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Regular Article |
a Department of Biological Sciences, University of Delaware, Newark, Delaware 19716-2590
ABSTRACT
The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.
environment, gamete biology, male reproductive tract, testis
INTRODUCTION
The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with bifunctional roles in mammalian fertilization. It has a hyaluronidase activity that is necessary for sperm to penetrate the cumulus cells surrounding the egg [1–3]. It has also been shown to have a zona pellucida-binding activity and is involved in the secondary binding of sperm to the egg [2, 4, 5]. Both transcription and translation of Spam1 in the testis are restricted to spermatids [6, 7], and the antigen is uniformly distributed on testicular sperm [6].
However, the protein migrates to specific domains on the sperm head during epididymal transit in guinea pigs [6], human [2], and mice [8]. In the latter, a biochemical maturation of Spam1 occurs during epididymal transit [8]. The relative molecular mass of mouse Spam1 decreases from 
74 kDa to 67 kDa as a result of progressive N-linked deglycosylation when sperm traverse the epididymis [8]. In addition, Western blot analysis shows that the dominant band in caudal sperm has a greater than fourfold higher intensity than that seen in caput sperm, and this is associated with a similar increase in Spam1 hyaluronidase activity [8].
Recently, we reported that in the mouse, Spam1 is also synthesized independently in the epididymis where it is found in membranous vesicles in the principal cells of epithelium in all three regions [9]. Epididymal Spam1 was also shown to have hyaluronidase activity and to be present in germ-cell deficient mice [9], underscoring the fact that its presence in normal mice is not a result of endocytosis. Because of the role of the epididymis in sperm maturation and storage, it is of interest to test whether epididymal Spam1 is released into the lumen, where it could facilitate the molecular interactions involved in sperm maturation and storage. To this end, we studied luminal fluid from the epididymis for the presence of Spam1 and used a culture system to determine if Spam1 is synthesized and released in vitro.
A second objective of this investigation was to determine whether epididymal and testicular Spam1 are under different transcriptional regulation. With primer extension analysis, we previously showed that there are two transcription initiation sites for Spam1 in mouse testis: positions +1 and -56 [10]. Position +1 is the predominant site, whereas -56 is only a minor one, and its usage eliminates a cAMP-responsive element (CRE) sequence, which is a transcription factor binding site at -57, and which is used by a number of haploid-expressed spermatogenic genes [11]. The presence of two transcription start sites, as well as the location and limited usage of the second site in the testis, suggest that testicular and epididymal Spam1 may have different predominant start sites and may be under different transcriptional control. With 5' rapid amplification of cDNA ends (5' RACE)/nested polymerase chain reaction (PCR), we now confirm that there are at least two transcription initiation sites in mouse testis and provide evidence that the start site at position +1 is not used in the diploid expression of Spam1 in the epididymis. This suggests that Spam1 is under different transcriptional regulation in testis and epididymis.
MATERIALS AND METHODS
Animals and Reagents
Sexually mature male ICR outbred mice provided by Harlan Sprague-Dawley, Inc. (Indianapolis, IN) were used in all studies. All chemicals were purchased from Sigma Chemical Company (St. Louis, MO) or Fisher Scientific Company (Malvern, PA) unless otherwise specified.
