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Male Reproductive Tract |
Department of Biological Sciences,3 University of Delaware, Newark, Delaware 19711
Department of Anatomy and Cell Biology,4 McGill University, Montreal, Quebec, Canada H3A 2B2
Centre Hospitalier de l'Universite Laval,5 Ste Foy, Quebec, Canada G1V 4G2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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efferent ducts, epididymis, hyaluronidase activity, male reproductive tract, prostate, seminal vesicles, sperm, vas deferens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A second function of SPAM1, which is a single chain glycosyl phosphatidylinositol-linked glycoprotein, resides in the carboxy terminus of the molecule, which confers on it the ability to bind to the zona pellucida after the acrosome reaction [3, 7]. Thus, the protein is important in sperm-zona pellucida secondary binding that is mediated by molecules lining the inner acrosomal membrane [2]. A third role for SPAM1 in fertilization involves the HA receptor domain of the molecule found in the N-terminus, where it is flanked by the two hyaluronidase domains [3]. The HA receptor is involved in the Ca2+ signaling that accompanies acrosomal exocytosis [3].
More recently, a role for SPAM1 in sperm maturation has been postulated based on the finding that the protein is synthesized in the epididymis. Deng et al. [8] showed that both Spam1 mRNA, detectable by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), and protein expression occur in the principal cells of all three regions of the murine epididymis. The murine protein, Spam1, was also shown to have the characteristic hyaluronidase activity at neutral pH [8]. Epididymal expression of Spam1 was confirmed using cultured mouse epididymal epithelial cells, and Spam1 was shown to be released in vitro as well as in vivo [9]. Later, it was shown that epididymal Spam1 is secreted in both a soluble and an insoluble form (epididymosomes), which contains an intact lipid anchor [10]. This suggests that the secreted form can bind to the plasma membrane of maturing sperm [10].
Our lab has also shown that the dual expression pattern of Spam1 in the murine testis and epididymis is evolutionarily conserved. Rats [11], macaques, and humans [12] were recently shown to express SPAM1 in the epididymis, bringing the number of such species to four in two mammalian classes. In humans and macaques, SPAM1 mRNA was detected by laser microdissection/reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ transcript hybridization in all three regions of the epididymal epithelium, and the protein expression was detected by Western and confirmed by immunohistochemistry [12]. While analyzing the epididymal tissues on commercially prepared slides with assorted histological sections of human reproductive tissues, preliminary evidence was obtained for the expression of human SPAM1 in the seminal vesicle [12]. Additionally, SPAM1 protein was found in the vas deferens by Western, but the analysis of its hyaluronidase activity in this organ was inconclusive [12]. Finally, SPAM1 transcripts have been found in the human prostate [13], but nothing is known of the protein expression in this organ.
The objective of the present study was to use the mouse model to gain information on the extent of expression of Spam1 in all the regions of the excurrent ducts and the male accessory organs, the prostate, and the seminal vesicles. Specifically, we wanted to know if Spam1 is expressed in the most proximal and distal regions of the extratesticular pathway, having already identified its presence in the epididymis [8]. It must be pointed out that the report that Spam1 is not essential for fertilization in the mouse [14] does not diminish the biomedical relevance of the protein or the model. While the protein may be redundant in the mouse model, this is not the case in humans, who lack the presence or function of two closely related and closely linked testicular hyaluronidase orthologs (HYAL5 and HYALP1) that are present in the mouse [13, NCBI database].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult (>90 days old) CD-1 or ICR mice were obtained from Charles River Laboratories (Montreal, QC) and Harlan Inc. (Indianapolis, IN). The studies were approved by the Animal Care Committee at the University of Delaware and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised 1985). Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (MTC Pharmaceuticals, Hamilton, ON) and killed using carbon dioxide.
Reagents and Antibodies
Rabbit antipeptide mouse Spam1 generated from a 15-mer oligopeptide (C-terminal #381–395) for Spam1 was prepared by Zymed (San Francisco, CA). The specificity of this antipeptide was previously confirmed [8]. Goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) was purchased from Cedarlane (Hornby, ON). All other reagents are from Sigma (St. Louis, MO) unless otherwise indicated.
