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Immunology |
Department of Biological Sciences,2 University of Tokyo, Tokyo 133-0033, Japan
Department of Histology and Cell Biology,3 Nagasaki University School of Medicine, Nagasaki 852-8523, Japan
Department of Experimental Nursing,4 Faculty of Nursing, Fukuoka Prefectural University, Tagawa 825-8585, Japan
Torry Pines Institute for Molecular Studies,5 San Diego, California 92121
Department of Anatomy, Kyorin University School of Medicine,6 Mitaka, Tokyo 181-8611, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain (C4BP) are expressed at significant levels in the guinea pig and mouse epididymis in an androgen-dependent manner. Here, we analyze the murine C4bpa gene and show that epididymal and liver C4BP mRNAs are generated from a single-copy gene and that the epididymal C4BP mRNAs are transcribed from novel transcription start sites located approximately 100 base pairs downstream from those used in the liver. Furthermore, in an immunohistochemical study using rabbit anti-mouse C4BP antiserum, we demonstrated that C4BP is localized in the stereocilia and Golgi apparatus of the epididymal epithelial cells and the surfaces of spermatozoa in the lumen in the region from the distal caput to the cauda but not in the proximal caput region. Indirect immunofluorescence of the isolated spermatozoa demonstrated that C4BP is localized preferentially on the head region of the spermatozoa, and immunoelectron microscopy located C4BP on the plasma membrane and the outer acrosomal membrane. These results indicate that epididymal C4BP is synthesized in the epithelial cells and secreted into the lumen in a region-restricted manner and is taken up to the sperm membranes on passage through the epididymis. Many epididymal proteins are secreted from the epithelial cells in a region-specific and androgen-dependent manner and are considered to contribute to sperm maturation. Our findings suggest a novel function of C4BP as one such epididymal secretory protein.
immunology, epididymis, gene regulation, male reproductive tract, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the reproductive organs, tissue distribution of the membrane-type regulators, such as DAF and MCP, as well as CD59, which regulates the formation of the membrane-attack complex, are well analyzed. They are detected on spermatozoa and on the epithelium of the female reproductive organs [2–6]. These regulators have been thought to be necessary for protection of those cells, because functionally active complement is present in the female reproductive tract fluids, such as ovarian follicular fluid and cervical mucus [7]. However, the recent literature suggests that some RCA proteins are involved in the reproductive system rather than in the complement system. For example, human MCP expressed on spermatozoa is suggested to be involved in egg-sperm recognition, because it is located on the inner acrosomal membrane [8]. The findings that guinea pig, mouse, and rat MCP is preferentially expressed on spermatozoa also suggest a restricted function of MCP in reproduction [9, 10]. Spermatozoa are also the major expression site for certain isoforms of DAF in adult guinea pigs [6, 11] and mice [12]. In addition, a C4BP-related protein has been identified as a sperm protein in mice [13] and guinea pigs [14]. This protein, called sp56 in mice and AM67 in guinea pigs, is structurally related to the chain of C4BP (C4BP), and its gene (designated Zp3r) is closely linked to the C4BP gene (C4bpa) in the RCA region [15]. They are located within the acrosomal region and are thought to interact with oocytes after the acrosome reaction occurs. Furthermore, we demonstrated recently that C4BP mRNA is expressed in guinea pig epididymis in an androgen-dependent manner [16].
The C4BP is a large, heterogeneous plasma protein (Mr 540 000–590 000) and has been isolated and/or cloned from several animals [16–24]. The major isoform of human C4BP is composed of seven chains of Mr 70 000 and one ß chain of Mr 30 000, which bind covalently to each other via the C-terminal regions. The and ß chains consist of eight and three SCR domains, respectively, with a C-terminal region containing two cysteines. In mice, the chain is composed of six SCRs with a C-terminal region, and several chains bind each other noncovalently, because the C-terminal region lacks cysteines and the ß chain gene has become a pseudogene. The C4BP binds to the activated complement component C4b and also to C3b, although very weakly, through the chain, and it works mainly in the classical pathway [25, 26]. The chain is also known to contain the binding sites for heparin [27, 28], serum amyloid protein (SAP) [29], and the surface proteins of such bacteria as Streptococcus pyogenes [30] and Neisseria gonorrhoeae [31], although heparin and SAP possess many other ligands, such as fibronectin and zymozan. In addition, one recent report shows that C4BP binds to low-density lipoprotein receptor-related protein [32]. The binding sites for these molecules in C4BP are located close to the binding site of C4b and C3b, but they are not identical [30, 33]. Furthermore, it has been demonstrated that C4BP directly binds to the cell surface of some ovarian adenocarcinoma cell lines in a functionally active form [34]. On the other hand, the ß chain, which contains no binding sites for complement proteins, has been reported to bind to anticoagulant protein S, suggesting another role for the ß chain in the regulation of anticoagulation [35].