Cell Cultures
Epididymal epithelial cell cultures were initiated using a dual chamber environment to incubate epithelial cells with fibroblasts according to the method of Carballada and Saling [12], with slight modifications. In brief, 4-wk-old mice were killed by cervical dislocation, and epididymal regions were removed under sterile conditions. To establish primary fibroblast cultures, epididymal tubules from corpus and cauda epididymides were separately isolated in Dulbecco PBS (without Ca2+ or Mg2+). Under the stereomicroscope, the tubules were flushed with a "drawn-out" Pasteur pipette, longitudinally split, and washed to remove sperm. The tissues were then minced and treated with 0.025% collagenase in RPMI-1640 containing 10% fetal bovine serum (FBS) at 37°C for 30 min. After vortexing, both the cells and the sedimented tissue bits were collected by centrifugation and washed free of enzyme with PBS. The pellets were then resuspended in s-RPMI (RPMI-1640 containing 10% FBS, 10 nM insulin, 20 nM hydrocortisone, 5 µg/ml transferrin, and 1 µg/ml retinol), seeded in a 24-well plate, and incubated in 5% CO2 at 37°C. Culture medium was changed every 2 days. When cells were confluent (after around 10 days), the medium was switched to serum-free defined medium (s-RPMI without FBS but with 5 ng/ml basic fibroblast growth factor [bFGF] and 0.1% bovine BSA) for 3 days before coincubation with epithelial cells.
For epithelial cell cultures, epididymal tubules of corpus and cauda from 4-wk-old mice were isolated and washed to remove sperm as described above. The tissues were minced and treated with 0.025% collagenase in RPMI-1640 containing 0.1% BSA at 37°C for 2 h. Tissue bits were then collected by centrifugation and incubated in 0.5% trypsin in 4x pancreatin solution for 1 h on ice, and then warmed by hand to room temperature and incubated for another 1 h at this temperature. The enzyme mixture was removed and the tissues were resuspended in PBS and vortexed to allow tissues to settle out from plaques of epithelial cells. The suspension containing free epithelial cells was then centrifuged to collect the cells, which were washed with PBS. The washed cells were resuspended in SFDM (s-RPMI without FBS but with 0.1% BSA and 1 µg/ml epidermal growth factor [EGF]) supplemented with 200 nM testosterone and 1 µM dihydrotestosterone, and plated at 0.6–1 x 106 cells/ml on inserts (Whatman Scientific Ltd., Maidstone, UK) freshly coated with a thin layer of Matrigel (Collaborative Research, Bedford, MA) diluted 1:5. The inserts with plated epithelial cells were then placed in the wells in which fibroblasts from the corresponding tissue were growing. The cultures were maintained in 5% CO2 at 32°C, and culture medium was changed every 2 days.
Immunocytochemistry and Standard and Confocal Fluorescence Microscopy
Corpus and caudal epithelial cells cultured for 3 days were fixed on inserts with 4% (w/v) paraformadehyde in 1x PBS (pH 7.5) for 30 min and blocked for 30 min in 2% BSA in 1x PBS (pH 7.5). Rabbit polyclonal antibody against cytokeratin (diluted 1:75 in 1x PBS containing 1% BSA; INC Biomedicals, Aurora, OH) for detection of cytokeratin or rabbit antipeptide mouse Spam1 antiserum (diluted 1:400 in 1x PBS containing 1% BSA; Zymed, San Francisco, CA) for detection of Spam1 was added to the fixed cultures on the inserts. The antipeptide antiserum was generated from a 15-mer oligopeptide (C-terminal #381–395) specific for Spam1 [9]. After incubation in a humid atmosphere for 2 h at room temperature, inserts were washed three times for 3 min in 1x PBS, then incubated with a secondary antibody: (fluorescein isothiocyanate [FITC]-conjugated goat anti-rabbit immunoglobulin [Ig] G diluted 1:320 in 1x PBS containing 1% BSA) for 30 min at 4°C. Control was incubated with preimmune rabbit serum as the primary antibody followed by detection with the FITC-conjugated second antibody. After the inserts were washed three times for 3 min in 1x PBS, the membranes were removed and mounted on slides in 
-phenylenediamine antifade with 1.5 µg/ml of 4' 6-diamidino-2-phenylindole (DAPI) for standard fluorescence microscopy. The specimens were examined and imaged with a Zeiss Axiophot (Carl Zeiss, Oberkochen, Germany) using the appropriate FITC filter set or with a Zeiss LSM 510 confocal microscopy using the 488-nm and 647-nm lines of the Ar/Kr laser to image FITC.
