| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
acrosome reaction, fertilization, gamete biology, sperm, spermatogenesis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CRES is also distinguished from the cystatins by its unique tissue and cellular localization. In contrast to the ubiquitous expression of the cystatin C gene, we have shown that in mice, the Cres gene is highly expressed in the proximal caput epididymidis, round spermatids, and anterior pituitary gonadotroph cells, suggesting specific roles within the reproductive and neuroendocrine systems [5–7]. Furthermore, studies of mouse spermatozoa show that CRES protein is localized in the sperm acrosome and is released following the acrosome reaction [8]. These studies taken together suggest that CRES may perform important roles as a protease inhibitor within the sperm acrosome either before or during the fertilization process.
To determine whether CRES is conserved among species, we screened a human testis library for CRES cDNA. The human CRES protein, similar to findings in mice, contains the four conserved cysteine residues and the single consensus site, but lacks the other two consensus sites that are necessary for inhibition of cysteine proteases [1]. These observations suggest that CRES likely performs unique functions in humans as in mice. To date, however, studies have not been carried out to examine the expression of the CRES gene in human tissues and, in particular, to determine whether CRES protein is present in human spermatozoa. These studies are essential, because there can be considerable evolutionary divergence in fertilization molecules between species [9, 10], and therefore it is important that we establish whether CRES is present in spermatozoa from a species other than mouse. If so, this would suggest a conserved role for CRES in sperm function, fertilization, or both. The studies presented herein were performed to address these questions.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All studies involving human subjects were approved by the Texas Tech University Health Sciences Center Institutional Review Board. Semen was obtained from healthy adult donors by masturbation following 48–72 h of sexual abstinence. Semen samples were allowed to liquefy for 30 min at 37°C, followed by semen analysis that included measurement of semen volume, sperm count, and evaluation of the percentage of motile spermatozoa. Ejaculates were washed by adding 10 times the volume of Dulbecco modified PBS and centrifugation at 300 x g for 10 min. Fixed and flash-frozen human testes and epididymides were obtained from the Brain and Tissue Bank for Developmental Disorders, University of Maryland, Baltimore, MD (National Institutes of Health contract NO1-HD-1-3138).
Northern Blot Analysis
Total RNA was isolated from human testis and epididymal tissue using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. RNA was separated on a 1% agarose gel containing 1x borate buffer pH 8.2, and 0.66 M formaldehyde. The RNA samples were heated at 95°C for 2 min and then loaded onto the gel and electrophoresed. To verify equal loading of RNA in each lane of the gel, ethidium bromide was included in the RNA sample. The gels were washed extensively in water to remove formaldehyde before transferring them to nylon membrane (Nytran; Schleicher and Schuell, Keene, NH). Blots containing intact mRNA isolated from various pooled human tissues were purchased from Invitrogen. For all Northern blots, membranes were prehybridized for 2 h at 42°C in hybridization buffer containing 50% formamide, 5x SSC (0.075 M sodium citrate and 0.75 M NaCl pH 7.0), 0.2 mg/ml salmon sperm DNA, 0.4 mg/ml yeast RNA, 50 µg/ml BSA, 0.1% SDS, and 12.5 mM sodium phosphate buffer pH 6.6, followed by hybridization overnight at 42°C in the presence of 1 x 106 cpm probe/ml of hybridization buffer. Blots were probed with a 32P-labeled human CRES cDNA prepared using a random primer labeling method (Prime-It II; Stratagene, La Jolla, CA). After hybridization, the blots were washed in 2x SSC (0.03 M sodium citrate and 0.3 M NaCl pH 7.0) at room temperature for 10 min followed by washing in 2x SSC and 1% SDS at 42°C three times for 15 min each, and then one or two times for 15 min at 65°C before exposure to film.