Tissue Preparation and In Situ Transcript Hybridization
CD-1 mice (n = 3 per group) were anesthetized with sodium pentobarbital, and the testes were perfused through the left ventricle with 4% paraformaldehyde, 0.1% glutaraldehyde, and 3% dextran sulfate in 0.05 M phosphate buffer (pH 7.4) for 15 min. Following perfusion, the efferent ducts and epididymis, were removed and immersed in the same fixative for 5 h at 4°C. The tissues were then cut into small blocks of approximately 8 mm3, embedded in 2.5% melted agarose (at 60°C), and cut into 60-µm-thick frontal sections with a vibrotome. Groups of 10 sections were collected in autoclaved vials and washed three times in RNase-free 0.05 M phosphate buffer (pH 7.4) at room temperature. Glycine (1 M) was added to the buffer to neutralize aldehyde groups.
An antisense RNA probe was generated from PCR of a 342-base pair (bp) fragment (nt 1704–2046) uniquely found in the 3'UTR of the murine Spam1 cDNA. It was radioactively labeled with tritium-UTP (NEN, Boston, MA) to a specific activity of 1–2 x 107 cpm/µg, using an in vitro transcription system (Roche Molecular Biochemicals, Indianapolis, IN).
Prehybridization and hybridization procedures were performed as described previously [15]. Briefly, the sections were transferred from the phosphate buffer to the prehybridization buffer containing 4x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 1x Denhardt solution for 1 h at room temperature with gentle agitation. The sections were then immersed in hybridization buffer containing 1 ml of 8x SSC, 1 ml of deionized formamide, 100 µl of Sarkosyl (2.3 mg/ml), 200 µl of 1.2 M phosphate, and 1.50 µg per vial of 3H-labeled Spam-1 antisense probe (specific activity 1.47 x 107 cpm/µg) or 1.50 µg per vial of a 3H-labeled control sense probe (specific activity 1.57 x 107 cpm/µg). After hybridization overnight at 40°C, the sections were rinsed sequentially at the same temperature in 4x SSC and 0.1x SSC for 1.5 h. Following the washes, the sections were quickly dehydrated in 50%, 70%, 90%, and 100% ethanol. Sections were then coated with NTB2 emulsion and exposed for 14 days for autoradiographic detection of the hybridization signal.
Preparation of Total RNAs from Vas Deferens, Prostate, and Seminal Vesicle
Total RNAs from the vas deferens, prostate, and seminal vesicle of mice 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 h at 37°C, followed by phenol/chloroform extraction and ethanol precipitation.
RT-PCR
First-strand Spam1 complementary DNA (cDNA) synthesis from 2 µg of total RNA was performed with a SuperScript Preamplification System (Gibco, Rockville, MD) under the conditions recommended by the manufacturer. Control experiments were performed simultaneously without the addition of RT. One microliter of each reverse transcription product was subjected to PCR amplification using two pairs of primers designed from the mouse Spam1 cDNA sequence (GenBank Accession No. U33958) (forward: nt 1650–1669, reverse: nt 2029–2049; and forward: nt 5–40, reverse: nt 188–222). The PCR reactions were performed under the following conditions: 94°C for 2 min; 35 cycles at 94°C for 1 min, 61°C for 2 min, and 72°C for 2 min; 72°C for 10 min; and 4°C hold. Total RNAs extracted from testis was used along with the samples as positive controls. The PCR products were resolved on 1% agarose gel and stained with ethidium bromide, and the experiments were repeated twice.
Sequencing of the RT-PCR Products
The RT-PCR products were cloned into pSTBlue-1 vector according to the manufacturer's instructions (Novagen, Madison, WI). Several clones of each product were isolated and sequenced.
Tissue Preparation and Immunohistochemical Staining for Light-Microscopic Viewing
The reproductive tracts of three CD-1 mice were fixed for 10 min by intracardiac perfusion with Bouin fixative via the left ventricle. Following perfusion, efferent ducts, epididymis, and vas deferens were removed and immersed in Bouin fixative for an additional 24 h. The tissues were then dehydrated in graded ethanol and embedded in paraffin. Sections (5 µm thick) were cut and mounted on glass slides. They were then deparaffinized with Histo-Clear (National Diagnostics), then rehydrated in a series of ethanol solutions (100%, 95%, 70%, 70%, 70%, 70%, 50%). Hydrogen peroxide (1% v/v) was added to the second 70% ethanol bath to destroy endogenous peroxidase that may interfere with the HRP labeling. Lithium carbonate (1% w/v) was added to the third 70% ethanol bath to neutralize residual picric acid from the Bouin fixative. The slides were then placed in a 5-min glycine bath (300 mM) to block free aldehyde groups. To uncover antigenic sites on the tissues, the slides were then microwaved in an antigen-retrieval solution (1.8 mM citric acid, 8.2 mM sodium citrate, pH 6.0) for 10 min, through a series of boiling cycles. After cooling, slides were blocked with 10% goat serum in TBS (20 mM Tris-HCl buffered saline containing 0.1% BSA, pH 7.4) and incubated for 15 min at 37°C. Slides were washed in TBS-Tween (0.1% Tween in TBS) before incubating for 90 min with Spam1 antipeptide primary antibody (1:500). This was followed by a second goat serum incubation for 15 min and further TBS-Tween washings. Goat anti-rabbit secondary antibody (1:200), conjugated to HRP, was applied to the sections and incubated for 30 min. Slides were again washed in TBS-Tween and immersed for 10 min in DAB solution (TBS with 0.05% diaminobenzidine tetrahydrochloride, 0.1 M imidazole, 0.03% hydrogen peroxide, pH 7.6). Rinsing with distilled water and counterstaining with methylene blue followed. Finally, the slides were rehydrated in a series of ethanol solutions and Histo-Clear. Coverslips were mounted using Permount.