Preparation of Protein Extracts from Sperm, Epididymal Luminal Fluids, Cultured Epithelial Cells, and Conditioned Media
Protein extracts from caudal sperm were prepared according to the method of Cherr et al. [13] and Deng et al. [9]. In brief, minced caudal epididymides were incubated in 2 ml of sperm suspension buffer (50 mM Tris, 20 mM EDTA, 1 mM p-hydroxy-mercurobenzenzoate, 5 mM N-ethylmaleimide, 1 mM benzamidine pH 7.2) for 10 min at room temperature with gentle agitation to disperse the sperm. The sperm suspension was centrifuged at 500 x g for 2 min to pellet the tissues, and sperm were collected by centrifuging at 1000 x g for 10 min. Protein extracts were prepared by lysing sperm with a solubilization buffer (62.5 mM Tris-HCl, 10% glycerol, 1% SDS, pH 6.8) at 4°C in a final sperm concentration of 1 x 105 sperm/µl. The suspension was vigorously vortexed for 3 min and then centrifuged at 10 000 x g for 10 min, and the supernatant containing proteins was collected.
Epididymal luminal fluids were collected from six 8-wk-old mice. The epididymides were isolated and divided into caput, corpus, and cauda. Each segment was minced into 1.5 ml Whittingham buffer and gently shaken for 10 min to permit dispersal of the luminal content. After the tissue pieces were allowed to settle, the upper fraction containing sperm and luminal fluid was collected and centrifuged at 3500 x g for 20 min at 4°C. The supernatant was then collected and the protein was precipitated with 10% trichloroacetic acid (TCA) and recovered in sample buffer. Media collected every 2 days from cultured epithelial cells up to Day 8 were subjected to centrifugation at 10 000 x g for 5 min to remove any floating cells and particulate material. The proteins were precipitated from the supernatant with 10% TCA and recovered in sample buffer. Cultured epithelial cells on inserts were collected by brief trypsinization and the protein extracts were prepared by lysing cells with the solubilization buffer.
SDS-PAGE and Western Blot Analysis
One-half of each protein sample recovered was exposed to reducing conditions (heated at 99°C for 4 min in the presence of 100 mM dithiothreitol [DTT]), subjected to 15% SDS-PAGE, and transferred to nitrocellulose membrane according to standard protocols. The membranes were blocked with 5% nonfat milk in washing buffer (150 mM NaCl, 50 mM Tris·Cl pH 7.5, 0.2% gelatin [w/v], 0.02% [v/v] Tween-20) overnight at 4°C (with shaking), then incubated with rabbit antipeptide Spam1 antiserum (Zymed) diluted 1:1000 in blocking buffer. Membranes were incubated sequentially with anti-rabbit IgG biotin-conjugated (1:150 000 in blocking buffer) and streptavidin-horseradish peroxidase (HRP; 1:1250 in blocking buffer; Zymed). Positive staining was detected using the HRP color development reagent, 4-chloro-1-naphthol (4CN; Bio-Rad, Hercules, CA) according to the manufacturer's protocol. Protein extracts from 1 x 105 caudal sperm and culture medium with 1% BSA were analyzed along with the samples as the positive and negative control, respectively.
Hyaluronic Acid Substrate Gel Electrophoresis
Hyaluronidase activities in luminal fluids of caput, corpus, and cauda, media collected from cultured epithelial cells on Days 2–6, and from the cells on Day 8 were measured using hyaluronic acid substrate gel electrophoresis (HASGE) performed as described by Guntenhoner et al. [14] and Deng et al. [9]. Briefly, hyaluronic acid from bovine vitreous humor was added to 15% SDS-polyacrylamide gel at a final concentration of 0.15 mg/ml. After electrophoresis, gels were incubated at room temperature for 2 h in PBS containing 3% Triton X-100 on a rocking platform to remove SDS. They were then incubated in 100 mM sodium acetate (pH 7.0) at 37°C for 24–48 h. To visualize the digestion of the hyaluronic acid, gels were stained with 0.5% Alcian blue in 3% acetic acid for at least 2 h, destained in 7% acetic acid, and then counterstained with Coomassie brilliant blue G-250. Undigested hyaluronic acid is stained with Alcian blue and shows a dark blue background against an unstained area with digested hyaluronic acid. The gels were scanned with a laser densitometer. Protein extracts from 1 x 105 caudal sperm and muscle were analyzed along with the samples as positive and negative controls, respectively.
