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a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Alternatively, several of these enzymes may perform functions within the acrosome prior to the acrosome reaction. Indeed, many acrosomal enzymes including acrosin [14], ß-galactosidase [15], and 
-L-fucosidase [16] undergo specific intraacrosomal processing resulting in the conversion of inactive precursor forms into enzymatically active forms. The processing of acrosomal enzymes may be an integral part of the sperm maturation process and likely requires specific hydrolases and/or proteases that are functional at the acidic pH in the sperm acrosomes. It is of interest, therefore, that while the sperm acrosome contains a remarkable number of enzymes that function at different times during sperm maturation and fertilization, these enzyme activities appear to be tightly controlled to avoid inappropriate processing. Several mechanisms by which enzyme activity in the sperm acrosome may be regulated include the presence of enzymes in an inactive, precursor state as discussed above, neutral or basic pH optima, the sequestering of enzymes in different compartments of the acrosome [17, 18], and enzymes that exhibit high substrate specificity [3]. Another possible means by which acrosomal enzyme activity may be regulated is through the binding of specific inhibitors. However, to date little is known regarding the presence or role of protease inhibitors in the sperm acrosome.
In previous studies we identified a unique gene termed CRES (cystatin-related epididymal spermatogenic) that is a member of the cystatin superfamily of cysteine protease inhibitors [19]. The cystatin superfamily consists of three families including the intracellular stefins and the secreted cystatins and kininogens [20]. Although in vitro studies have established that the cystatins are cysteine protease inhibitors with substrate specificities against papain-like cysteine proteases such as the cathepsins B, S, H, and L [21], the in vivo function of these proteins is not well understood. Recently, several cystatin and cystatin-like proteins have been identified that exhibit gene structure, amino acid sequences, and/or expression patterns distinct from those of the classic cystatin proteins [22, 23]. The identification of these cystatin proteins has led to the premise that there are new cystatin families or subgroups of families that have evolved to perform tissue-specific functions distinct from the housekeeping-type functions of the classic cystatin proteins. Recent genomic cloning and chromosomal localization studies suggest that the CRES gene represents a new subgroup in the family 2 cystatins (see Note Added in Proof, reference 1). The CRES protein is distinct from other cystatin families in that it exhibits highly restricted expression in the reproductive tract suggesting roles in reproduction. CRES protein is present in the elongating spermatids in the testis and is secreted by the proximal caput epididymal epithelium [19, 24]. Recently, we have determined that CRES protein is also expressed by the anterior pituitary gonadotrophs (see Note Added in Proof, reference 2).
The presence of CRES protein in the testicular germ cells as well as in the proximal caput epididymal lumen prompted us to examine whether CRES protein was associated with spermatozoa. The studies presented herein show by indirect immunofluorescence, immunogold electron microscopy, and Western blot analysis that CRES protein is present in the sperm acrosomes. Moreover, CRES protein is released following the acrosome reaction. These studies suggest that CRES may perform roles in the regulation of intraacrosomal protein processing or alternatively may be involved in the fertilization process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Male retired breeder ICR mice were purchased from Harlan (Indianapolis, IN). Mice were housed under a constant 12L:12D cycle and were allowed free access to food and water. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guidelines for Care and Use of Experimental Animals. Animals were killed by cervical dislocation after asphyxiation in CO2. The epididymides were removed and divided into five regions: 1, proximal caput; 2, mid-caput; 3, distal caput; 4, corpus; 5, cauda epididymidis [19]. Epididymal spermatozoa were collected by mincing tissues on ice in Dulbecco's PBS, pH 7.4, containing the protease inhibitors 10 mM iodoacetamide, 0.5% aprotinin, and 0.1% PMSF (PBS-PI). Spermatozoa were purified from contaminating epididymal epithelial cells by Percoll step gradient centrifugation (20%, 30%, 40% in PBS, 1 ml of each) at 3000 x g for 10 min at 4°C. After washing of the pelleted spermatozoa in 1 ml of PBS and centrifugation at 500 x g, 10 min, spermatozoa were resuspended in PBS and used for either indirect immunofluorescence, immunoelectron microscopy, or Western blot analysis.