Reverse Transcription-Polymerase Chain Reaction
Total RNA (2.5 µg) from human caput, corpus, and cauda epididymides was incubated in a reverse transcription (RT) reaction buffer containing 5 mM MgCl2, 50 mM KCl, 10 mM Tris pH 8.3, 0.5 mM deoxynucleotide triphosphates (dNTPs), 20 U RNasin (Promega, Madison, WI), and 2.5 µM oligo(dT) (Promega) in a final volume of 25 µl for 30 min at 37°C in the presence of 2.5 units of RNase-free DNase I (Roche, Indianapolis, IN). After heat inactivation of DNase I at 75°C for 5 min, an aliquot was removed for polymerase chain reaction (PCR) amplification as a no-RT control to confirm the absence of contaminating DNA. Murine leukemia virus reverse transcriptase (50 U; Applied Biosystems, Foster City, CA) was added to the remainder, and RT was carried out at 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min. Three microliters of each RT and no-RT reaction was amplified by PCR in separate reactions using primers that recognize human CRES cDNA. The PCR master mix contained 10 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.25 mM dNTPs, 0.5 µM forward (5'-CAAAGAGAGCGAGGACAAGTATGTC-3') and reverse (5'-GGAAGAGCCTCCCACCTGCCATT-3') primers and 1.25 units of Taq DNA polymerase (Sigma Chemical Company, St. Louis, MO). After an initial denaturation at 95°C, PCR was carried out at 95°C for 45 sec, at 64°C for 25 sec, and at 72°C for 1 min for 40 cycles using a minicycler (MJ Research Inc., Watertown, MA). RT-PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing 1x Tris acetate EDTA. The identity of the RT-PCR products was confirmed by cloning the RT-PCR products into the pGEM-T-Easy vector (Promega) followed by sequencing.
In Situ Hybridization
In situ hybridization was carried out as described previously [11] using human testis tissue sections and human CRES antisense and sense (control) RNA probes. To prepare the RNA probes, a CRES cDNA containing the full-length human CRES sequence was digested with either BamHI (antisense) or XhoI (sense) to linearize the DNA followed by phenol/chloroform extraction and ethanol precipitation. Riboprobes were generated using the Riboprobe Gemini system (Promega). Briefly, 1 µg of linearized DNA was incubated with 100 µCi of 35S-UTP, 10 mM dithiothreitol (DTT), 40 units of RNasin, 2.5 mM dNTPs, and 20 units of T3 or T7 RNA polymerase in the presence of 1x transcription buffer in a final reaction volume of 20 µl for 1 h at 37°C. The template DNA was removed by the addition of 1 unit of RQ1 RNase-free DNase (Promega) in the presence of 40 units of RNasin and 20 µg of tRNA, and incubation at 37°C for 15 min. The labeled RNA was then phenol/chloroform extracted in the presence of 5 mM DTT, and the aqueous phase was removed and ethanol precipitated by adding 2.5 M ammonium acetate and a 5x volume of 100% ethanol and incubation on dry ice for 10 min. Following centrifugation, the RNA pellet was washed with 70% ethanol, allowed to air dry, and then resuspended in 50 µl of 10 mM DTT and counted in a scintillation counter. Because the RNA probes were expected to be less than 1 kilobase (kb) in size, probes were not hydrolyzed before hybridization.
Preparation of Recombinant Human CRES Protein and Antibody Production
Recombinant human CRES protein was prepared as described previously for mouse CRES protein [6]. Briefly, a human CRES cDNA lacking the signal peptide was generated by PCR from a plasmid containing the full-length human CRES cDNA. The forward and reverse primers contained BamHI and HindIII sequences to allow in-frame cloning into the pQE9 expression vector (Qiagen, Chatsworth, CA) The amplified products were cloned into the pGEM-T-Easy vector (Promega), digested with the appropriate restriction enzymes, and ligated into the BamHI/HindIII sites of pQE9, resulting in CRES sequences that were downstream of a 6x histidine coding sequence. The His-CRES fusion protein was expressed in Escherichia coli M15[pREP4] and purified from inclusion bodies by nickel affinity chromatography following the manufacturer's protocol (Qiagen). The eluted His-CRES protein was extensively dialyzed and then used for polyclonal antibody production.