In the case of the vas deferens, the sections were treated with an FITC-conjugated goat anti-rabbit secondary antibody and imaging was done using the multiphoton confocal microscopy, as previously described [16].
Preparation of Protein Extracts from Efferent Ducts, Vas Deferens, Prostate, the Seminal Vesicle and Its Fluid, and Caudal Sperm Tissues and Seminal Vesicle Fluid
Efferent ducts, vas deferens, prostate, and seminal vesicles were minced separately in PBS buffer containing 1 mM phenylmethylsulphonyl fluoride. Initially, the efferent ducts and vas deferens were minced in PBS warmed to 37°C and sperm allowed to swim out. The tissues were then washed at least three times with PBS (centrifuged at 500 x g for 2 min) to remove adhering sperm, as assessed by microscopic analysis of the washes. The proteins were extracted by manually homogenizing the tissues (using a mortar and pestle) with a solubilization buffer at 4°C. The suspension was centrifuged at 10 000 x g for 10 min at 4°C, and the supernatant containing the proteins was collected.
Sperm
To collect sperm, cauda epididymides were thoroughly minced and incubated in 5 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) at 37°C to disperse the sperm and allow them to swim out. 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 at 4°C. Protein extracts were prepared by lysing sperm with a solubilization buffer (62.5 mM Tris-HCl, 10% glycerol, 1% sodium dodecyl sulfate [SDS], pH 6.8), at 4°C. The suspension was vigorously vortexed for 4 min and then centrifuged at 10 000 x g for 10 min, and the supernatant containing proteins was collected.
The protein concentrations of all samples were determined with a biocinchoninic acid protein assay kit (Pierce, Rockford, IL), using different concentrations of BSA) as standards. An equal mass (40 µg for Western blot analysis or 60 µg for hyaluronic acid substrate gel electrophoresis [HASGE]) of each protein sample was used for all experiments.
SDS-PAGE and Western Blot Analysis
Each protein sample was exposed to reducing conditions (heated at 99°C for 4 min in the presence of 100 mM dithiothreitol), subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane according to standard protocols. Protein extracts from caudal sperm, skeletal muscle, and bovine serum albumin were analyzed along with the samples as controls. Western blotting was performed with the WesternBreeze Chemiluminescent Immunodetection Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. In brief, the membrane was blocked for 30 min at room temperature, then probed with Spam1 antipeptide antiserum diluted 1:1000 in blocking solution. The membranes were washed and the secondary antibody, alkaline-phosphatase-conjugated anti-mouse IgG, was applied. The protein was visualized using the chemiluminescence substrate provided with the kit.
Hyaluronic Acid Substrate Gel Electrophoresis
Hyaluronidase activity was measured using HASGE performed as described [8, 17]. Briefly, hyaluronic acid from bovine vitreous humor was added to 15% SDS-polyacrylamide gel at a final concentration of 0.15 mg/ ml. Gels were run at 15 mA constant current until the leading dye (bromophenol blue) had migrated near the bottom. After electrophoresis, gels were incubated at room temperature for 2 h in 1x 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. Images showing the digestion of the hyaluronic acid by hyaluronidase activity in each sample were captured by scanning the gels, using a laser densitometer. Protein extracts from caudal sperm were used as a positive control and were analyzed along with the samples to determine their ability to digest hyaluronic acid. The experiments were repeated twice.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Due to the small size of the mouse efferent ducts that are embedded in a large amount of fat, the mRNA was best studied by in situ hybridization. An investigation of the presence of Spam1 transcript in histological sections of the efferent ducts and the epididymal regions, studied as positive controls, revealed synthesis of the mRNA in both organs. Radioautography of sections hybridized with the 3H-labeled Spam-1 antisense RNA probe demonstrated the strongest reaction in the distal segment of the efferent duct. Staining was confined to the epithelial cells of the distal segment of the efferent ducts and the epididymis. The epithelial lining the body and cauda epididymis showed lower levels of reaction (Fig. 1). The control sense probe did not yield radioautographic reaction in any of these ducts (Fig. 1).