A crude preparation of testicular spermatozoa and spermatids was obtained by mincing testes on ice in PBS-PI followed by Percoll step gradient centrifugation (20%, 30%) at 3000 x g for 10 min at 4°C. A pure population of testicular spermatozoa was obtained by ligating the efferent ducts near the testis and 18–20 h later carefully cutting near the site of ligation and gently extruding the spermatozoa and rete testis fluid into 50 µl of PBS-PI. Approximately 5 x 105 spermatozoa were obtained from each efferent duct-ligated mouse.
Indirect Immunofluorescence
Approximately 1.5 x 104 spermatozoa were air dried onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and circled with a Pap pen to allow small incubation volumes. Sperm cells were fixed and permeabilized with 95% methanol:5% glacial acetic acid for 30 min at -20°C. After fixation, spermatozoa were washed with PBS containing 0.2% Tween 20 (PBS-T) and incubated with 5% goat serum in PBS-T for 1 h at 37°C in a humidified chamber to block nonspecific binding. The spermatozoa were then incubated with a polyclonal rabbit anti-mouse CRES antiserum (1:400 in PBS-T supplemented with 5% goat serum) [24] or a polyclonal rabbit anti-guinea pig proacrosin antiserum (1:200 in PBS-T supplemented with 5% goat serum) [25] for 1 h at 37°C. The spermatozoa were washed three times in PBS-T for 10 min at room temperature and then incubated with a Texas Red-conjugated goat anti-rabbit secondary antibody (1:100) (Jackson Immunoresearch, West Grove, PA) for 1 h at 37°C. The spermatozoa were washed three times in PBS-T for 10 min at room temperature and once in PBS, pH 8.5. Control samples included spermatozoa incubated with preimmune serum (1:400), secondary antibody alone (1:100), and CRES antiserum (1:400) that had been preincubated with the His-CRES antigen for 45 min at 4°C (blocked antiserum). The slides were inverted onto coverslips containing 15–20 µl of mounting medium containing 92 mM Tris, pH 8.5, 18.5% dimethyl sulfoxide (DMSO), 23% methanol, and 0.092 mg/ml Mowiol 4–88 (Fisher Scientific) to which 1 mg/ml final p-phenyl diamine (Sigma Chemical Co., St. Louis, MO) was added immediately before use, and slides were allowed to cure overnight. The spermatozoa were then examined using an Olympus (Tokyo, Japan) BX-60 microscope equipped with epifluorescence.
Immunoelectron Microscopy, Postembedding Labeling
Mouse caput and cauda spermatozoa prepared as described previously were fixed in 4% paraformaldehyde for 30 min at room temperature. Spermatozoa were dehydrated in 50% ethanol for 10 min and then twice in 75% ethanol for 45 min by gentle resuspension and centrifugation at 500 x g for 10 min between each step. The final sperm pellet was embedded in LR white resin (medium grade; Electron Microscopy Sciences, Fort Washington, PA) at 56°C overnight. Ultrathin sections were cut and mounted onto nickel grids by the Texas Tech University Health Sciences Center Electron Microscopy core facility. Sections were incubated with CRES, preimmune, or proacrosin antiserum diluted 1:25 in PBS-T containing 5% goat serum; they were then incubated with a goat anti-rabbit IgG antibody conjugated to 10-nm colloidal gold particles (1:25) (Amersham, Arlington Heights, IL). Between each step the grids were washed three times with PBS-T for 10 min at room temperature. The sections were counterstained with uranyl acetate and lead citrate and then examined using a H600 Hitachi (San Bruno, CA) electron microscope.
Induction of Sperm Acrosome Reaction
Induction of the sperm acrosome reaction was carried out as described previously [26]. Briefly, mouse cauda epididymal spermatozoa were obtained by mincing cauda epididymides in Earle's modified medium 199 (M199; Gibco BRL, Grand Island, NY) supplemented with 2 mg/ml BSA and 30 µg/ml sodium pyruvate (Sigma). The spermatozoa were diluted, and approximately 4 x 106 cauda spermatozoa in 200 µl of the supplemented M199 medium were allowed to capacitate for 1 h at 37°C in a humidified atmosphere of 5% CO2. The upper fraction containing motile spermatozoa was carefully removed, and 3.5 x 106 spermatozoa were incubated with either 10 µM final concentration of the calcium ionophore A23187 (Sigma) to induce the acrosome reaction or an equivalent volume of the vehicle DMSO as the control. Spermatozoa were incubated for 1 h under the same conditions as described for capacitation and then examined for CRES and proacrosin proteins by immunofluorescence analysis as described above. Spermatozoa were considered acrosome reacted by the absence of proacrosin staining.
