Identification of Low Density Lipoprotein Receptor-related Protein-2/megalin as an Endocytic Receptor for Seminal Vesicle Secretory Protein II*

The low density lipoprotein receptor-related protein-2/megalin (LRP-2) is an endocytic receptor that is expressed on the apical surfaces of epithelial cells lining specific regions of the male and female reproductive tracts. In the present study, immunohistochemical staining revealed that LRP-2 is also expressed by epithelial cells lining the ductal region and the ampulla of the rat seminal vesicle. To identify LRP-2 ligands in the seminal vesicle, we probed seminal vesicle fluid with 125I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solid-phase binding assays were used to show that the SVS-II bound to the receptor with high affinity (K d = 5.6 nm). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation.

I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solidphase binding assays were used to show that the SVS-II bound to the receptor with high affinity (K d ‫؍‬ 5.6 nM). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125 I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation.
The low density lipoprotein receptor-related protein-2/megalin (LRP-2) 1,2 has been shown to be widely expressed by cells lining the reproductive tracts of male and female rodents (1)(2)(3). For example, it is expressed by epithelial cells of the efferent ducts, epididymis, and vas deferens of the male and by the germinal layer and epithelium of endometrium and oviduct of the female (1)(2)(3)(4). The function of LRP-2 in the reproductive tracts is not yet known, but an endocytic function can be inferred from its apical surface localization and presence within endocytic compartments including coated pits, endocytic vesicles, and early endosomes of efferent duct cells and principal cells of the epididymis (2).
During transit through the epididymis, spermatozoa undergo a maturational process that includes morphological and physiological changes (e.g. acquisition of sperm motility and alterations in the properties of the plasma membrane). A widely held view is that the secretory and absorptive activity of the epithelial cells lining the efferent ducts, epididymis, and accessory glands (e.g. seminal vesicles) profoundly influences sperm maturation (5)(6)(7). Given the prominent expression of LRP-2 by efferent duct and epididymal epithelial cells, it has been hypothesized that LRP-2-mediated catabolism of proteins present in the epididymal fluid and/or associated with spermatozoa surfaces may be a contributing factor to spermatozoa maturation (2). Indeed, many LRP-2 ligands are found in the epididymis, seminal vesicle, and seminal plasma such as apolipoprotein J/clusterin (2), apolipoprotein B (8), apolipoprotein E (9), tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor-I (10) and have been speculated to be involved in sperm maturation and fertilization.
We report here that LRP-2 is also expressed by epithelial cells of the seminal vesicles. In the rat, the seminal vesicles are two coiled tubular glands connected by a short duct to an excretory tubule that is anatomically equivalent to the ampulla of humans and other primates and connected to the ejaculatory duct (11). The seminal vesicles produce proteins that become covalently cross-linked by a transglutaminase and form the fibrin-like seminal coagulum. In rodents, the seminal coagulum forms the copulatory plug, whereas in humans, seminal coagulum is degraded within the female reproductive tract by proteases of prostatic origin, such as prostate-specific antigen (PSA), a member of the kallikrein family of serine proteinases (12,13). The major components of the seminal coagulum belong to a multigene family (14,15) that includes proteins such as rat seminal vesicle secretory proteins II, IV, and V (SVS-II, IV, and V, respectively) (16,17), mouse seminoclotin (14), and human seminogelins I and II (18). In the following study, we present evidence indicating that a major rodent seminal vesicle secretory protein and transglutaminase substrate, SVS-II, is a novel LRP-2 ligand.