In the previous report [16], we also demonstrated that murine epididymis express, in an androgen-dependent manner, the transcripts that hybridize with C4BP cDNA. In the present report, we confirmed that these species are transcribed from the C4bpa gene and detected the protein in the epididymis by Western blot analysis and immunohistochemistry. Our results suggest a novel function of C4BP as an epididymal secretory protein involved in sperm maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA was isolated from various tissues of 9-wk-old BALB/c mice using the guanidine thiocyanate/CsCl method. Northern blot analysis was performed using the glyoxal-denaturing method as previously described [11, 36]. The mouse C4BP cDNA fragment of 0.9 kilobase (kb), covering from the signal peptide to the SCR5 region, was used as a probe. For C3 and C4, 0.4- and 1.4-kb fragments, respectively, containing a thiolestel region were used. Probes were labeled with [-32P]dCTP using the Rediprime II DNA Labeling System (Amersham Biosciences Corp., Piscataway, NJ).
Molecular Cloning of the Chain of Murine Epididymal C4BP
The cDNA clones encoding the chain of the murine epididymal C4BP were obtained by reverse transcription-polymerase chain reaction (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE) methods using the isolated epididymal total RNA. The nucleotide sequences were determined using an ABI377 sequencer (Applied Biosystems, Foster City, CA).
Analysis of Genome Structure
Isolation of the mouse C4bpa gene was performed by PCR amplification and screening of a murine genomic library (129SrJ strain) constructed in Lambda FIXII (Stratagene, La Jolla, CA), which was provided by Dr. R.A. Wetsel (University of Texas-Houston, Houston, TX). Four positive clones among the 5 x 105 screened clones were plaque-purified, and the phage DNA was isolated using standard methods. The inserts were digested with the appropriate restriction enzymes, subcloned into pBluescript SKII vectors (Stratagene), and sequenced.
For genomic Southern blot analysis, 5 µg of genomic DNA were digested to completion with the EcoRI, BamHI, and HindIII restriction enzymes and electrophoresed through a 1% agarose gel. After transfer to a Hybond-N membrane (Amersham Biosciences), they were hybridized with the probes encoding the SCR3 and SCR4 regions, which were labeled with [-32P]dCTP as described above.
RNase Protection Assay
Five or ten micrograms of total RNA were hybridized with [-32P]CTP-labeled RNA probes using the ribonuclease protection assay kit RPAII (Ambion, Inc., Austin, TX) according to the manufacturer's directions. Labeled probes were obtained using the RiboProbe in vitro transcription system (Promega Corp., Madison, WI). Two fragments including the promoter and 5'-untranslated (UT) regions were cloned into pGEM3Zf(+), linearized, and used to synthesize the probes.
Western Blot Analysis of the Epididymal Fluid
Epididymal fluid was obtained from the epididymal tracts of the 9-wk-old mice by suspending the minced tissues in 30 µl of PBS. Two microliters of the supernatant were separated by 8% SDS-PAGE under nonreducing conditions, then transferred onto a Hybond-P membrane (Amersham Biosciences), and treated with 1:2000 diluted rabbit anti-mouse C4BP antiserum. The antiserum, which was raised against a recombinant mouse C4BP, fused with the transmembrane segment of CR2 [26]. Bands were visualized using enhanced chemiluminescence detection reagents (Amersham Biosciences). Mouse serum was used as a reference.
Immunohistochemistry
Tissue samples of 9-wk-old mice were fixed in 4% formaldehyde in PBS overnight and embedded in paraffin using standard procedures. Sections (thickness, 5 µm) were placed on silane-coated glass slides and dewaxed with toluene, then rehydrated with serial ethanol solutions. The sections were preincubated with normal goat immunoglobulin (Ig) G (500 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) dissolved in 1% BSA/PBS for 1 h and then incubated overnight with the rabbit anti-mouse C4BP (1:400) antiserum as described above. After being washed with 0.075% Brij35 in PBS, sections were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG for 1 h, and sites of HRP binding were visualized with H2O2 and 3,3'-diaminobenzidine-4HCl. The sections were counterstained with methyl green. As a negative control, some sections were incubated with normal rabbit serum instead of with the specific antiserum.