To prepare acrosomal protein extracts for Western blot analysis, spermatozoa were induced to undergo the acrosome reaction as described above. The spermatozoa were then centrifuged at 10 000 x g for 10 min and resuspended in Laemmli buffer (acrosome-reacted spermatozoa). The supernatant solution was ultracentrifuged at 50 000 x g for 60 min at 4°C to separate the acrosomal soluble fraction (supernatant) and acrosomal membranes (pellet). Laemmli buffer was added to both the soluble and membrane fractions, and protein samples were separated by SDS-PAGE followed by Western blot analysis.
Sequential Extraction of Sperm-Associated Proteins
To examine the association of CRES protein with spermatozoa, sequential extraction of caput and cauda epididymal spermatozoa was performed as described by Rankin et al. [27]. Briefly, caput and cauda spermatozoa were collected from the epididymides of 14 mice and diluted into 2 ml of PBS-PI. The sperm samples were divided into thirds to avoid overloading the Percoll gradients and centrifuged through step Percoll gradients as described above to remove contaminating epididymal epithelial cells. The sperm pellets were gently resuspended in 300 µl of PBS-PI, and sperm concentrations were determined. A small volume of the cauda sperm suspension was removed and diluted with PBS-PI so that the total number of spermatozoa undergoing sequential extraction was identical between caput and cauda spermatozoa (approximately 1 x 107 total spermatozoa). After centrifugation of the spermatozoa at 500 x g for 10 min, the spermatozoa were resuspended in 100 µl of PBS-PI and then centrifuged again. The supernatant solution was removed and the sperm pellet resuspended in 100 µl of low-salt buffer (PBS-PI with 5 mM EDTA, pH 7) and extracted for 30 min at room temperature. The resulting supernatant solution, following a 500 x g spin for 10 min, was designated the low-salt extract. The sperm pellet then underwent a high-salt extraction (PBS-PI with 5 mM EDTA, 0.5 M NaCl, pH 7) followed by Triton extraction (PBS-PI with 0.1% Triton X-100, 5 mM EDTA, pH 7) using the same incubation volume, time, and centrifugation conditions as described for the other extractions and each time saving the supernatant solution. The sperm pellet was then extracted in 100 µl of 2% SDS for 5 min at 95°C followed by centrifugation at 10 000 x g for 5 min (SDS extract), and the final sperm pellet was resuspended in 100 µl Laemmli buffer. After each extract supernatant solution was obtained, it was immediately centrifuged at 10 000 x g for 5 min to remove residual sperm/cellular debris and then desalted into distilled water using Centricon-10 columns (Amicon Co., Danvers, MA). The final concentrates were brought to 100 µl and subjected to SDS-PAGE followed by Western blot analysis. Since there was some loss of spermatozoa with each centrifugation step, the absolute number of spermatozoa remaining was determined after each extraction, and extracts representing approximately equal numbers of spermatozoa were loaded in each lane during SDS-PAGE.
