 ml (10 8 cells) of noncapacitated, capacitated and acrosome reacted samples were centrifuged in a microfuge at 75006g for 5 mins at room temperature, the supernatant was discarded. The resulting sperm pellet was resuspended in 500 ml extraction medium (2% SDS, 28% sucrose, 12.4 mM N,N,N9,N9-tetramethylethylenediamine and 185 mM Tris-HCl, pH 6.8 [18] and immediately incubated for 5 mins at 100uC. After centrifugation at 75006g for 5 mins, the supernatant was recovered. The concentration of proteins in the supernatant was measured using the Micro BCA protein assay reagent kit. To the supernatant, 2-mercaptoethanol and glycerol were added to a final concentration of 5 and 1% respectively. Finally, extracts were incubated at 100uC for 5 mins and then stored at 220uC until used for Western blot analysis [17]. The cytosol from different epididymal region was prepared following the method previously standardized in our laboratory [12]. Briefly, the tissue was homogenized in 50 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 1.5 mM thioglycerol and 0.1 mM TPCK, 0.1 mM TLCK, 1 mM PMSF, 10 mg/100 mL leupeptin as protease inhibitors. The homogenized mixture was centrifuged at 120006g for 20 mins at 4uC, supernatant collected and centrifuged at 300006g for 30 mins at 4uC. The post 300006 g supernatant was subjected to ultracentrifugation at 1000006g for 1 hr at 4uC. The post 1000006g supernatant, termed as 'cytosol', was collected and used for Western analysis.
The plasma membranes and soluble components of the acrosome were fractionated from the sperm pellet following the described method [5,20]. Briefly, sperm were resuspended at a concentration of 20610 6 /ml in 0.625% Triton X-100 and 0.15 M NaCl, 5% sucrose, protease inhibitor cocktail and 20 mM sodium acetate, pH 5.2 (5 mins, 4uC). The sperm suspension was further homogenized by passing through a 26-gauge syringe needle (twice). The sperm suspension was separated into soluble and insoluble fractions by centrifugation (10 0006 g, 10 mins, 4uC). Each fraction was subjected to protein quantification using BCA protein estimation and to SDS-PAGE/immunoblotting. Peripheral plasma membrane proteins from intact caudal epididymal sperm were extracted by treatment with a sucrose solution (320 mM) containing 1 mM ATP, 1 mM EDTA, and 0.2 mM Na-p-tosyl-L-lysine chloromethylketone hydrochloride (AES). Approximately 70610 6 mouse sperm were incubated in 0.5 ml of AES for 20 mins at 4uC. Sperm were then centrifuged (6006 g, 10 mins, 4uC), and the collected AES supernatant was concentrated for immunoblotting [20].

Raising antibody against p14
Antibody was raised against p14, purified from goat cauda epididymal cytosol, by immunizing 1-year-old white albino rabbit with the protein [21]. All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, Registration Number: 95/99/CPCSEA).
In first shot, 100 mg of the protein was mixed with Freund's complete adjuvant (1:1) and intramuscular injection was given. The subsequent three booster doses were given 10 days apart with Figure 1. Presence of p14 in caprine sperm cells and in epididymal epithelial tissue. Rabbit polyclonal antisera were raised against p14 protein (antigen), purified by affinity purification procedure and was used for Western blotting. (A) Western blot analysis of p14 in total tissue extracts from caput (Cap), corpus (Cor) and cauda (Cau) of the epididymis and vas deferens (Vd). Blot was probed with monoclonal antibody against actin to assess protein loading. (B) Western blot analysis showing p14 in total protein extracts of sperm from caput (Cap), corpus (Cor) and cauda (Cau) regions of the epididymis and vas deferens (Vd). Actin was used as loading control. (C) The immunohistochemical staining to show the expression pattern of p14 in the epithelial cells and lumen of the caput, corpus and cauda epididymis. Preimmune serum at the same condition showed no immunoreactivity. All the analysis was performed four times. doi:10.1371/journal.pone.0030552.g001 each time. Fifth shot (intravenous) with pure antigen only was given one month following the fourth one.
Rabbit was bled from marginal ear vein 10 days following fourth and fifth shot and serum was collected for subsequent study [22].
The immunoglobulin of the immune serum was precipitated twice with 50% ammonium sulfate. The final precipitate was dissolved in 0.01 M PBS, pH 7.0, and excess ammonium sulfate was removed by dialysis against the same buffer. The immunoglobulin fraction obtained after the salt fractionation was subjected to DEAE-cellulose chromatography. Unbound protein peak containing IgG was eluted with 0.01 M phosphate buffer at pH 7.0. This purified anti-p14 antibody was used for subsequent immunological studies.