The spermatozoa were collected from the epididymis and washed twice with PBS. The pellets were fixed as described above and treated with normal goat serum to block nonspecific protein binding, followed by incubation with anti-mouse C4BP antiserum (1:200 dilution in PBS) overnight at 4°C. For the immunofluorescence method, the specimens were subsequently incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L; Molecular Probes, Inc., Eugene, OR) for 60 min at room temperature. The specimens were observed with a confocal laser-scanning microscope (MRC-1024K; Japan Bio-Rad Laboratories, Tokyo, Japan).
For the electron-microscopic immunohistochemistry, the sections of the epididymal tissue specimens were incubated with the HRP-labeled anti-rabbit IgG for 24 h after the incubation with the anti-mouse C4BP antiserum, then visualized by 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2 in Tris-HCl buffer (pH 7.6). The sections were postfixed with 1% OsO4 and then embedded in Epon 812 (Oken Shoji Co., Tokyo, Japan). The thin sections (thickness, 80 nm) were prepared, stained with lead citrate, and observed with an electron microscope (JEM-1010; JEOL, Tokyo, Japan).
Analysis of Human Male Reproductive Organs
Human tissue samples, except for prostate, were derived from two or three men for each sample who were treated for prostate cancer by castration, and the prostate specimens were obtained from two patients with bladder tumor who were treated with total cystoprostatectomy. The mean age of the patients was 46.6 yr (range, 26–66 yr), and none had received previous hormonal treatment. All specimens were removed during operation from the patients with informed consent and showed no tumor invasion. For Northern blot analysis, samples were quickly frozen in liquid nitrogen and stored at -130°C. The RNAs were extracted as described above, and Northern blot analysis was performed using the 0.7-kb human C4BP cDNA fragment covering SCR4 through SCR7 as a probe. For immunohistochemical analysis, tissue samples were fixed in 4% formaldehyde in PBS (pH 7.4) for 24 h. After being permeated by sequential incubation in PBS containing 10%, 20%, and 30% sucrose, samples were embedded into OCT compound (Sakura Fine Technical Co. Ltd., Tokyo, Japan) and quickly frozen in liquid nitrogen. Samples were then sectioned (thickness, 10 µm), mounted on slide glass, and air-dried. Sections were immersed in PBS, then treated with normal donkey serum (Jackson ImmunoResearch Laboratories) to block nonspecific protein binding, followed by incubation overnight at 4°C with 1:100 diluted sheep anti-human C4BP antiserum (Biogenesis Ltd., Poole, U.K.). Specimens were subsequently incubated with mixtures of Alexa Fluor 488 donkey anti-sheep IgG (H+L), Alexa Fluor 568 phalloidin for labeling of F-actin, and TO-PRO-3 iodide (Molecular Probes) for labeling of nuclei for 60 min at room temperature. Stained specimens were studied using a confocal laser-scanning microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study the tissue distribution of murine C4BP as well as the complement components C4 and C3 that interact with C4BP, Northern blot analysis was performed using mRNAs obtained from various mouse tissues in the normal condition (Fig. 1). Significant expression of C4BP mRNA was observed in epididymis as well as in liver but not in female reproductive organs. The mRNAs of both C4 and C3 were detected in the epididymis, but the expression levels were very low and were not affected by castration (data not shown).
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Because both 1.9- and 3.0-kb species of the epididymal C4BP mRNAs were a little smaller than those of liver, the primary structure of epididymal C4BP was determined by isolating the epididymal C4BP cDNA clones by RT-PCR, 5'-RACE, and 3'-RACE amplification methods. Sequencing of the isolated clones indicated that the nucleotide sequences of the epididymal C4BP cDNAs were completely identical to that of liver C4BP cDNA regarding signal peptide, six SCR, C-terminal, and the 3'-UT regions, but the 5'-UT regions were shorter (EMBL/GenBank/DDBJ accession no. AB094486).