Western Blot Analysis
Samples were separated by 17.5% SDS-PAGE followed by transfer to nitrocellulose membrane (Protran; Schleicher and Schuell, Keene, NH). Blots were incubated for 1 h at room temperature in 5% milk in PBS-T followed by an overnight incubation at 4°C with either a polyclonal rabbit anti-mouse CRES antiserum (1:5000), an affinity-purified polyclonal rabbit anti-mouse CRES antibody (1:100, 1.3 µg/ml), or a polyclonal rabbit anti-guinea pig proacrosin antiserum (1:2000) in 2% milk in PBS-T. The following day the blots were washed extensively in 2% milk in PBS-T and incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (BioSource International, Camarillo, CA) at 1:40 000 dilution in 2% milk in PBS-T. Control blots were incubated with preimmune serum (1:5000), secondary antibody alone (1:40 000), or affinity-purified CRES antibody that had been blocked by incubation with His-CRES antigen. After incubation with the secondary antibody, the blots were washed several times in 2% milk in PBS-T, incubated with SuperSignal reagent (Pierce, Rockford, IL) for 5 min, and then exposed to film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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In previous studies, we demonstrated that CRES protein is present in the elongating spermatids in the testis and in the epithelial cells and luminal fluid of the proximal caput epididymidis [24]. These observations suggested that CRES protein may not only be present during germ cell development but also may interact with spermatozoa during epididymal transit. In the present studies, indirect immunofluorescence was carried out to determine whether CRES protein was associated with epididymal spermatozoa. Mouse spermatozoa obtained from five regions of the epididymis were fixed, permeabilized, and incubated with a polyclonal rabbit anti-mouse CRES antibody followed by a Texas Red-conjugated goat anti-rabbit secondary antibody. As shown in Figure 1, a fluorescent signal representing CRES protein was localized to the sperm heads and appeared to be restricted to the acrosomal region in both proximal caput and cauda epididymal spermatozoa. No fluorescent signal was detected in the sperm midpiece or tail. A comparison of the fluorescent staining pattern between proximal caput and cauda epididymal spermatozoa suggested a slight difference in the distribution of CRES protein between the two sperm populations. CRES protein in the proximal caput epididymal spermatozoa was more diffusely distributed and was localized to both the anterior and posterior acrosomal regions, whereas CRES protein in the cauda epididymal spermatozoa was primarily restricted to the anterior acrosomal region. Examination of spermatozoa from the mid-caput, distal caput, and corpus epididymidis also showed acrosomal localization of the CRES protein, with the protein distribution gradually becoming more restricted to the anterior acrosomal region in spermatozoa from progressively more distal epididymal regions (data not shown). The specificity of the CRES antiserum for the CRES protein was demonstrated by the absence of a fluorescent signal in spermatozoa incubated with preimmune serum or CRES antiserum that had been preincubated with CRES antigen (block) (Fig. 1). Spermatozoa incubated with secondary antibody alone also did not exhibit a fluorescent signal (data not shown). Also, spermatozoa that went through the immunofluorescence procedure without incubation with primary or secondary antibodies showed the same faint sperm-associated background staining as in the preimmune and blocked controls, suggesting that the faint signal is due to sperm autofluorescence (data not shown). As a marker for sperm acrosomes, cauda epididymal spermatozoa were incubated with a polyclonal rabbit anti-guinea pig proacrosin antibody that has been shown to recognize both proacrosin and acrosin proteins [25]. As shown in Figure 1, the fluorescent signal representing proacrosin was restricted to the anterior region of the sperm acrosomes. These observations suggest that CRES protein, like proacrosin, is present in the sperm acrosomes.
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Because the epididymal spermatozoa examined in Figure 1 were permeabilized by methanol:glacial acetic acid prior to incubation with the CRES antibody, both intraacrosomal and plasma membrane-associated CRES protein would be detected. Therefore, fixed, nonpermeabilized proximal caput and cauda epididymal spermatozoa were examined for CRES protein by indirect immunofluorescence. Only a weak signal was observed that did not appear to be notably different from the background signal present in nonpermeabilized spermatozoa incubated with preimmune serum (data not shown). These observations suggest that CRES protein localization in spermatozoa is intraacrosomal rather than associated with the plasma membrane. This was further supported by indirect immunofluorescence analysis of testicular spermatozoa. Spermatozoa were obtained from mouse testes either by mincing of the tissue followed by Percoll gradient centrifugation or by collection of spermatozoa from the rete testis following efferent duct ligation. Spermatozoa obtained by tissue mincing represented a mix of testicular spermatozoa and germ cells, while spermatozoa obtained from efferent duct-ligated mice represented a relatively pure population of spermiated, testicular spermatozoa. As shown in Figure 2, CRES protein was detected in the acrosomes of fixed, permeabilized testicular spermatozoa and exhibited a localization similar to that in proximal caput epididymal spermatozoa. Specifically, CRES protein localization was diffuse and was present in both the anterior and posterior regions of the acrosomes. Interestingly, some of the cells in the mixed testicular spermatozoa-germ cell fractions (Fig. 2, top) showed an even more dispersed pattern of CRES protein localization in the sperm acrosomal region, probably due to incomplete condensation of the acrosomes. These testicular cells may be immature spermatids, since the differential interference contrast (DIC) images show the sperm heads to be swollen compared to those of the testicular spermatozoa obtained by efferent duct ligation (Fig. 2, bottom).