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RT-PCR assays, using Spam1-specific primers generated from the 3' UTR, yielded a 342-bp product (the identical size expected from the testis) in the vas deferens, the prostate, and the seminal vesicle (Fig. 2, A and C). Complete identity with Spam1 cDNA originating from the testis was revealed by cloning and sequencing of the product. Because no 342-bp product was obtained in the absence of RT in the samples, amplification of the product is not due to DNA contamination. A second pair of primers was used for RNA from the accessory organs. This primer pair, which is unable to amplify DNA because they flanked exon I, which is 4.1 kb, generated the expected product size (200 bp; Fig. 2B) for the prostate and the seminal vesicle. This product was shown to be Spam1 by sequencing.
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Spam1 Protein Is Localized Similarly to the Transcripts
The presence of Spam1 transcript in the efferent ducts and vas deferens was confirmed by immunohistochemistry. The efferent ducts showed a pronounced apical staining in the nonciliated cells, while there was no staining of the ciliated cells. Again, the strongest reaction was observed in this region of the excurrent ducts. In the lumen of the epididymis, the spermatozoa were strongly stained by the Spam1 antibody (Fig. 3). Control preimmune serum did not produce peroxidase reaction in any of these ducts.
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When the vas deferens was analyzed by confocal microscopy, the epithelium was shown to be strongly immunopositive, while the control treated with the preimmune serum had no reaction. The staining was confined to the epithelium and immunopositive material could also be seen within the lumen, indicating that the protein is secreted in the vas deferens (Fig. 4).
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Western blot analysis confirmed the immunohistochemical data for the efferent ducts and vas deferens showing that the protein is synthesized in both organs (Fig. 5A). The 67-kDa Spam1 protein was absent from the skeletal muscle and bovine serum albumin, two negative controls, but present in sperm used as a positive control. Expression of Spam1 protein, similar to the transcript, was higher in the efferent ducts compared with the epididymis (data not shown). The 67-kDa isoform of Spam1 was also observed in the accessory organs, the prostate, and the seminal vesicle and was secreted in the seminal vesicle fluid (Fig. 5C). In the vas deferens, Spam1 was shown to have hyaluronidase activity at pH 7.0, as revealed by HASGE (Fig. 5B), similar to sperm. However, no activity could be detected for the efferent ducts (Fig. 5B), the prostate, or the seminal vesicle (data not shown) after several repeated experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Consistent with Spam1 mRNA in the efferent ducts is the presence of the protein that was immunolocalized to the nonciliated epithelial cells. This confirms that Spam1 is synthesized in large amounts in this region of the excurrent duct. Immunopositive cells displayed the protein throughout the cytoplasm with a concentration in the apical region reflecting the secretory nature of the protein. The immunohistochemical finding was corroborated by the results from Western analysis, which showed the efferent ducts to share the same isoform with the vas deferens and sperm (Fig. 5A) as well as the epididymis [8] and to have the most intense staining along the excurrent ducts. When protein extracts of the efferent ducts were analyzed for hyaluronidase activity at pH 7.0, no activity was detected, suggesting that Spam1 in the efferent ducts, unlike the other regions of the excurrent ducts, may play a nonenzymatic role. It is possible that, in the efferent duct, the catalytic domain of Spam1 may be inactivated by a molecular interaction because the same isoform of the protein is found in all regions of the excurrent duct.
This novel finding for expression of Spam1 in the efferent ducts suggests a new role for this secretory protein during sperm maturation. Based on the knowledge that the primary physiological function of the efferent ducts is that of concentrating the sperm fluid by reabsorbing most of the luminal fluid as sperm pass from the testes [18, 19], it is likely that Spam1 plays a role in this activity. In support of this role is the finding that Spam1 is enriched on the apical (but not basolateral) membrane of the nonciliated cells, where it could readily function in fluids reabsorption. However, its direct role in this activity in the efferent ducts, which is highly likely not to involve hyaluronidase activity, remains to be demonstrated.