Analysis of the Mouse C4bpa Gene
To study whether the liver and epididymal C4BP mRNAs are derived from a single-copy gene, the overlapping genomic clones encoding mouse cbpa gene were isolated (Fig. 2A). Four clones (
1, 2, 3, and 5) were isolated from the mouse genomic library, and three fragments (P1, P2, and P3) were obtained by PCR amplification. The restriction enzyme maps and nucleotide sequences indicated that these clones and fragments are derived from a single-copy gene. In addition, we performed genomic Southern blot analysis using two 0.2-kb fragments corresponding to SCR3 and SCR4, respectively, as probes (Fig. 2B). Both probes detected only one band in all three lanes, which contained genomic DNA digested with EcoRI, BamHI, and HindIII, respectively. These results indicate that the murine C4bpa gene is a single-copy gene.
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Analysis of the Promoter Region
To determine the transcription start site of the liver and epididymal transcripts, an RNase protection assay was performed using the liver and epididymal RNA with probes synthesized from the 5'-UT and the 5'-flanking regions (Fig. 3A, probes 1 and 2). Although the probes detected multiple bands in both tissues, all bands detected in the epididymis were different from and smaller than those detected in the liver, indicating that the transcription of the C4bpa gene is differentially regulated in the liver and epididymis. The short length of the 5'-UT region of the epididymal transcripts is compatible with the result of the cDNA cloning and Northern blot analysis.
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The major transcription start sites used in the epididymis (termed Et1 and Et2) are located 93- and 128-base pairs, respectively, downstream from the major transcription site of the liver (termed Lt) (Fig. 3B). Although the consensus sequences for the androgen-response elements (ARE; 5'-AGAACANNNTGTTCT-3') were not found within 1.5 kb upstream of Et1 and Et2 in the mouse C4bpa gene, one ARE-like sequence and five TGTTCT half-sites were found in the first intron (Fig. 3B, underlined). Because it has been reported that some proteins contain functional AREs or TGTTCT half-sites in the first intron [37, 38], these sequences may be involved in androgen regulation. Further analyses are needed to confirm this point.
The alternative use of promoters found in the mouse and guinea pig [16] C4bpa genes are schematically summarized in Figure 4 with the human C4BPA gene [39]. The promoter region used in the liver is highly conserved between these species. However, the positions of transcription start sites used in the mouse epididymis are totally different from those of guinea pigs, in which two epididymis-specific promoters are found in the first intron (Fig. 4, Et-a and Et-b) [16]. The first intron of the guinea pig and of mouse C4bpa genes showed no similarity, and no homologous regions with the guinea pig epididymis-specific promoters were detected in the first intron of the murine C4bpa gene. This indicates that the mechanism of epididymal C4BP expression is not conserved between guinea pigs and mice.
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Western Blot Analysis
The expression of epididymal C4BP was confirmed by Western blot analysis using the epididymal fluid with rabbit anti-mouse C4BP antiserum (Fig. 5). Epididymal C4BP was detected in the position similar to that of serum C4BP, but with a somewhat smaller molecular weight, indicating that liver and epididymal C4BP undergo similar, but not identical, posttranslational modifications.
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Immunohistochemistry
Sections of epididymis from a 9-wk-old mouse were stained with the same antiserum used in the Western blots. In the efferent ductules (Fig. 6A) and the proximal caput epididymis (Fig. 6B), the interstitial region and the blood vessels were highly stained, possibly because of the existence of serum C4BP, whereas the epithelial cells and spermatozoa in the lumen were not stained. However, in the distal caput region (Fig. 6C), the microvilli, called stereocilia, in the apical regions of the epithelial cells and the supranuclear, well-developed Golgi apparatus in those cells were significantly stained. In addition, the spermatozoa in the lumen were also well stained. In the cauda epididymis (Fig. 6D), the microvilli and the spermatozoa in the lumen were stained as well. The liver, in which serum C4BP is synthesized and secreted, was used as a positive control (Fig. 6E). These results indicate that C4BP is also synthesized in the epididymal epithelial cells and is secreted into the lumen in a region-restricted manner, mainly in the distal caput region, and then interacts with spermatozoa during their passage through the epididymis.