Preparation of Total RNAs from the Epididymis
Total RNAs from testes, and caput, corpus, and caudal epididymides were extracted using Tri-Reagent according to the manufacturer's protocol. RNA samples were further treated with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN; final concentration of 5–10 U/ml) for 2–4 h at 37°C, followed by phenol/chloroform extraction and ethanol precipitation.
Rapid Amplification of the 5' cDNA End
Amplification of the 5' Spam1 cDNA end was performed using the GeneRacer Kit (Invitrogen, Carlsbad, CA). Briefly, 3–5 µg total RNA was treated with calf intestinal phosphatase (CIP) to remove the 5' phosphates. Dephosphorylated RNA was further treated with tobacco acid pyrophosphatase (TAP) to remove the 5' cap structure. The GeneRacer RNA Oligo (0.25 µg) was then ligated to the decapped RNA using T4 RNA ligase. Complementary DNA was generated using avian myeloblastosis virus reverse transcriptase (RT) and random primers (N6). One microliter of diluted (twofold) cDNA was subjected to PCR to amplify the 5' end using a reverse gene-specific primer designed from the mouse Spam1 cDNA sequence (GenBank accession number U33958; nt 188–222) and the GeneRacer 5' primer provided with the kit. Nested PCR using the same gene-specific primer and GeneRacer 5' nested primer was performed. After gel-purification, the PCR product was cloned into pCR 4-TOPO TA vector according to the manufacturer's instructions (Invitrogen). Several clones were isolated, plasmid DNA was prepared, and inserts were sequenced.
To confirm the results of the 5' RACE, PCR was performed using the RACE-ready cDNA template. The forward primer was designed from mouse Spam1 promoter sequence (GenBank accession number AF154126; nt -56 to -38), according to sequence information obtained from primer extension analysis [10]. The reverse primer (nt 188–222 of cDNA sequence) is identical to that used in the 5' RACE. The PCR products were then cloned using the TOPO TA Cloning Kit (Invitrogen) and sequenced.
RESULTS
Detection of Cytokeratin in Cultured Epididymal Epithelial Cells
To confirm that our cultures were from epithelial cells, an indirect immunocytochemistry assay was performed using cytokeratin as a marker. Epididymal epithelial cell preparations from the cauda (Fig. 1A) and corpus (Fig. 1C) stained positively and specifically for this marker.
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Synthesized and Released Spam1 from Epididymal Epithelial Cells In Vivo and In Vitro
In cultures that were fixed 3 days after plating, indirect immunofluorescence assays showed intense fluorescence in epididymal epithelial cells of both the corpus (Fig. 2B) and cauda (Fig. 2E) when anti-Spam1 antibody was used. On the other hand, control cultures that were incubated only with preimmune rabbit serum followed by detection with the FITC-conjugated secondary antibody have no fluorescence staining (Fig. 2A, corpus; Fig. 2D, cauda), which indicates that the signals detected in the test slides are specific. Confocal images from vertical Z-section slices showed Spam1 to be present on the apical surface of the cells (Fig. 2C, corpus; Fig. 2F, cauda), in a position to be released from the lumen.