To produce a polyclonal antibody against the human His-CRES fusion protein, a New Zealand rabbit was injected with 1 mg of His-CRES protein in 1.5 ml of Freund complete adjuvant. After 1 mo, the rabbit was boosted by an i.m. injection of 500 µg of His-CRES protein in 1.5 ml of Freund incomplete adjuvant. The CRES antiserum was affinity-purified by chromatography on His-CRES-conjugated Sepharose.
Western Blot Analysis
Approximately 5–10 x 107 washed spermatozoa were lysed directly in 200 µl of Laemmli buffer. Powder from freeze-dried human testis was resuspended in 2 ml of antibody lysis buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.2% SDS, 0.1 mg/ml PMSF, and 0.01 mg/ml of aprotinin and sonicated for 15 sec on ice. After centrifugation at 13 000 x g for 10 min, the supernatant was recovered and stored at -20°C. For CRES protein analysis, lysates representing approximately 1 x 107 spermatozoa or 20 µg of testis protein were separated under reducing conditions on a 17.5% SDS polyacrylamide gel followed by transfer to PVDF membrane (Immobilon-P, Millipore, Bedford, MA). For proacrosin analysis, lysates were separated under reducing conditions on a 10% SDS polyacrylamide gel and transferred to nitrocellulose membrane (Schleicher and Schuell). Blots were incubated for 1 h with Tris-buffered saline (50 mM Tris-HCl pH 7.4 and 200 mM NaCl) containing 0.2% Tween-20 (TBS-T) and 5% (w/v) nonfat dry milk at room temperature, followed by incubation with a polyclonal rabbit anti-guinea pig proacrosin antiserum (1:20 000) for 1 h [12] or an affinity-purified polyclonal rabbit anti-human CRES antibody (0.2 µg antibody/cm2 membrane) at 4°C overnight. Control blots were incubated overnight at 4°C with preimmune serum (1:5000) or anti-human CRES antibody that had been preincubated with human CRES recombinant protein (block). The blots were washed extensively in TBS-T and incubated with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Biosource, Camarillo, CA) at 1:40 000 for 1 h at room temperature. The blots were washed several times in TBS-T, incubated with Supersignal Reagent (Pierce, Rockford, IL) for 5 min, and then exposed to film.
Endoglycosidase F Treatment
To determine whether the human CRES protein possessed N-linked carbohydrate residues, 20 µg of testis protein or 2–5 x 106 spermatozoa were denatured in Laemmli buffer containing 1% ß-mercaptoethanol and incubated with 500 units of N-glycosidase F (PNGase, New England Biolabs Inc., Beverly, MA) in the presence of 1% NP40 for 2 h at 37°C. SDS was then added to an 8% final concentration to solubilize any potential protein aggregates followed by SDS-PAGE and Western blot analysis.
Induction of the Acrosome Reaction
Washed spermatozoa were gently resuspended in 2 ml of Biggers, Whitten, and Whittingham medium (BWW) [13] and centrifuged at 300 x g in a 15-ml snap-cap centrifuge tube. The resulting sperm pellet was overlaid with 2 ml of BWW containing 30 mg/ml of human serum albumin (HSA fraction V powder; Sigma), and the tubes were inclined at 45°C and incubated at 37°C in an atmosphere of 5% CO2 and 95% air for 4 h to allow capacitation. The upper fraction containing motile spermatozoa was centrifuged at 300 x g for 10 min, and the pellet was resuspended in BWW without HSA. To induce the acrosome reaction, spermatozoa were incubated for 50 min with a final concentration of 30 µM thapsigargin (Sigma) or an equivalent volume of the vehicle, dimethyl formamide/100% ethanol (3:1 DMF/EtOH) as a control. An aliquot of spermatozoa was air-dried on a glass slide and subjected to immunofluorescence analysis as described below to confirm acrosomal status. The remainder of the sample was centrifuged at 500 x g, and the pellet was resuspended in 50 µl of reducing Laemmli buffer. The supernatant containing proteins released during the acrosome reaction was precipitated with 10% trichloroacetic acid, washed with cold acetone, and then resuspended in reducing Laemmli buffer. The sperm pellet and supernatant were analyzed for CRES and proacrosin/acrosin proteins by SDS-PAGE followed by Western blot analysis.