EXPERIMENTAL PROCEDURES
Proteins-LRP-2 was purified from porcine kidney using RAP-Sepharose affinity chromatography as described previously (19). Human 39-kDa receptor-associated protein (RAP) was expressed as a glutathione S-transferase fusion protein in bacteria and was purified according to Williams et al. (20). SVS-II was purified from rat seminal vesicle as described by Wagner and Kistler (16). Subfragments of rat SVS-II corresponding to the N-terminal domain (from residues 25 to 107), 13-residue-repeat domain central domain (from residues 108 to 311), and C-terminal domain (from residues 312 to 414) were expressed in bacteria as fusion proteins with GST using the pGEX2T vector (Amersham Pharmacia Biotech) using primers described in Table I. Fusion proteins were purified by glutathionine-Sepharose (Amersham Pharmacia Biotech) chromatography as described by the manufacturer. Fibulin-1 was purified from human placenta as described in Argraves et al. (21). LRP-2 and SVS-II were labeled with [ 125 I]iodine (Amersham Pharmacia Biotech) using Iodogen (Pierce Chemical Co., Rockford, IL) to specific activities of 7-8 and 2-10 Ci/g, respectively (ϳ0.222 MBq/g and ϳ0.279 MBq/g, respectively).
Antibodies-LRP-2-Sepharose affinity chromatography was used to isolate anti-LRP-2 IgG from a polyclonal serum (rb6286) derived from a rabbit immunized with porcine kidney LRP-2 prepared as described previously (22). The bound IgGs were eluted using both high and low pH buffers and dialyzed against TBS (150 mM NaCl, 50 mM Tris, pH 7.4). After dialysis, the IgG preparation was applied to a column of RAP-Sepharose (1.0 mg of protein/ml of resin) and the unbound IgG selected on protein G-Sepharose. Control IgG was purified from normal rabbit serum using protein G-Sepharose.
Mouse monoclonal antibody 3C3 to human LRP-2 was prepared by fusing NS1 myeloma cells with spleen cells from Balb/c mice immunized with human LRP-2 purified from urine (23). Hybridomas were selected based on production of immunoglobulin G antibodies reactive with both human and rat LRP-2 by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Isolation of Rat Seminal Vesicle Fluid-Sexually inactive, male Sprague-Dawley rats (age 6 -8 months) were killed by CO 2 inhalation, and their seminal vesicles were removed. The fluid contents of the seminal vesicles were released immediately into an ice-cold solution of spermine and spermidine (Sigma) (each at 5 mM in water). The fluidcontaining solution was centrifuged for 10 min at 10,000 ϫ g, and the supernatant was recovered. Protein concentration in the clarified fluid was estimated using a commercial protein assay system (Bio-Rad).
Immunohistochemistry-Isolated rat seminal vesicles were dissected into four parts along their proximal to distal axis. Each segment was placed in Bouin's fixative (EM, Diagnostic Systems, Gibbstown, NJ) for 4 h at room temperature and then placed in 70% ethanol for 18 h at 4°C. The fixed tissue specimens were dehydrated, embedded in paraffin, and sectioned at 5 M thickness. Immunohistochemical staining was carried out using a Sequenza coverplate system (Shandon Lipshaw, Pittsburgh, PA) with polyclonal LRP-2 IgG (rb6286) and normal rabbit IgG (each at 2 g/ml). An Elite Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and the chromogenic substrate DAB (Sigma) were used for antibody detection. Sections were counter-stained with hematoxylin (Shandon Lipshaw).