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Localization of CRES Protein in the Sperm Acrosomes by Immunogold Electron Microscopy
Electron microscopy was performed using immunogold postembedding labeling to confirm CRES protein localization within the sperm acrosomes. Proximal caput and cauda epididymal spermatozoa were embedded, and ultrathin sections were incubated with the polyclonal rabbit anti-mouse CRES antiserum, preimmune serum, or polyclonal rabbit anti-guinea pig proacrosin antiserum followed by a secondary antibody conjugated to colloidal gold. The micrographs shown in Figure 3 support our observations at the light microscopic level and show that CRES protein is localized in the sperm acrosomes with no specific labeling detected in the sperm nucleus, midpiece, or tail regions. Furthermore, the gold particles appeared to be distributed throughout the acrosomal contents, suggesting that CRES protein localization is intraacrosomal and does not appear to associate preferentially with the acrosomal membranes. Since it was difficult to determine whether comparable regions of the sperm acrosome were represented in the sections of proximal caput and cauda epididymal spermatozoa, we were unable to confirm by immunogold labeling whether CRES protein localization differed between the two sperm populations. Control sections incubated with preimmune serum (Fig. 3) or secondary antibody alone (data not shown) showed only background levels of gold particles with no specific localization in the spermatozoa. Cauda spermatozoa incubated with the rabbit anti-guinea pig proacrosin antiserum showed a distribution of proacrosin similar to that of CRES protein. Proacrosin was distributed throughout the acrosomal contents (Fig. 3).
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Identification of CRES Proteins in Testicular and Epididymal Sperm Extracts
We previously identified two predominant CRES proteins of 19 and 14 kDa in whole testicular and epididymal tissue lysates by Western blot analysis [24]. Recently, we identified two additional CRES forms of 17 and 12 kDa and determined that the 19- and 17-kDa proteins were the result of N-linked glycosylation of the 14- and 12-kDa CRES forms (see Note Added in Proof, reference 2). To determine which CRES form(s) was associated with spermatozoa, Western blot analysis was performed using whole sperm extracts prepared from testicular and epididymal spermatozoa. For comparison, whole epididymal and testis tissue lysates were also examined. As expected, the predominant CRES proteins of 19 and 14 kDa were present in the epididymal and testis tissue lysates (Fig. 4, A and B). Lower levels of the 17- and 12-kDa CRES proteins were also detected in the epididymal tissue (Fig. 4B). Examination of the sperm extracts showed that both the 19- and 14-kDa CRES proteins were present in testicular and proximal caput epididymal spermatozoa (Fig. 4, A and B). Minor amounts of the 17-kDa CRES protein were also detected in the proximal caput epididymal spermatozoa; however, because of the intensity of the chemiluminescence signal, it was difficult to determine whether the 17-kDa CRES protein was also present in testicular spermatozoa. Overall, the relative levels of CRES protein present in the testicular sperm extracts appeared higher than the levels present in a similar number of spermatozoa from the proximal caput epididymidis (Fig. 4A). Extracts prepared from spermatozoa from the mid-caput to the cauda epididymidis contained primarily the 14-kDa CRES protein with barely detectable levels of the 19- and 17-kDa CRES proteins (Fig. 4B).
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To ensure that the CRES proteins observed in the testicular sperm extracts were not due to CRES protein present in rete testis fluid, the supernatant solution obtained after gentle centrifugation of testicular spermatozoa was examined by Western blot analysis. No detectable CRES protein was present in the crude rete testis fluid sample (data not shown). Similarly, no detectable CRES protein was present in the Percoll solution surrounding the epididymal spermatozoa (data not shown). The specificity of the CRES antiserum for the 19-, 17-, 14-, and 12-kDa CRES proteins present in tissue and sperm extracts was demonstrated by the absence of these proteins when preimmune serum was used (Fig. 4C). Similarly, when the affinity-purified CRES antibody was preincubated with the antigen (block), the CRES proteins were not detected (data not shown). In addition to the CRES proteins, minor levels of 29- and 24-kDa proteins were detected in tissue and sperm extracts. Since these proteins were not detected by the preimmune serum or by a blocked CRES antibody (data not shown), they may possess cross-reacting epitopes with the CRES antibody or may represent CRES protein complexes resistant to the reducing agents. A 57-kDa protein detected in the epididymal sperm extracts, however, was also detected by the preimmune serum, suggesting that it represents nonspecific binding (Fig. 4C).