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To confirm the binding of C4BP to the surface of spermatozoa, the epididymal spermatozoa were immunostained and studied with a confocal laser-scanning microscope (Fig. 7A). Significant labeling was observed on the surfaces of the head region of the spermatozoa from the distal caput to the cauda region of the epididymis. Furthermore, electron microscopy using thin sections of the immunostained epididymal tissue not only confirmed the localization of C4BP in the head region but also indicated that C4BP is located on the outer acrosomal membrane as well as the plasma membrane of the spermatozoa (Fig. 7B). These results suggest that C4BP is not loosely attached to the sperm surfaces but is taken up to the sperm membranes selectively in the head region.
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Expression of Human C4BP in the Epididymis
To determine whether human epididymis expresses C4BP, we performed Northern blot analysis (Fig. 8A) and immunohistochemistry (Fig. 8B). Human C4BP mRNA was not detected in any of the male reproductive organs. Immunohistochemistry showed significant staining in blood vessels and the interstitial region and weak staining in the cytoplasm of the epithelial cells, possibly because of the existence of serum C4BP, but none in the stereocilia and spermatozoa in the lumen. These results suggest that human epididymal epithelial cells secrete little or no C4BP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA is the same as that of liver C4BP mRNA and that they are transcribed from different transcription start sites in the murine C4bpa gene. The transcription is differentially regulated—that is, androgen-dependent in the epididymis and constitutive in the liver. Next, we demonstrated immunohistochemically that epididymal C4BP is synthesized in the epididymal epithelial cells and secreted into the lumen in a region-restricted manner and that it binds to the surface membranes of the spermatozoa in transit.
We have previously reported that the guinea pig C4bpa gene also possesses androgen-dependent, epididymis-specific promoters as well as the liver-specific promoters [16]. However, the epididymis-specific promoter regions are differentially located on the mouse and guinea pig C4bpa genes, whereas the liver-specific promoter regions are highly conserved between these species, the human [39], and the rat [40]. This indicates that the transcription mechanism of the C4BP gene in epididymis has evolved differentially in the human and rat, although the epididymis, not the liver, is the major expressing organ of C4BP mRNA in both species. Interestingly, although the human epididymis expresses no C4BP mRNA, the first intron of the human C4BPA gene contains regions that are 60% and 70% identical to the two epididymis-specific promoter regions found in the guinea pig C4BP gene in the corresponding regions (data not shown). This finding suggests that the human C4BPA gene might possess, or might have possessed, additional promoters in the first intron.
In the immunohistochemical study, C4BP was detected in the epididymal epithelial cells and the spermatozoa in the lumen from the distal caput to the cauda regions, but not in the proximal caput region. These results indicate that epididymal C4BP is synthesized and secreted by the epididymal epithelial cells in a region-restricted manner interacting with the spermatozoa. The epididymis is a highly coiled duct that links the efferent ducts to the vas deferens, and it is the site of accumulation, maturation, and storage of spermatozoa. The epididymal luminal fluid contains many proteins that are different in composition from those of blood plasma and interstitial fluid. In addition, the epithelium is characterized by its distinct morphological changes along its length and by the region-restricted secretion of epididymal proteins, indicating that the functional changes occur along its length [41–44]. Although the precise role of each epididymal secretory protein is still to be clarified, it is generally thought that the main function of the epithelial cells of the proximal caput region is in taking up the fluid that leaves the testis from the lumen by pinocytosis to concentrate spermatozoa in the lumen. The cauda region is the principal site of sperm storage. The distal caput region shows the highest number of secreted proteins and is suggested to be the most active region for sperm maturation. The secreted proteins in this region are thought to interact with physiologically immature spermatozoa in the lumen, contributing to their acquirement of motility and fertilizing ability [42, 44–46]. Murine C4BP was identified as one such epididymal secretory protein.
Immunofluorescence and electron microscopic immunohistochemistry demonstrated that C4BP is located on the plasma membrane and the outer acrosomal membrane of the head region of the spermatozoa. This result not only confirmed the preferential binding of C4BP to sperm surfaces but also indicated the restricted localization on the spermatozoa. Some epididymal secretory proteins have been found to be regionally located on the spermatozoa. For example, rat HIS-50 binds only to the sperm head, whereas other proteins, such as rat 2D6, bind only to the tail [42]. However, the exact role of these proteins in spermatozoa remains unclear.