Indirect Immunofluorescence
Approximately 1 x 106 noncapacitated, capacitated, or acrosome-reacted spermatozoa were air-dried onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) that had been marked with a hydrophobic slide marker (PAP pen) to allow small incubation volumes. Sperm cells were fixed and permeabilized with 95% methanol/5% glacial acetic acid for 20 min at -20°C. After fixation, spermatozoa were washed with PBS followed by PBS containing 0.3% Tween 20 (PBS-T), and subsequently incubated with PBS-T containing 5% goat serum for 1 h at 37°C to block nonspecific binding. Spermatozoa were then incubated with affinity-purified rabbit anti-human CRES antibody (3.6 µg/ml) or a rabbit anti-guinea pig proacrosin antiserum (1:200) in PBS-T containing 5% goat serum for 1 h at 37°C. The spermatozoa were washed three times with PBS-T for 10 min at room temperature and then incubated with a goat anti-rabbit immunoglobulin G conjugated to Texas Red secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37°C. Control samples included spermatozoa incubated with preimmune serum (1:200) or CRES antibody that had been preincubated with recombinant human CRES protein for 45 min at 4°C (blocked antiserum). After incubation with secondary antibody and repeated washing with PBS, the slides were inverted onto coverslips containing Fluoromount G (Southern Biotechnology Associates, Birmingham, AL), and fluorescence was observed using a Zeiss (New York, NY) Axiovert 135 epifluorescence microscope. To quantify the percentage of spermatozoa that had undergone the acrosome reaction, approximately 100 spermatozoa were analyzed in each treatment group for the presence of proacrosin protein. Spermatozoa that lacked proacrosin were determined to have undergone the acrosome reaction.
To examine live, nonfixed spermatozoa for CRES protein, 5–10 x 106 spermatozoa that had been treated with either DMF or thapsigargin were centrifuged at 300 x g for 5 min and carefully resuspended in 200 µl of PBS containing 5% normal goat serum. The spermatozoa were allowed to bind to polylysine-treated slides (2 h at room temperature in a hydrated chamber). The slides were gently washed with PBS to remove unbound spermatozoa and then incubated with affinity-purified rabbit anti-human CRES antibody at 37°C as described above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To examine the tissue expression of the CRES gene, blots containing mRNA isolated from various human tissues were probed with the human CRES cDNA in Northern blot analysis. As shown in Figure 1A, a transcript of approximately 0.9 kb was detected in the testis but not in any other tissues examined, including lung, prostate, ovary, cervix, uterus, spleen, kidney, placenta, and brain. The commercially prepared tissue blots were tested for intact mRNA by the supplier, and thus the absence of CRES mRNA in tissues other than the testis is likely not due to degradation of the mRNA. Because we have previously determined that CRES is expressed at high levels in the mouse proximal caput epididymidis, Northern blot analysis was also carried out to examine CRES expression in human caput, corpus, and cauda epididymal regions. In contrast to the high levels of CRES expression in the testis, CRES mRNA was not detected in the epididymis by Northern blot analysis (Fig. 1B). RT-PCR analysis of the same RNA preparations, however, revealed CRES mRNA in all three epididymal regions, showing that indeed, CRES is present in human epididymis, but at lower levels compared with those in testis (Fig. 1C).