Flow cytometry analysis
The extent of p14 expression in goat sperm was determined from flow cytometric analysis (BD-FACS calibur) following established procedure. Sperm were stained with anti-p14 polyclonal antibody at a final concentration of 1:1000, and the secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat antibody against rabbit IgG (Sigma) at a dilution of 1:50. At least 10000 individual sperm per sample were analyzed for FITC fluorescence emission.

Western blot analysis
Western blot analysis of protein associated with spermatozoa was done following standard protocol. Briefly, equal amount of protein from extracts of the caput, corpus and cauda epididymis and spermatozoa from each section were resolved over 16.5% SDS-polyacrylamide gels, transferred onto PVDF membranes, probed with rabbit polyclonal antisera against p14 (1:1000), the bound IgG was detected with goat anti-rabbit-ALP (Sigma), immunoreactive band was visualized using NBT-BCIP as a chromogenic substrate for alkaline phosphatase. Similar procedure was followed in case of NC, C and AR samples.

Confocal Microscopy study
Immunohistochemistry was carried out for p14 localization and expression in different parts of the epididymis. Fixed epididymis samples were dehydrated in gradient ethanol, treated with xylene, and finally embedded in paraffin wax. Serial paraffin sections (5 mm) were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. To prevent background staining, sections were incubated in normal goat serum at room temperature for 20 mins after being washed in phosphate buffer saline (PBS, 0.01 M pH 7.2). Following fixation and permeabilization, incubation was continued for overnight at 4uC with polyclonal rabbit anti-p14 as primary antibody (1:1000 dilution) in PBS buffer (0.01 M, pH 7.2). After washing in PBS buffer, sections were treated with the green-fluorescein isothiocyanate (FITC) conjugated goat-anti-rabbit IgG which was previously incubated with primary antibody. Negative controls were run in parallel under identical conditions, but either in the absence of primary antibody or in the presence of normal rabbit serum. Finally, the sections were imaged using a Zeiss Confocal Microscope (Carl Zeiss, Germany).
Indirect immunofluorescence to localize p14 on noncapacitated, capacitated and acrosome reacted sperm cells The localization of p14 was determined by immunofluorescence with washed spermatozoa or cells incubated in capacitation/ acrosome reaction condition. In the first set of experiments, the spermatozoa were washed with PBS (pH 7.2) and fixed for 30 mins in 3.7% paraformaldehyde. After washing the cells with PBS, cells were permeabilized with 0.25% Triton-X-100 for 15 mins, washed thrice with PBS. Non-specific binding sites were blocked with 3% BSA in PBS for 1 hr at 37uC in a humid chamber. After blocking, cells were washed three times with PBS and incubated with either purified anti-p14 antibody or preimmune sera (negative control) for 2 hrs at 37uC. Following extensive washing in PBS, the cells were incubated for 2 hrs at room temperature with fluorescein isothiocyanate (FITC)-conjugated secondary antibody raised in goat. Next, cells were washed thrice with PBS; smear prepared on a glass slide with a drop of cell suspension and allowed to dry. 10 ml antifade mounting media (invitrogen) was added on the dried smear, covered with a coverslip and sealed by nail polish. Spermatozoa were examined by confocal microscopy (Zeiss laser scanning confocal microscope, LSM-510). Noncapacitated swim-up cells (NC), capacitated cells (C) and acrosome reacted cells (AR) were processed similarly to localize p14 there.
In another set of experiments, the presence of p14 was investigated in nonpermeabilized cells to measure the surface expression of the protein. After two washings, a 100-ml aliquot was incubated with the antibodies as described above for 2 hrs at room temperature. The cells were next washed twice by centrifugation (2506g, 10 mins), resuspended in PBS. The FITC-conjugated secondary antibody was added to the suspension, and the spermatozoa were incubated for 2 hrs. Finally the spermatozoa were washed twice in PBS by centrifugation (2506g, 10 mins), and mounted on slides for analysis as described above. Noncapacitated swim-up cells, capacitated cells and acrosome reacted cells were processed similarly.