Gel-blot Overlay Assay-Aliquots of rat seminal vesicle fluid, rat SVS-II, GST-SVS-II fusion proteins, GST, RAP, or fibulin-1 were electrophoresed with fibulin-1 and RAP on 4 -12% polyacrylamide gels (Novex, San Diego, CA) in the presence of SDS and under non-reducing conditions. The separated proteins were electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell), and unoccupied sites were blocked with 3% nonfat milk in TBS (pH 8.0) (blocking buffer). The membranes were incubated with 125 I-LRP-2 (1 nM) in blocking buffer containing 5 mM CaCl 2 and 0.05% Tween 20. To block the function of LRP-2, radiolabeled LRP-2 solution was preincubated with RAP (1 M) for 1 h at room temperature prior to incubation with the membranes. Following the incubation, the membranes were washed with TBS containing 0.05% Tween 20 and exposed to Kodak BioMax MR film (Eastman Kodak Company, Rochester, NY) at room temperature.
For non-radioactive probing, filters were incubated with unlabeled LRP-2 (1 nM) in blocking buffer containing 5 mM CaCl 2 and 0.05% Tween 20 for 3 h at room temperature. Bound receptor was detected using mouse anti-LRP-2 IgG (1 g/ml), anti-mouse IgG-HRP, and the ECL Plus Western blotting detection system (Amersham Pharmacia Biotech).
Protein Sequence Analysis-Seminal vesicle fluid, isolated in the presence of spermine and spermidine, was electrophoretically separated on a 4 -12% acrylamide gel in the presence of SDS, under nonreducing conditions. The gel was stained with 0.2% Coomassie Brilliant Blue R-250 (Bio-Rad) in 45% methanol, 10% acetic acid. The polypeptide band having an M r corresponding to that identified in seminal fluid by gel-blot overlay assay using radioiodinated LRP-2 as probe was excised. The gel slice was minced, and the pieces were washed and destained in 50% methanol for 18 h. The gel pieces were dehydrated in acetonitrile and rehydrated in 10 mM dithiothreitol, 0.1 M ammonium bicarbonate and reduced at 55°C for 1 h. The dithiothreitol solution was removed, and the sample was alkylated in 50 mM iodoacetamide, 0.1 M ammonium bicarbonate for 1 h at room temperature in the dark. The gel pieces were washed with 0.1 M ammonium bicarbonate and dehydrated in acetonitrile for 5 min. The acetonitrile was removed, and the gel slices were rehydrated in 0.1 M ammonium bicarbonate. The pieces were again dehydrated in acetonitrile and dried by vacuum centrifugation. The gel pieces were rehydrated in a solution of trypsin (12.5 ng/l) in 0.1 M ammonium bicarbonate and incubated on ice for 45 min. The trypsin-containing solution was removed, 0.05 M ammonium bicarbonate was added, and the slices were incubated for 18 h at 37°C. The proteolytic fragments were then extracted using 50% acetonitrile, 5% formic acid and dried to Ͻ20 l. The sample was then analyzed by capillary high performance liquid chromatography-electrospray tandem mass spectrometry using a Finningan-MAT TSQ7000 located in the W. M. Keck Biomedical Mass Spectrometry Laboratory (University of Virginia, Charlottesville, VA).
Solid Phase Binding Assay-Both homologous and heterologous ligand displacement assays were carried out as described previously (25). Radioiodinated proteins (1-2 nM) in TBS, 3% bovine serum albumin (U. S. Biochemical Corp.), 5 mM CaCl 2, 0.05% Tween 20 were incubated for 3 h at room temperature in breakaway microtiter wells (Dynatech, Chantilly, VA) coated with LRP-2, SVS-II, or ovalbumin (each coated at 3 g/ml) in the presence of increasing concentrations of the competitor. The wells were washed with TBS, 5 mM CaCl 2 , 0.05% Tween 20, and bound radioactivity was determined using a gamma counter. The computer program Ligand (26) was used to analyze the competition data and to determine dissociation constants (K d ) for receptor-ligand interactions.
Assay For Cellular Internalization and Degradation of 125 I-SVS-II-Ligand internalization and degradation by LRP-2 expressing cells was performed according to previously published methods (24,27). Briefly, RA-and Bt 2 cAMP-treated F9 cells were plated into 3.83-cm 2 wells at 1.5-1.75 ϫ 10 5 cells/well and allowed to grow for 18 h at 37°C, 5% CO 2 .