|
In situ hybridization experiments were next performed to determine whether CRES expression in the testis was restricted to a distinct cell population. Cross-sections of human testis were probed with an antisense RNA probe to the human CRES cDNA. As shown in Figure 2, A and B, CRES mRNA was present in clusters of germ cells within the seminiferous epithelium. Examination of the tissue sections at a higher magnification (Fig. 2, C and D) showed that the silver grains representing CRES mRNA were localized to clusters of germ cells with darkly staining nuclei located near the apical surface of the seminiferous epithelium, indicating that CRES is expressed in round and possibly early stages of elongating spermatids. Significant numbers of silver grains were not detected in any other cell types in the seminiferous tubules or within the peritubular or interstitial cells. The specificity of the RNA probe for the CRES mRNA was demonstrated by only background levels of silver grains in testis sections incubated with the sense strand CRES RNA probe (Fig. 2, E and F).
|
Identification of CRES Protein in Human Testis and Spermatozoa
To analyze CRES protein, lysates from human testis and ejaculated spermatozoa were separated by SDS-PAGE under reducing conditions and immunoblotted using antibodies against a bacterially expressed recombinant human CRES protein. A predominant 19-kDa and a minor 14-kDa protein were specifically detected with the CRES antibody, but not the preimmune serum, in the testis and spermatozoa (Fig. 3). In addition, a 29-kDa protein was observed in the testis, which may represent CRES protein dimers or a complex of CRES with another protein (Fig. 3B). We previously have observed a similar 29-kDa protein in Western blot analysis of mouse testis lysates [8]. The CRES antibody also detected a minor protein above 29 kDa in the human sperm lysates. However, this represents nonspecific binding of the CRES antibody because the protein was also detected by the preimmune serum (Fig. 3A).
|
Because we had previously determined that in mouse, the 19-kDa protein represented the N-linked glycosylated form of the 14-kDa CRES protein, studies were next carried out to determine whether similar N-linked glycosylation was present in the human CRES proteins. As shown in Figure 3B, protein lysates incubated with endoglycosidase F, which hydrolyzes most types of N-glycan chains from glycoproteins, resulted in a downward shift of the 19-kDa protein to the 14-kDa form, indicating that the higher molecular weight human CRES protein contains N-linked carbohydrates.
CRES Protein Is Present in the Sperm Equatorial Segment and Remains Associated after Induction of the Acrosome Reaction
A polyclonal rabbit anti-human CRES antibody was used in indirect immunofluorescence analysis to determine the localization of CRES protein in human spermatozoa. In methanol/acetic acid fixed and permeabilized spermatozoa, CRES protein was detected in the sperm equatorial segment with no immunostaining detected in other regions of the spermatozoa (Fig. 4). The lack of a detectable signal in spermatozoa incubated with preimmune serum or a blocked CRES antibody shows the specificity of the antibody for CRES protein.
|
To determine whether CRES was released from spermatozoa following the acrosome reaction, thapsigargin, a specific inhibitor of the microsomal calcium pump [14, 15], was used to induce the acrosome reaction, and spermatozoa were examined for CRES protein by immunofluorescence analysis. Immunofluorescence analysis using a polyclonal anti-guinea pig proacrosin antiserum was also performed to determine the presence or absence of the acrosomal marker, proacrosin. As shown in Figure 5, spermatozoa treated with thapsigargin (+Thaps) showed CRES immunostaining in the equatorial segment that was not different from that observed in capacitated spermatozoa exposed to DMF alone (+DMF). Examination of proacrosin in the same populations of spermatozoa, however, showed intense immunostaining in the sperm acrosomes of capacitated spermatozoa, but a dramatic loss of proacrosin from spermatozoa exposed to thapsigargin, indicating that indeed, the spermatozoa had undergone the acrosome reaction.