Microscopic analysis of sperm motility
Spermatozoa showing forward progression were identified under a phase contrast microscope at 4006 magnification. An aliquot of the freshly extracted sperm preparation (1610 6 cells) was incubated with anti-p14 antibody in a total volume of 0.5 ml RPS medium for 15 mins at room temperature before assessing the sperm motility with a haemocytometer. For the microscopic method of assay of sperm motility (expressed as %), all cells which showed some degree of motility (vibrating, progressive motility) was counted. Experiments were repeated four times.

Preparation of sperm sample for assay in CASA
The assay conditions were same as that of forward motility assay under phase contrast microscope. Total cell numbers were counted under a phase contrast microscope at 4006magnification in a hemocytometer. Spermatozoa (0.5610 6 cells) were incubated in the absence or presence of appropriate dilution of anti-p14

Sperm motility study following CASA
After preparation of sperm sample, analysis was performed using the CASA system (Version: 12, HTM-CEROS CASA System, Hamilton Thorne Research, Inc., Beverly, MA, USA) for assessing the sperm motility following incubation with anti-p14 antibody. Briefly, a 5 ml aliquot of prepared sperm sample was placed on a Mackler chamber. At least 200 spermatozoa were counted with CASA to evaluate the sperm motility variables including Average Path Velocity (VAP), Straight Line Velocity (VSL) and Curvilinear Velocity (VCL). The CASA settings were followed according to the manufacturer's instruction.

Rose Bengal Staining
The conventional ''acrosome reaction'' is based on the detection of the acrosomal glycoproteins following interaction with Rose Bengal dye [23] with following modification [24]. Following termination of acrosome reaction with 3% glutaraldehyde, the cell suspension was incubated at room temperature for 2 hrs. Samples were centrifuged at 8006g for 3 mins and the supernatant was aspirated. The pellet was resuspended in PBS and washed twice with the same medium. Smear was prepared with a drop of suspension and air-dried. The slides were stained with 0.8% Bismarck brown in deionized water (pH 1.8 with 2N HCl) at 37uC for 25 mins and rinsed with distilled deionized water. Finally the slides were stained for 25 mins in 0.8% Rose Bengal in 0.1 M cacodylate buffer, pH 6.0 for detection of the glycoprotein content of the intact acrosomal sac.
The slides were then washed with deionized water, dehydrated in increasing concentration of alcohol (%), cleared in xylene and mounted with paramount and cover slip. A total of 200-500 spermatozoa were evaluated and recorded as either ''acrosome reacted'' (un-intact) sperm (no colored spot on tip of sperm head) or ''acrosome un-reacted'' (intact) sperm (with a colored spot on tip of sperm head). The experiments were repeated four times.

Statistical analysis
Values are shown as standard error of mean (SEM) except otherwise indicated. Data were analyzed and, when appropriate, significance of the differences between mean values was determined by a Student's t test. Results were considered significant at p,0.05.