Sequential Extraction of CRES Proteins from Epididymal Spermatozoa
Immunofluorescence and immunogold analyses suggested that CRES protein localization was intraacrosomal rather than associated with the sperm surface. Sequential extraction of proximal caput and cauda epididymal spermatozoa followed by Western blot analysis was performed to confirm these observations. Similar numbers of proximal caput and cauda epididymal spermatozoa were sequentially extracted with low salt, high salt, 0.1% Triton X-100, 2% SDS, and Laemmli buffer containing 5% SDS and ß-mercaptoethanol. Although low levels of the 19- and 14-kDa CRES proteins were present in the low-salt and Triton X-100 extractions, 2% SDS was required to extract the majority of CRES protein from proximal caput epididymal spermatozoa (Fig. 5). Some CRES protein was also extracted from cauda epididymal spermatozoa after incubation in Triton X-100. However, in contrast to observations with proximal caput epididymal spermatozoa, Laemmli buffer rather than SDS alone was required to extract the majority of the CRES protein from cauda spermatozoa. While the predominant CRES protein extracted from cauda epididymal spermatozoa was the 14-kDa form, some 19-kDa CRES protein was also detected (Fig. 5). In the Triton X-100 extracts of both proximal caput and cauda epididymal spermatozoa, an intermediate-sized CRES protein of approximately 15 kDa was also observed that may reflect proteolytic processing of larger CRES forms.
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CRES Protein Was Released during the Acrosome Reaction
The presence of CRES protein in sperm acrosomes next raised the question whether CRES protein was released during the acrosome reaction. Therefore, cauda epididymal spermatozoa were induced to undergo the acrosome reaction by the addition of the calcium ionophore A23187. Indirect immunofluorescence was used to examine spermatozoa for the presence of CRES and proacrosin proteins before capacitation, 1 h following capacitation, and 1 h after induction of the acrosome reaction. The loss of proacrosin immunofluorescence in the cauda sperm acrosomes was used as an indication that spermatozoa had undergone the acrosome reaction. As shown in Figure 6, after 1-h capacitation the majority of the cauda epididymal spermatozoa contained CRES and proacrosin proteins and did not appear notably different from the noncapacitated cauda epididymal spermatozoa in Figure 1. However, after induction of the acrosome reaction with the calcium ionophore, the majority of spermatozoa exhibited a dramatically reduced fluorescent signal for both the CRES and proacrosin proteins. As indicated by the arrowheads in Figure 6, only a few spermatozoa contained detectable CRES and proacrosin, suggesting that these few spermatozoa had not undergone or had not completed the acrosome reaction.
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The percentage of spermatozoa undergoing the acrosome reaction was determined by examining approximately 100 spermatozoa by indirect immunofluorescence before and after induction of the acrosome reaction. As summarized in Table 1, after 1-h capacitation approximately 23% of the spermatozoa lacked detectable CRES and proacrosin immunofluorescence, suggesting that these spermatozoa had undergone a spontaneous acrosome reaction. However, 1 h after induction of the acrosome reaction with the ionophore, 92% of the spermatozoa lacked CRES and proacrosin. Spermatozoa that received carrier DMSO instead of the ionophore exhibited only a slightly higher number of acrosome-reacted spermatozoa (37%) than after 1 h of capacitation. This most likely represents spermatozoa that had spontaneously acrosome reacted during the additional hour of incubation in capacitating medium, rather than any direct effects of DMSO.
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Western blot analysis was next performed to determine the movement of CRES protein following the induction of the sperm acrosome reaction. Cauda epididymal spermatozoa were induced to undergo the acrosome reaction with the calcium ionophore using the same conditions as for indirect immunofluorescence analysis. The soluble, membrane, and sperm fractions were then collected and examined for CRES and proacrosin proteins. Whole cauda epididymal sperm extracts prepared from noncapacitated spermatozoa were examined for comparison. As shown in Figure 7, induction of the acrosome reaction with the calcium ionophore resulted in the release of CRES protein into the soluble fraction. Besides the 14-kDa CRES protein, which was the predominant form in the noncapacitated spermatozoa, additional CRES forms ranging from 12 to 19 kDa were identified in the soluble fraction. Also, not all CRES protein appeared to be released following the acrosome reaction, since the 14-kDa CRES protein was also detected in the acrosome-reacted spermatozoa (Fig. 7).