|
To get a quantitative assessment of acrosome-reacted spermatozoa following the treatments, approximately 100 cells from three separate immunofluorescence experiments were scored for proacrosin and CRES staining before and after capacitation and acrosome reaction. As summarized in Table 1, the majority (77%–94%) of the noncapacitated (control) and capacitated spermatozoa (capacitated in the presence or absence of the vehicle DMF) contained CRES and proacrosin proteins. However, only 10% of the spermatozoa retained proacrosin after thapsigargin treatment, whereas 92% of the spermatozoa retained CRES protein. To confirm the immunofluorescence studies, Western blots were performed to follow CRES and proacrosin proteins before and after induction of the acrosome reaction by thapsigargin. In capacitated cells, a 55-kDa proacrosin protein was detected only in the sperm pellet. Following the acrosome reaction, proacrosin was converted to 45- and 30-kDa forms that were detected only in the soluble fraction, indicating that proacrosin had been released from the sperm acrosome following exposure to thapsigargin (Fig. 6). In contrast, the 19- and 14-kDa CRES proteins were present in the sperm pellet before and after the acrosome reaction. These experiments confirm our immunofluorescence studies and demonstrate that CRES remains associated with the equatorial segment following the acrosome reaction.
|
|
CRES Is Exposed to the Extracellular Environment after Induction of the Acrosome Reaction
Studies were next carried out to determine whether CRES was present on the sperm cell surface. Capacitated spermatozoa were incubated with or without thapsigargin followed by CRES antibody without any prior fixation and permeabilization. As shown by immunofluorescence analysis in Figure 7, CRES protein was not detected in capacitated spermatozoa incubated with DMF alone (+DMF nonfixed). However, after treatment with thapsigargin (+Thaps nonfixed), about 50%–60% of acrosome-reacted spermatozoa exhibited a weak but specific CRES staining at the equatorial segment. Maximal levels of CRES immunostaining were observed when acrosome-reacted spermatozoa were treated with methanol/acetic acid before incubation with CRES antibody (+Thaps fixed). These results taken together indicate that CRES protein is not present on the sperm surface but becomes accessible to the extracellular environment following the acrosome reaction.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The studies presented here show that the CRES gene also exhibits tissue-specific expression in humans and is highly expressed in the testis. Similar to the findings in mice, in situ hybridization studies revealed CRES expression in the developing germ cells of the testis and, in particular, in round spermatids. We cannot rule out at this time that CRES mRNA may also be present in the early stages of elongating spermatids. These studies show, however, a striking conservation between mice and humans of CRES expression in postmeiotic germ cells.
In contrast, there appears to be a notable difference between mice and humans with regard to CRES expression in the epididymis. Unlike in mice, where CRES mRNA was easily detected in the proximal caput epididymidis by Northern blot analysis [5], RT-PCR was required to detect CRES mRNA in human epididymis. Furthermore, these studies showed CRES mRNA in all epididymal regions. We cannot rule out that the age or health of the individual from whom the tissue was acquired could contribute to the differences in CRES expression between the two species. However, Northern analysis of testis and epididymal tissue obtained from another individual (data not shown) showed the same pattern of CRES expression as in Figure 1B, supporting the notion that CRES mRNA levels are lower in human epididymis than in testis. The physiological significance of low levels of CRES mRNA in the epididymis is not known at this time. It is possible that CRES function in the epididymis may differ between humans and mice. The lack of regionalized expression for CRES in humans is also intriguing and may also reflect subtle differences in CRES function. However, because RT-PCR studies provide more qualitative than quantitative results, further studies using real-time PCR are required to accurately determine whether CRES expression is region-specific in human epididymis. Additional studies are also needed to examine CRES expression in other human tissues, including the anterior pituitary gland.