Because fluid reabsorption in the efferent ducts is under steroid hormone control [18, 20] and gene expression in the efferent ducts is known to be estrogen regulated [21], we searched a 2-kb region upstream from the transcriptional start site of Spam1 using the NCBI database for estrogen receptor elements (EREs). Using the DNASIS Mac version 3.5 program, a sequence with 60% homology to the human consensus sequence (5'-AGGTCAnnnTGACCT-3') was identified at –457 to –438. This putative ERE is consistent with the expression of Spam1 in the efferent ducts.
Our RT-PCR results revealed that Spam1 mRNA is present in the vas deferens. This is consistent with the finding of Spam1 protein by Western analysis, and this was confirmed by imunohistochemistry, which showed it to reside in the epithelium and to be secreted within the lumen. Unlike the efferent ducts, but similar to the three regions of the epididymis, Spam1 found in vas deferens was shown to have hyaluronidase activity at neutral pH, which is characteristic of sperm-surface Spam1 [8, 9]. The similarity of expression of Spam1 in the epididymis (which plays a role in sperm maturation) and the vas deferens, in the most distal region of the excurrent duct, suggests that the protein may be playing the same role in these two organs. Thus, the presence of Spam1 in the vas deferens underscores its role in sperm maturation, which has been more recently attributed to this organ [22]. As an endo ß-glycosidase, Spam1 in the epididymis and vas deferens may participate in the modification of sperm surface proteins that occurs during posttesticular maturation of sperm [23].
Both the prostate and the seminal vesicles were shown to express Spam1 mRNA when two pairs of primers were used and the products generated were verified by sequencing. Spam1 protein was also present in these tissues as well as the seminal vesicle fluid, as revealed by Western analysis. The finding of Spam1 expression in the murine prostate correlates with the finding of the mRNA in the human prostate [13] and the protein in the human seminal vesicle [12]. Similar to the efferent ducts, Spam1 in prostate and seminal vesicle showed no hyaluronidase activity at pH 7.0. Hyaluronidase activity of Spam1 has been shown to be regulated by the glycosylation levels of the protein [24, 25]. However, the absence of its activity in the efferent ducts and the accessory organs is unlikely to reflect different glycosylation levels compared with the epididymis and vas deferens because all the organs share the Spam1 isoform with a molecular mass of 67 kDa. Thus, similar to the efferent ducts, inactivation of hyaluronidase activity in the accessory organs is a likely explanation.
It is unclear what role Spam1 may be playing in the prostate and seminal vesicles as their fluids mix with the sperm fluid. However, Spam1 has been shown to be expressed and secreted in all three regions (vagina, uterus, and oviduct) of the female reproductive tract [16]. Its expression is cyclic, with the highest levels seen in proestrus and estrus, and the protein abruptly disappearing in metestrus [16]. Thus, the arrival of sperm/seminal fluid in the female tract coincides with the highest expression of Spam1 in the female. It is therefore possible that Spam1 that is secreted in the accessory organs complements or enhances that in the female tract, contributing to posttesticular maturation and physiological activity of sperm during their journey from the testis to the oviduct. Whether Spam1 in the accessory organs accomplishes this by modification of the spermatozoa or by enriching the chemical milieu of the sperm fluid with protective agents is unknown. In this vein, it could serve as an antimicrobial agent because some epididymis-secreted sperm-binding proteins have been shown to have antimicrobial activity [26, 27].
The findings in the present study allow us to conclude that Spam1 is present in all three regions of the murine extratesticular pathway and the accessory organs, where it may perform different functions. With these findings and those in the murine female reproductive tract [16], as well as those for its expression in human normal breast [28], prostate, fetal, and placental cDNA libraries [29], it may be appropriate to refer to SPAM1 as the reproductive hyaluronidase rather than as the sperm hyaluronidase.
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FOOTNOTES |
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2 Correspondence. FAX: 302 831 2281; pdeleon{at}udel.edu
Received: 1 April 2004.
First decision: 26 April 2004.
Accepted: 20 May 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  K. A. Miller, M. Shao, and P. A. Martin-Deleon Hyalp1 in Murine Sperm Function: Evidence for Unique and Overlapping Functions With Other Reproductive Hyaluronidases J Androl, January 1, 2007; 28(1): 67 - 76. [Abstract] [Full Text] [PDF]
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