Results
Presence of p14 in caprine sperm cells and in epididymal epithelial tissue p14 was previously purified and biochemically characterized from goat cauda epididymis [12]. With this background knowledge, we have examined the presence of p14 by Western blot analysis in caprine epididymis and epididymal spermatozoa using a polyclonal anti-p14 antibody raised in rabbit in our laboratory. The sensitivity and specificity of the antibody were previously verified by Western blot analysis. As evident from Figure 1A, p14 expression was distinctly observed and was found to gradually increase in the protein extract obtained from the caput, corpus and cauda epidydimis region as well as in the vd. Similar expression pattern was also observed in the sperm protein extracts obtained from the caput, corpus, cauda and vd regions though the change in the expression level was not very prominent ( Figure 1B).

Localization of p14 in epididymal epithelial tissue and spermatozoa
The western blot analysis was further verified using immunohistochemical studies.The positive signal was localized to epididymis and was present both in luminal sperm cells and epithelial cells ( Figure 1C). Confocal microscopic analysis of live and fixedpermeabilized spermatozoa suggested that for live cells (Figure 2), p14 was present on anterior acrosomal region only in caput and corpus spermatozoa, whereas in cauda and vd spermatozoa, it was found to be localized on anterior as well as post acrosomal region. In fixed-permeabilized cell (Figure 3), p14 was found to be localized only in anterior acrosomal region of caput. In corpus, cauda, vd spermatozoa on the other hand the protein was present in anterior as well as post-acrosomal region. FACS analysis showed the expression of p14 in 70-80% of live as well as fixedpermeabilized cells from all epididymal part (Figure 4).

Presence of p14 in plasma membrane and soluble fraction of acrosome
As p14 was found to be present on sperm surface as well as intracellular region (above), we next sought to investigate its expression level in the different subcellular region. Western blot analysis revealed that although the protein is mostly cytosolic, it was found to be expressed in relatively low level in sperm membrane also ( Figure 5A). Significant level of expression of the protein in epididymal plasma was also detected ( Figure 5B). When plasma membranes and soluble components of acrosome were extracted from sperm with 0.625% Triton-X-100 solution for immunoblotting, low level of expression of p14 was observed in the soluble part but was absent in detergent insoluble matrix fraction ( Figure 5C). Immunoblot with protein extracted from peripheral plasma membrane of washed caudal spermatozoa following treatment with 320 mM sucrose solution containing 1 mM EDTA and 1 mM ATP (AES) revealed low level of expression of this protein ( Figure 5D).

Effect of capacitation and acrosome reaction on the distribution of sperm p14
Following determination of the expression status of p14, we next explored the changes in its distribution pattern during capacitation and acrosomal reaction. On incubating the spermatozoa under capacitating conditions, a change in the immunofluorescence pattern was observed. In non-permeabilized cells, three different patterns were found ( Figure 6A). The majority of the cells showed  Figure 6B). The pattern of distribution and number of cells showing this pattern did not change significantly with increase in capacitation time (data not shown). When capacitated cells were challenged with the calcium ionophore, A23187, to acrosome react, the pattern of labeling changed in the anterior acrosomal region of fixed/permeabilized sperm cells compared with cells incubated under capacitating conditions ( Figure 7A). In nonpermeabilized spermatozoa, a weak signal ( Figure 7B) was observed in 29.363.8%(n = 4) of cells. On the other hand, 69.765.8% of the cells showed almost no labeling in any region of spermatozoa cell.