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Using antipeptide Spam1 antibody, a band of about 67 x 103 Mr was detected on immunoblots of proteins extracted from cultured epididymal cells collected on Day 8, conditioned medium collected on Days 2–6 (Fig. 3A), as well as epididymal luminal fluids from fresh tissue (Fig. 3B). The size of Spam1 (67 x 103 Mr) in all epididymal samples was similar to the size of this protein in caudal sperm (Fig. 3, A and B). The expression of Spam1 in luminal fluid varied with the region, with corpus and caput showing the highest and lowest expression, respectively (Fig. 3B). It is highly unlikely that the band in the luminal fluid is due to contamination from sperm cells ruptured during its collection. As we have shown [9], a band due to sperm contamination would require 105 sperm broken and present in the fluid. This finding documents synthesis and release of Spam1 in the epididymis both in vivo and in vitro. The intensities of the bands indicate that the release of Spam1 by cultured epididymal epithelial cells varied with time (Fig. 3A). The signal was maximal in medium collected on the second day of culture, gradually decreased after 4 days (Fig. 3A), and could not be detected on Day 8; although it was found in cells at that time.
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Epididymal Spam1 Demonstrates Hyaluronidase Activity
In order to further confirm the synthesis and release of Spam1 in vitro in the epididymis and verify its enzymatic activity, HASGE assays were performed on protein extracts of epididymal luminal fluids, cultured epididymal cells, and conditioned media. At neutral pH, all samples demonstrate hyaluronidase activity at 67 x 103 Mr, which comigrates with the hyaluronidase activity from control caudal sperm (Fig. 4), and is characteristic of Spam1 [8, 15]. The band intensities in culture media collected from Days 2–6 suggest that Spam1 hyaluronidase is maximal on Day 2 of culture and gradually decreases after 4 days of culture (Fig. 4A). This correlates with the amount of protein measured by Western blot (Fig. 3A). Spam1 hyaluronidase activity in caput luminal fluid is lower compared with that in the corpus and caudal regions (Fig. 4B).
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Transcriptional Start Site of Epididymal Spam1
The transcription start site of Spam1 in testis, and caput, corpus, and caudal epididymides was determined by 5' RACE. Two transcription start sites in the testis were found to be located at positions +1 and -56, respectively (Table 1). However, in the epididymis, transcription start sites were identified at -47 (caput) and -20 (corpus and cauda; Table 1). The sequences of RT-PCR products of 5' RACE ready cDNA revealed the presence of the transcripts that included -56 in all three epididymal regions (Table 1). This suggests that the start site at position -56 may be also used in the epididymis, but these clones may not have been identified in 5' RACE due to their low abundance. It should be noted that the RT-PCR product with the -56 position in the transcript does not result from a genomic DNA contaminant, because primers 5' of this region in the promoter failed to amplify a product for the RACE-ready templates but did so for the BAC control DNA (data not shown).
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DISCUSSION
Spam1 Is Synthesized and Released in Mouse Epididymal Epithelial Cells
During their transit through the epididymis, spermatozoa undergo biochemical and morphological changes to acquire motility and the ability to fertilize an oocyte in vivo [16–18]. Much of the biochemical changes that occur during this maturation process involve plasma membrane molecules. These changes are believed to be of critical importance because the sperm surface is involved in intercellular and intracellular aspects of fertilization [19]. Previous studies in our laboratory have demonstrated that Spam1, a surface protein known to be synthesized in spermatids [7], shows quantitative differences in caput and caudal mouse sperm [8]. Recently, we reported that Spam1 and its transcript are present in all three regions of the epididymis of both wild-type and germ cell-deficient mice [9]. These data suggest that epididymal Spam1 may be a secretory protein that is involved in posttesticular maturation and storage of sperm.