The primary role of epididymal secretory C4BP likely is in sperm maturation rather than complement regulation in this tissue, because C4BP is only expressed in adult mice in an androgen-dependent and region-restricted manner. In addition, it has been assumed that the blood-epididymal barrier exists in the epididymal epithelial cells. The tight junctions between the epithelial cells isolate the luminal environment from blood to provide an optimal environment for sperm maturation and survival. This seems to be important to prevent the invasion of immune components, such as antibodies, and complement components from blood plasma, both because spermatozoa acquire many different surface antigens during their development and because exposure of these surface antigens to the immune system would mount an immune response [46]. Furthermore, we confirmed that the epididymal epithelial cells synthesize C4 and C3 mRNA at very low levels under normal conditions. Therefore, epididymal C4BP does not seem to be involved in regulation of the complement system in this organ.
It remains probable, however, that C4BP on spermatozoa may act as a complement regulator in the female reproductive tract where active C3 is detected. In this context, testicular spermatozoa have been reported to express some membrane-type complement regulators, such as MCP, DAF, and CD59. Among them, DAF and CD59 are expressed on the whole surface of the spermatozoa [3, 6], whereas C4BP is located only in the head region of the spermatozoa. Therefore, DAF and CD59 seem to be more useful for protection of the spermatozoa. Recently, it has been reported that male mice depleted of CD59b, which is one of the two isotypes of mouse CD59 and highly expressed in testis [47], show a progressive loss of fertility after 5 mo of age associated with immobile dysmorphic and fewer sperm cells [48]. The authors speculated that such sperm abnormalities may be caused by the susceptibility of the sperm to autologous complement attack, although they also noted that another functional role of CD59b in reproduction cannot be conclusively ruled out.
On the other hand, MCP has been detected only in the inner acrosomal membrane and is suspected to have a role in sperm-egg recognition [8, 49, 50]. In addition, sp56, which is structurally related to C4BP, has been detected in the acrosomal region and is also considered to be involved in sperm-egg interaction. These findings suggest that certain RCA proteins composed of SCR domains have evolved for reproduction in addition to their roles in the complement system. Our findings that C4BP may contribute to sperm maturation as an epididymal secretory protein provides a new link between the complement system and reproduction, supporting the intriguing idea of a reproductive function of these proteins.
It remains to be determined what molecules C4BP binds to on spermatozoa. It is not likely that activated forms of complement components C4 and C3 are the ligands for epididymal C4BP, because their concentrations in this tissue seem to be low. It has been reported that guinea pig C4BP coeluted with apexin during the isolation of fertilin from epididymal spermatozoa using an antifertilin affinity column [51]. Although the relationship among these proteins remains unknown, C4BP may react with these molecules in the epididymis. Alternatively, C4BP may bind to spermatozoa directly, as has been demonstrated recently in some ovarian adenocarcinoma cell lines [34].
To our knowledge, this is the first observation that shows C4BP as an epididymal secretory protein. In guinea pigs, it has been reported that C4BP mRNA is significantly expressed in the epididymis similar to mice [16], but no direct evidence has been obtained. In other species, such as the rat, boar, and stallion, in which many epididymal secretory proteins have been identified, C4BP has not been detected so far. In humans, no detectable C4BP is synthesized and secreted in the epididymis (Fig. 8), although the gene possesses the regions homologous to the guinea pig epididymal-specific promoters. It has been reported that human seminal plasma contains C4BP at a very low level [7]. Many epididymal secretory proteins are known to be species specific as well as tissue specific [45]. In addition, RCA proteins, including C4BP, are characterized by their low sequence identities between species, and many isoforms or isotypes have developed differentially in each species [9, 10, 16, 36]. Therefore, the functions of C4BP may have evolved differentially in each species. Further analyses are necessary to clarify the role of epididymal C4BP, and a targeting experiment is in progress in our laboratory.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 12 June 2003.
First decision: 7 July 2003.
Accepted: 4 August 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain gene in epididymis. J Immunol 2001 166:4570-4577- and ß-chains provides structural background for lack of complex formation with protein S. J Immunol 1994 153:4190-4199[Abstract]- and ß-chains: structural and functional relationships among human, bovine, rabbit, mouse, and rat proteins. J Immunol 1997 158:1315-1323[Abstract]-chains of C4b-binding protein mediate complex formation with low density lipoprotein receptor-related protein. J Biol Chem 2002 277:2511-2516
, new members of an epididymis-specific family of androgen-regulated proteins in the human. Endocrinology 2000 141:1245-1253
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