Expression level of p14 during capacitation and acrosome reaction at different time periods
Next, to address how the distribution of p14 changes upon induction of capacitation, p14 level was checked in PPM, total membranes and soluble components of the acrosome, and whole cell lysate during capacitation. The result showed that during first hour of capacitation, p14 level increased in peripheral plasma membrane ( Figure 8A) and possibly in acrosome ( Figure 8B) but its level in whole cell lysate remained the same ( Figure 8C) in noncapacitated and capacitated cells. Protein level in these membrane fractions and whole cell lysate did not change significantly with increase in capacitation time from 1 hr to 3 hrs. All these findings ( Figure 6 and Figure 8) together confirmed that upon induction of capacitation, significant changes occur in the membrane expression level of p14. In acrosome reacted cells, p14 level was found to be decreased when compared with 3 hrs capacitated cells ( Figure 8D).
Effect of anti-p14 antibody on the forward motility retarding ability of p14 Microscopic Analysis. Previously it was reported from our laboratory that incubation of sperm cells with p14 inhibit forward motility of caprine spermatozoa in a concentration dependent manner [22], however its presence on sperm surface was not known. In the present study, sperm cells were incubated with anti-p14 antibody to block sperm surface p14 and monitored the number of progressively motile sperm cells with haemocytometer. The result showed that p14-antibody at a dilution of 1:500 increased the number of progressively motile sperm cells from ,42% (in control pre-immune serum treated cell) to ,70% ( Figure 9, Table 1). Low motile sperm populations were taken for microscopic assay to determine the motility promoting activity of p14 antibody.
Analysis by CASA. Addition of 1:500 dilution of anti-p14 antibody to the sperm preparation caused significant increase in sperm forward motility. Two different doses, 1:500 and 1:1000 dilutions of anti-p14 antibody were used to see the effect on motility variables of CASA. There was approx. 26% increase in VAP, 26% in VSL and 19.4% in VCL at higher concentration of the antibody ( Figure 10, Table 2).

Effect of anti-p14 antibody on ionophore induced acrosome reaction
Effect of anti-p14 antibody on sperm acrosome reaction was studied by Rose Bengal staining method. Incubation of sperm cells  with anti-p14 antibody, before induction of acrosome reaction, decreased the number of acrosome reacted cells from 68.063.6% in negative control to 38.763.5% in antibody treated sample ( Figure 11A, Table 3). Acrosome intact or unreacted cells were identified by a red colored spot on the tip of sperm head. Acrosome reacted cells did not show any such spot ( Figure 11B). Acrosome reaction was also assessed by chlortetracycline assay and similar result was obtained (data not shown). The calculated percentages are given as the mean 6 SD of at least four experiments.

Discussion
Mammalian sperm-egg interactions are mediated by specific complementarity of proteins, glycoproteins, and carbohydrates present at the surface of gametes [25,26]. Before spermatozoa can interact with the zona pellucida, which is an extracellular matrix surrounding the egg, they are subjected to post-testicular modifications yielding male gametes that exhibit forward motility and oocyte-binding ability [27]. Epididymal secretory products are involved in the acquisition of fertilizing ability by mammalian spermatozoa. Although many epididymal proteins have been described, the function of the majority of them in sperm physiology remains to be clarified. After epididymal transit, male gametes undergo other complex surface transformations that occur when they encounter accessory gland secretions during ejaculation as well as within the female genital tract [28]. Physiological modifications of the spermatozoa within the female reproductive tract have been extensively studied and collectively defined as capacitation [29]. Capacitation represents the completion of sperm maturation that confers on mammalian sperm the acquisition of fertilization competence either in vivo or in vitro. As spermatozoa progress through capacitation, dramatic changes occur in the sperm membrane and in surface protein distribution [30,31]. In vivo, sperm egg interaction triggers Ca 2+ influx in sperm, which in turn activates a series of biochemical events leading to the phenomenon of membrane fusion [32]. It can be induced in vitro in capacitated spermatozoa by incubation with solubilized zona pellucida, progesterone, epidermal growth factor, or by Ca 2+ /2H + /ionophore A23187 [33].
Recently we have reported the isolation and characterization of a14 kDa protein (p14) from goat epididymal cytosol [12]. In the present work the localization of p14 in spermatozoa has been described. It has been observed that in total epididymal tissue, p14 level is increased gradually while in individual sperm cell, the level remains the same from caput to vd. This clearly indicates that p14 level specifically rises in epididymal epithelial tissue during epididymal transition of spermatozoa. Immunohistochemical study confirms its presence in luminal sperm cells and epithelial  cells of epididymis. Indirect immunofluorescence of non-permeabilized spermatozoa reveals the localization of the protein at the anterior acrosomal region of the head in caput and corpus spermatozoa, while in cauda and vd spermatozoa, it is observed in anterior and post acrosomal region. This suggests that p14 is expressed at the sperm surface, facing the extracellular milieu. A strong signal, however, has been detected on the anterior region of the head of fixed-permeabilized caput spermatozoa, on the anterior as well as post acrosomal region of corpus, cauda and vd spermatozoa. Our findings thus strongly supports that a population of p14 is also expressed intracellularly which is also evident from the FACS analysis. Subcellular localization experiments reveal that the protein is mainly cytosolic, although a significant level of expression is also observed in membrane. The detail physiological significance of why p14 is located more or less in all compartments of the sperm head needs further investigation. As the interior of the acrosome is compartmentalized biochemically and morphologically, a given protein of the acrosomal lumen may be considered as a soluble constituent or an acrosomal matrix component depending upon whether the protein is solubilized following extraction with Triton X-100 under conditions that block proteolysis. Now in our study when membranes and soluble components of acrosome were extracted from sperm with 0.625% Triton X-100 solution for immunoblotting, results indicate that the protein is present in the detergent extract with complete absence in the insoluble matrix fraction. This suggests that along with cytosol, the protein resides in the membrane and/or the soluble fraction of acrosome but not in the acrosomal matrix.  Table 3. Effect of anti-p14 antibody on caprine sperm acrosome reaction.