In the present study, we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to further investigate the synthesis and secretion of Spam1 in the epididymis, as well as the enzymatic activity of this protein in vivo and in vitro. The epithelial cells used in culture were isolated from the corpus and caudal epididymis, regions showing the highest expression in vivo [9] and ones that play an important and separate role in sperm posttesticular maturation [20–22]. The cells cultured in the presence of testosterone, dihydrotestosterone, and factors released from their corresponding fibroblasts displayed positive staining for cytokeratin, a recognized marker of epithelial cells [12, 23]. Immunocytochemical studies and confocal microscopy localized Spam1 to the cultured epididymal epithelial cells from both regions, demonstrating the endogenous synthesis of Spam1 in the epididymis. The finding that the immunoreactivity is specifically located on the surface of the epithelial cells, as revealed from Z-sections of vertical slices from confocal images (Fig. 4, C and F), suggests that the protein is released primarily from the apical surface of these polarized cells. Although secreted Spam1, which is likely to contain its GPI-anchor bound to the membrane as are other secretory epididymal proteins such as CD52 [24], is found in the luminal fluid, it is also possible that it may be transferred directly from the epididymal cells to sperm. Transfer of a GPI-anchored protein by cell-to-cell interaction has been shown to occur between erythrocytes and endothelial cells [25].
The Spam1 protein was also detected by Western blot analyses in extracts from the cultured epithelial cells at 8 days after plating, as well as in serum-free conditioned medium recovered from Day 2 to Day 6 after plating. The levels of protein recovered in the medium showed a decline with time, similar to that reported by Carballada and Saling [12] for protein CP27. When luminal fluid from fresh caput, corpus, and caudal epididymides was analyzed by Western blot the protein was found in all regions. These observations confirm that Spam1 is independently synthesized in epididymal epithelial cells and is released into the lumen where it is likely to be involved in sperm-epididymis interactions during sperm transit. Consequently, the data support our previous hypothesis that quantitative differences of Spam1 in caput and caudal sperm may be a result of acquisition of the protein by sperm during epididymal transit [8, 9]. Epididymal expression of Spam1 was shown to be region-specific with the corpus and caput consistently showing the highest and lowest amount, respectively [9]. Although the analysis in the present study was qualitative, the finding in Figure 3 is consistent with a region-dependent expression, because similar quantities were loaded on the gel. Our findings also suggest that epididymal Spam1 may be involved in both sperm maturation and storage, which occurs in the cauda. There is indirect evidence that epididymal Spam1 may accumulate in sperm storage because Rb(6.16)-bearing sperm, which carry a Spam1 mutant (unpublished observations), have increased fertilizing ability when they reside in the male tract for prolonged periods [26–28].
Consistent with the results from Western blot, the HASGE assay demonstrates that at neutral pH, epididymal Spam1 has hyaluronidase activity, which is characteristic of caudal sperm [8, 15]. Importantly, there was a correlation between the amount of the protein expressed and the level of hyaluronidase activity of the different regions. For example, the corpus showed both the highest amount of protein and the highest level of hyaluronidase activity. These findings confirm our previous report that epididymal Spam1 is enzymatically active at physiological pH [9]. The finding that neutral hyaluronidase activity is shared by both epididymal and sperm hyaluronidase, along with the identical molecular mass (67 x 103 Mr), argues in support of epididymal hyaluronidase being Spam1. The presence of the enzyme activity at physiological pH in epididymal cells is consistent with the protein being a membrane-bound insoluble hyaluronidase that is found on sperm rather than being from a lysosomal source. Taken together, the data confirm the synthesis of physiologically active Spam1 in the epididymis and demonstrate that it is released in the epididymal duct.
Transcriptional Regulation of Epididymal Spam1
Gene transcription can be regulated by various mechanisms, including RNA initiation, alternate splicing, RNA elongation attenuation, and RNA stability, as discussed by Weisinger et al. [29, 30]. Five-prime RACE using mouse testicular and epididymal mRNA was conducted to determine the transcription initiation sites. Sequencing of 5' RACE products showed that there are two transcription initiation sites for Spam1 in mouse testis; positions +1 and -56. This confirms previous results from our laboratory using primer extension analysis [10]. The findings indicate that the start site at +1 is not used in the epididymis. However, RT-PCR results indicate that the site at -56 may be used in the diploid expression of epididymal Spam1, because transcripts include this nucleotide. This site may, however, be infrequently used with transcripts from the three regions occurring in low abundance because they were not readily detectable in 5' RACE. These longer transcripts might have been detected had more clones been sequenced.