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Acrosin is present in spermatozoa as a proenzyme that is converted to an active enzyme with a lower molecular weight during the acrosome reaction [28]. Western blot analysis showed that proacrosin was present in the noncapacitated spermatozoa as a 55–60-kDa protein that was processed to an active form of approximately 32 kDa and was released into the soluble fraction during the acrosome reaction (Fig. 7). Some of the 32-kDa acrosin protein was also detected in the acrosomal membrane fraction. Similar to observations for CRES protein, not all proacrosin was processed and released from the spermatozoa after induction of the acrosome reaction, as the 55–60-kDa protein was also detected in the acrosome-reacted sperm fraction. These observations are in good agreement with previous studies examining acrosin processing and release after induction of the acrosome reaction [28]. These studies suggest, therefore, that CRES protein is similar to acrosin and is released during the acrosome reaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Examination of isolated testicular and epididymal spermatozoa by indirect immunofluorescence suggest that CRES protein localization changes in the acrosome as spermatozoa undergo maturation in the epididymis. In the testicular and proximal caput epididymal sperm acrosomes, CRES protein was dispersed and was present in the anterior and posterior regions of the acrosome. Spermatozoa from the cauda epididymidis, however, showed CRES protein to be primarily present in the anterior region of the acrosome. A similar condensation of protein localization during epididymal transit has been observed for other acrosomal proteins. For example, the 90-kDa intraacrosomal antigen MN7 is more restricted in its localization in the acrosomes of mature guinea pig cauda epididymal spermatozoa than in the immature caput epididymal spermatozoa [29]. This condensation of acrosomal proteins is likely correlated with the remodeling and final organization of the sperm acrosome as spermatozoa undergo the maturation process in the epididymis.
Although our studies indicate that CRES protein localization is intraacrosomal, the question whether CRES protein is also on the sperm surface is more difficult to answer. Indeed, we have previously shown that in addition to expression of CRES protein in the testicular germ cells, CRES protein is synthesized and secreted exclusively by the proximal caput epididymal epithelium [24]. The luminal CRES protein accumulates in the lumen of the mid-caput but disappears by the distal caput epididymidis. Therefore, it is possible that luminal CRES protein may associate with the plasma membranes of spermatozoa in the proximal caput epididymidis.
While this is intriguing, several lines of evidence suggest that the major pool of CRES protein associated with spermatozoa is intracellular rather than on the sperm surface. First, immunofluorescence and Western blot analyses showed that notable levels of CRES protein were associated with spermatozoa prior to their entrance into the epididymis and to any exposure to luminal CRES proteins. Furthermore, CRES proteins levels did not increase in proximal caput epididymal spermatozoa exposed to the epididymal lumen but rather appeared to be lower than the levels of protein in testicular spermatozoa. This may reflect maturational changes in the organization of the sperm acrosome and its contents. Also, permeabilization of testicular and epididymal spermatozoa was required to detect a strong signal by immunofluorescence, suggesting that the majority of CRES protein was intracellular. Finally, immunogold electron microscopy and the differential extraction studies suggest that CRES protein localization is primarily intraacrosomal. The requirement of ß-mercaptoethanol for the extraction of CRES protein from cauda but not from proximal caput epididymal spermatozoa likely reflects the high degree of sulfhydryl cross-linking that occurs in spermatozoa during epididymal transit. Therefore, while it appears that the majority of CRES protein associated with spermatozoa is intraacrosomal, we cannot rule out at this time the possibility that there may be a transient interaction of luminal CRES protein with the sperm surface in the proximal caput epididymidis.