Treatments
Acrosome reacted sperm (%), mean ± SD Western blot analysis of protein extracted from peripheral plasma membrane with AES solution confirms its presence in this membrane fraction. During capacitation, p14 level is found to be increased in peripheral plasma membrane and probably in acrosome also. However, its level in whole cell lysate remains the same indicating no new synthesis or breakdown of p14 during this process. Indirect immunofluorescence with live cells confirms modification in the surface distribution pattern of p14 during sperm capacitation. Majority of the capacitated cells (66%) show strong labeling only on the anterior acrosomal region. Other cells show either strong labeling in anterior acrosomal region and faint labeling in post-acrosomal region (26%) or faint labeling only in post-acrosomal region. The pattern does not change significantly with time. In the fixed-permeabilized capacitated cells, a decrease in the signal intensity is observed in the anterior acrosomal region of few cells but majority of cells show labeling in anterior as well as post-acrosomal region. All these findings together strongly suggest the change of distribution of p14 upon induction of capacitation thereby confirming the involvement of this protein in the process of caprine sperm capacitation. On the other hand, when capacitated spermatozoa is challenged to acrosome react, the signal is found to be decreased significantly in most permeablized and non-permeabilized cells indicating loss of the protein from sperm plasma membrane/outer-acrosomal membrane. Thus, all our findings cumulatively suggest that p14 is mainly cytosolic, also associated with the outer acrosomal membrane, or associated with the plasma membrane, but not with the inner acrosomal membrane. Capacitation reaction induces a change in the distribution of the protein from post acrosomal to anterior acrosomal surface. During acrosome reaction p14 is lost from membranes over the anterior head region which again confirms that the protein is not present on inner acrosomal membrane (IAM). One of the major reasons of male infertility is due to low/no sperm motility (asthenospermia) [13,34]. Previously, it has been reported from our laboratory that incubation of sperm cells with p14 inhibits forward motility of caprine spermatozoa in a concentration dependent manner [22]. In the present study anti-p14 antibody has been used to block sperm surface p14. The results from CASA and microscopic analysis show that blocking sperm surface p14 enhances the progressive motility of sperm cells. Although no clear mechanism in support of this event is known till now, a change in serine-threonine phosphorylation of some spermatozoal proteins which might be associated with sperm motility could be responsible for this (results not shown). Treatment of sperm cells with anti-p14 antibody, before induction of acrosome reaction with Ca 2+ -ionophore A23187 decreases the number of acrosome reacted cells from ,68% in negative control to ,39% in antibody treated sample. Therefore it is suggested that p14 is probably involved in the acrosomal membrane fusion event. Blocking p14 by anti-p14 antibody may somehow hinders this fusion, thus decreases the number of acrosome reacted cells. However further investigations are required to delineate the exact biochemical mechanism in detail.