Epididymal transcripts that were readily detectable with 5' RACE had start sites at -47 (caput) and -20 (corpus and cauda), as shown in Table 1 and Figure 5. The data therefore suggest that there are multiple transcription start sites for Spam1, with region-specific sites in the epididymis. Whether or not these start sites in the epididymis are related to the region-specific differences in the expression of Spam1 [9] is unclear.
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The usage of the predominant start site at position +1 in the testis accommodates at -57 a transcription binding site, CRE (Table 1), for the transcription factor called CREM, which is used by a number of spermatid-expressed genes [11]. Although in the epididymis, start sites at -47 and -20 do not eliminate CRE, CRE's position relative to the start site becomes uncharacteristic [11] as shown in Table 1; thus, epididymal Spam1 is likely not to be regulated by CREM and would be under a different transcriptional regulation than testicular Spam1. It must be noted that although there are different start sites for testis and epididymal transcripts (Table 1), the size of the protein remains approximately the same in all tissues (Fig. 3) because the translation start site is at position 313 in exon II (Fig. 5).
Because epididymal secretory proteins are under the control of androgens [12, 31], we searched the available 770-bp sequence of the Spam1 promoter (GenBank accession number AF154126) that was published earlier from our laboratory [10] to determine the presence of one or more androgen responsive elements (AREs). Our results showed two sequences at -336 to -312 and -507 to -486 that have a 48% and 54% homology, respectively, to the consensus sequence (5'-GGA/TACANNNTGTTCT-3') reported as a binding site for the androgen receptor [31, 32]. The hexamers flanking the NNN in these sequences had homologies that ranged from 66% to 83%. It is possible that cloning of a longer promoter region may reveal AREs with a greater degree of homology to the consensus sequence. However, we noted the presence of two GATA-like sequences (at -113 to -108 and -190 to -185) that are seen in the regulation of gene expression in the epididymis [33]. In conclusion, the present study not only confirms our previous finding that Spam1 is synthesized in the epididymis, but extends them by showing that Spam1 is released in the luminal fluid, where it may effect posttesticular maturation, storage, and function of sperm. Transcript analysis indicates that epididymal and testicular Spam1 are under different transcriptional regulation.
ACKNOWLEDGMENTS
We are grateful to Dr. Patricia Saling at Duke University Medical Center (Durham, NC) for sharing her detailed protocol for the mouse epithelial cell culture system with us. We also thank JoAnne Julian in the Department of Biological Sciences for advice and assistance with the cultures.
FOOTNOTES
1 Supported by National Science Foundation grant 997480 to P.A.M.-D. 
2 Correspondence: FAX: 302 831 2281; pdeleon{at}udel.edu
Accepted: July 5, 2001.
Received: May 23, 2001.
REFERENCES
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  H. Chen, G. Griffiths, D. S. Galileo, and P. A. Martin-DeLeon Epididymal SPAM1 Is a Marker for Sperm Maturation in the Mouse Biol Reprod, May 1, 2006; 74(5): 923 - 930. [Abstract] [Full Text] [PDF]
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 E. Kim, D. Baba, M. Kimura, M. Yamashita, S.-i. Kashiwabara, and T. Baba Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass PNAS, December 13, 2005; 102(50): 18028 - 18033. [Abstract] [Full Text] [PDF]
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H. Zhang and P. A. Martin-DeLeon Mouse Spam1 (PH-20) Is a Multifunctional Protein: Evidence for Its Expression in the Female Reproductive Tract Biol Reprod, August 1, 2003; 69(2): 446 - 454. [Abstract] [Full Text] [PDF]
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 H. Zhang and P. A. Martin-Deleon Mouse Epididymal Spam1 (PH-20) Is Released in the Luminal Fluid With its Lipid Anchor J Androl, January 1, 2003; 24(1): 51 - 58. [Abstract] [Full Text] [PDF]
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