Our studies of CRES protein in human testis and sperm lysates also show several similarities as well as a striking dissimilarity with that observed in mice. Specifically, CRES protein is present in human and mouse tissues as 19-kDa and 14-kDa forms. In both species, the 19-kDa protein represents the N-linked glycosylated form of the 14-kDa protein. In addition to the 19- and 14-kDa proteins, CRES proteins of 17 and 12 kDa are also present in mouse tissues; however, the nature of these lower forms is not known [7, 8]. Although it is not apparent on the blots shown, overexposure of the Western blots revealed these lower forms in human tissues as well (data not shown). There are also some interesting differences between mouse and human CRES proteins. For example, the amount of the glycosylated 19-kDa form relative to the 14-kDa protein is higher in human testis than in mouse testis, where both proteins appear to be present in similar amounts [6]. Perhaps the most profound difference between mouse and human CRES proteins is their distinctive localizations in spermatozoa. Whereas CRES localization is intracellular in both species rather than associated with the sperm surface, CRES in mice is present within the acrosomal cap [8], whereas in human spermatozoa, CRES is located in the equatorial segment. Furthermore, following induction of the acrosome reaction, the majority of CRES is released from the sperm acrosome in mice [8], whereas it remains associated with the equatorial segment in humans. Because the calcium ionophore A23187 was used to induce the acrosome reaction in mice and thapsigargin was used to induce the acrosome reaction in humans, we cannot rule out subtle differences in the physiology of the induced acrosome reactions and thus possible effects on CRES protein. However, the fact that acrosin, a marker for the loss of the acrosomal matrix, was released under both acrosome induction conditions, indicates that the acrosome reaction occurred and suggests that the difference in the fate of CRES protein following the acrosome reaction truly reflects species differences.
The functional role of CRES, a protease inhibitor, in the sperm equatorial segment, is currently not known. The equatorial segment has long been postulated to be important in the late fusion step between spermatozoa and egg in mammalian fertilization [16]. So far, only a few proteins have been found to localize specifically to the equatorial segment of spermatozoa in different species, including M29 [17], oscillin [18], equatorin [19], and fertilin ß in mice [20]. Antibodies to equatorin did not affect sperm motility, zona pellucida adhesion, or penetration, but inhibited fertilization [21]. The human sperm protein SP10 also localizes to the equatorial segment as well as the inner acrosomal membrane and acrosomal matrix [22]. Antibodies to SP-10 inhibited adhesion of human spermatozoa to the hamster egg plasma membrane but did not inhibit binding to the human zona pellucida [23]. In humans, protein C inhibitor (PCI), a serine protease inhibitor that exhibits potent inhibitory activity against acrosin [24], has been shown to be redistributed from the plasma membrane overlying the acrosomal head to the equatorial segment following the induction of the acrosome reaction [25]. In zona-binding assays, addition of exogenous PCI blocked sperm-egg binding in a concentration-dependent manner, suggesting an important role for this inhibitor in fertilization, possibly by its modulation of acrosin activity [25]. These studies taken together support a functional role for equatorial segment proteins in fertilization events.
Because CRES is not associated with the sperm surface but becomes accessible to the extracellular environment during the acrosome reaction suggests that CRES protein may function during later stages of sperm/egg interaction. Whether the exposure of CRES to the sperm surface mirrors a physiological consequence of the acrosome reaction or is an effect of the relatively high concentrations of thapsigargin used to induce the acrosome reaction is not known at this point. However, other equatorial epitopes become accessible to the sperm surface after ionophore treatment [26]. Similarly, SP10 cannot be detected on the surface of acrosome-intact spermatozoa but is detected after the induction of acrosome reaction [23].
Studies have clearly demonstrated an important role for proteases, in particular serine proteases, in the fertilization process. For example, nonphysiological protease inhibitors such as soybean trypsin inhibitor and p-nitrophenyl p-guanidinobenzoate have been shown to inhibit sperm adhesion to and penetration of the zona pellucida [27, 28]. Although the potential targets of these inhibitors are not known, it has been proposed, as for PCI, that they may modulate acrosin activity [25, 29]. In contrast, the substrate specificity of the trypsin-inhibitor-sensitive site identified by Benau and Storey [28] indicated activity that was not due to acrosin, but rather to the involvement of other sperm-associated serine proteases in fertilization. Indeed, several recent studies support this hypothesis. In Cynops sperm, a tryptic protease was identified that hydrolyzed synthetic substrates containing dibasic residues and that induced Xenopus egg activation [30]. Also, studies using spermatozoa from acrosin-deficient mice showed inhibition of fertilization in vitro when the trypsin/acrosin inhibitor p-aminobenzamidine was added, suggesting the role of proteases other than acrosin in fertilization [31]. Finally, zinc metalloproteases have also been shown to play important roles in gamete fusion [32, 33].