Western blot analysis of spermatozoa from the testis and from the five regions of the mouse epididymis showed a striking difference in the CRES protein forms present in spermatozoa. While the levels of the 14-kDa CRES protein remained relatively constant during epididymal transit, there was a loss of the predominant 19-kDa and the minor 17-kDa CRES proteins from spermatozoa isolated from regions distal to the proximal caput epididymidis. There may be several possible mechanisms by which the levels of the higher molecular weight CRES proteins are reduced in these spermatozoa. We have previously determined that the 19- and 17-kDa CRES proteins are the glycosylated forms of the 14- and 12-kDa proteins and that they contain high mannose residues (see Note Added in Proof, reference 2). Therefore, one possibility is that active glycohydrolases in the sperm acrosome may deglycosylate the higher molecular weight CRES proteins. Glycohydrolases that have been shown to be active in the sperm acrosome include -D-mannosidase, ß-D-mannosidase, -L-fucosidase, ß-D-galactosidase, and many others [3]. However, if the 19- and 17-kDa CRES proteins were deglycosylated, one would expect to see an increase in the levels of the nonglycosylated 14- and 12-kDa proteins. A second possibility is that the glycosylated forms of CRES protein are particularly sensitive to proteolysis and therefore may be degraded by specific proteases that become active within the sperm acrosome during epididymal transit. A third possibility is that during epididymal transit the 19- and 17-kDa proteins may become compartmentalized or complexed to other proteins within the acrosome, rendering them inaccessible to the CRES antibody even under reducing SDS-PAGE conditions. The possibility that some of the higher-molecular weight CRES proteins do become masked during epididymal transit is supported by our observation that, following induction of the acrosome reaction and the release of the acrosomal contents from cauda spermatozoa, the 19- and 17-kDa CRES proteins were observed in the soluble fraction. Similarly, treatment of cauda spermatozoa with Triton X-100 showed the presence of the larger CRES forms.
The localization of CRES protein in the sperm acrosomes raises interesting possibilities with regard to its function. CRES protein is similar to several other acrosomal proteins in its dual localization in the sperm acrosomes and epididymal fluid. For example, several hydrolytic enzymes including ß-D-galactosidase [15, 30], -L-fucosidase [16], ß-N-acetyl-hexosaminidase [31, 32], and -D-mannosidase [3, 32] are also present in epididymal fluid and sperm acrosomes. Most of these enzymes have a broad pH optimum and are active at both acidic and neutral pH [16, 33]. Therefore, these proteins may not only function in the sperm acrosome during epididymal maturation but also may be involved in the modification of epididymal sperm surface proteins. One possible role of CRES protein may be to regulate these specific processing events both within the acrosome and in the epididymal luminal fluid.
CRES protein is also similar to other acrosomal proteins such as proacrosin/acrosin in its behavior following the acrosome reaction. While CRES protein is released after the acrosome reaction, Western blot analysis showed that some CRES protein remained associated with the acrosome-reacted spermatozoa. At this time we cannot rule out the possibility that the CRES protein detected in the acrosome-reacted spermatozoa may in fact represent the small proportion of spermatozoa that had not undergone the acrosome reaction. This conclusion would support the immunofluorescence data showing that a small percentage of the spermatozoa remained positive for CRES protein following the acrosome reaction. It is equally possible, however, that after the acrosome reaction, spermatozoa retain a proportion of CRES protein that is detectable only by the more sensitive chemiluminescence technique and not by indirect immunofluorescence. If CRES protein does remain associated with spermatozoa after the acrosome reaction, this raises the possibility that CRES, like proacrosin, may be associated with the acrosomal matrix as well as present in the acrosomal contents. It has been proposed that the acrosomal matrix and its associated proteins regulate the differential release of hydrolytic enzymes during the acrosome reaction [34, 35]. It has also been postulated that the acrosomal matrix retains a population of hydrolytic enzymes following the acrosome reaction to maintain a pool of hydrolytic activity at the site of sperm-zona binding [34]. Therefore CRES protein, a putative protease inhibitor, may be present at sites of high proteolytic and hydrolytic activity to regulate the release and activity of enzymes that are critical for fertilization.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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1. Cornwall GA, Hsia N, Sutton HG. Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene. Biochem J 1999; (in press).
2. Sutton HG, Fusco A, Cornwall GA. CRES protein colocalizes with LHß protein in mouse anterior pituitary gonadotropes. Endocrinol 1999; (in press).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gail A. Cornwall, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990; cbbgc{at}wpoffice.net.ttuhsc.edu
Accepted: February 2, 1999.
Received: December 7, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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-L-fucosidase. Biol Reprod 1993; 48:1228–1238.[Abstract]This article has been cited by other articles:
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