Although the inhibitory activity of CRES against acrosin has yet to be tested, we propose that CRES and likely other protease inhibitors are present in the sperm acrosome to regulate the activities of proteases other than acrosin. Because the acrosome is an active site of proprotein processing, one possibility is that CRES may regulate proteases involved in processing events. The presence of the prohormone convertase family member PC4 in testicular germ cells [34] and the observation that PC4-null mice exhibit impaired fertility [35], supports an important role for these proprotein processing proteases in fertilization. Indeed, several acrosomal proteins such as fertilin ß undergo important proteolytic processing during sperm transport in the epididymis [36], whereas other acrosomal proteins such as Sp17 [37] and sperm-borne oocyte-activating factor [38] are processed later during the fertilization process. Perhaps the function of CRES in the human sperm equatorial segment is to modulate proteases that activate acrosomal proteins directly or indirectly involved in sperm-egg fusion events. Studies are currently underway to assess the role of CRES during mouse and human fertilization to ultimately address these questions.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by National Institutes of Health grants HD33903 (to G.A.C.), T32-HD07271 (to N.H.), and HD35166 (to D.M.H.); and by grants from the French Foreign Affairs Ministry (to P.S.), the Texas Tech University Health Sciences Center of Excellence in Reproductive Sciences (to G.A.C. and D.M.H.), and the South Plains Foundation and Houston Endowment (to G.A.C.). 
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; gail.cornwall{at}ttmc.ttuhsc.edu
Accepted: April 2, 2002.
Received: January 28, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-trace) and chicken cystatin. FEBS Lett 1986 170:370-374This article has been cited by other articles:
|
|
 |
|
  M. J. Wolkowicz, L. Digilio, K. Klotz, J. Shetty, C. J. Flickinger, and J. C. Herr Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion J Androl, May 1, 2008; 29(3): 272 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, C. A. Putnam-Lawson, H. Knapp-Hoch, P. J. Friel, D. Mitchell, R. Hively, and M. D. Griswold Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis Biol Reprod, November 1, 2005; 73(5): 872 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Tohonen, J. Frygelius, M. Mohammadieh, U. Kvist, L. J. Pelliniemi, K. O'Brien, K. Nordqvist, and A. Wedell Normal Sexual Development and Fertility in testatin Knockout Mice Mol. Cell. Biol., June 15, 2005; 25(12): 4892 - 4902. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A Soler-Garcia, R. Maitra, V. Kumar, T. Ise, S. Nagata, R. Beers, T. K Bera, and I. Pastan The PATE gene is expressed in the accessory tissues of the human male genital tract and encodes a secreted sperm-associated protein Reproduction, April 1, 2005; 129(4): 515 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hsia, J. P. Brousal, S. R. Hann, and G. A. Cornwall Recapitulation of Germ Cell- and Pituitary-Specific Expression With 1.6 kb of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene Promoter in Transgenic Mice J Androl, March 1, 2005; 26(2): 249 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Cornwall, A. Cameron, I. Lindberg, D. M. Hardy, N. Cormier, and N. Hsia The Cystatin-Related Epididymal Spermatogenic Protein Inhibits the Serine Protease Prohormone Convertase 2 Endocrinology, March 1, 2003; 144(3): 901 - 908. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
CRES). B) Carbohydrate analysis of CRES protein in 6 µg human testis lysate and 1 x 107 spermatozoa. Lysates from testis and spermatozoa were incubated in the absence (-) or presence (+) of N-glycosidase F (Endo F). The samples were then examined by Western blot analysis as in A. The arrowheads indicate the 19-kDa and 14-kDa forms of CRES protein