3548-3528
a Restriction site sequences (SmaI and EcoRI) and residues added to maintain reading frame are underlined.
b The numbers indicated correspond to nucleotide residues within the rat SVS-II sequence (GenBank™ accession number J05443).
pre-incubation medium was removed, and medium containing the radioiodinated proteins (1 nM) plus competitors was added and incubated for 5 h at 37°C, 5% CO 2 . The conditioned culture medium was treated with trichloroacetic acid (final concentration 10%) and centrifuged at 10,000 ϫ g for 10 min. The amount of radioactivity present in the supernatant was taken to represent the amount of degraded SVS-II (28). The cell layer was washed three times with cold (4°C) Dulbecco's phosphate-buffered saline and then treated with 0.5 mg/ml trypsin, 50 g/ml proteinase K, and 0.5 mM EDTA (all from Sigma) in Dulbecco's phosphate-buffered saline for 2-4 min at 4°C. The released cells were pelleted by centrifugation at 1,300 ϫ g for 15 min, and the amount of radioactivity in the cell pellet was measured.

Immunohistological Analysis Reveals that LRP-2 Is Expressed by Seminal Vesicle Epithelial Cells Located Proximal to
the Duct-We have recently found that LRP-2 was expressed on apical surfaces of epithelial cells lining the efferent ducts and in the intermediate zone, proximal head, corpus, and caudal regions of the epididymis (2). In the present study, we have examined LRP-2 expression in the accessory sex gland, the seminal vesicle. As shown in Fig. 1, using affinity-selected LRP-2 antibodies, pronounced immunoperoxidase staining was observed on the apical regions of epithelial cells covering the duct of the gland. In addition, LRP-2 staining was also strong in the apical portion of the epithelial cells lining the ampulla (Fig. 1C). This staining also appeared to occur in budding vesicles (Fig. 1C, arrow). Very weak LRP-2 staining was seen in the underlying stroma lying adjacent to the LRP-2-expressing epithelial cells. Examination of distal segments of the gland showed negligible LRP-2 staining in both the epithelial cells and the underlying stromal cells (data not shown). The results indicate that LRP-2 is expressed by epithelial cells lining the duct of the seminal vesicles and may be present within secreted vesicular particles.

LRP-2 Binds to a 100-kDa Protein Present in Seminal Vesicle
Fluid-The expression of LRP-2 in the seminal vesicle prompted us to examine seminal vesicle fluid for the presence of LRP-2-binding proteins. Seminal vesicle fluid collected in the presence of the polyamines spermine and spermidine, to prevent coagulation, was electrophoretically separated by SDSpolyacrylamide gel electrophoresis (under non-reducing conditions) and transferred to nitrocellulose membranes. The membrane-bound proteins were probed using 125 I-LRP-2. As shown in Fig. 2, the radiolabeled receptor bound to a polypeptide with an M r of 100,000 ( Fig. 2A). Other minor LRP-2binding bands were also apparent in the profile. As a control, the 125 I-LRP-2 was shown to bind to a known LRP-2 ligand, the FIG. 1. Immunohistochemical localization of LRP-2 within rat seminal vesicle epithelial cells located proximal to the duct. Rat seminal vesicle (panels A-D) and rat kidney tissue sections (panels E-F) were incubated with affinity purified rabbit anti-LRP-2 IgG (panels A, C, and E) or pre-immune rabbit IgG (panels B, D, and F) followed by goat anti-rabbit-horseradish peroxidase and the chromogenic substrate DAB. The tissue sections were counter-stained using hematoxylin. (29 -31). Fibulin-1, a non-LRP-2-binding protein did not exhibit binding to the radiolabeled receptor in this assay.

39-kDa RAP
To evaluate the specificity of the binding interaction between the receptor and the 100-kDa protein, duplicate filters were incubated with 125 I-LRP-2 in the presence of RAP, an antagonist of the ligand binding activity of LRP-2 (19). As shown in Fig. 2C, RAP completely blocked binding of radioiodinated LRP-2 to the 100-kDa protein. The results indicate that seminal vesicle fluid contains a 100-kDa protein that binds to LRP-2. Consistent with all other LRP-2 ligands (19), the binding of the 100-kDa protein to LRP-2 was inhibited by RAP.

Identification of the 100-kDa LRP-2-binding Protein as Seminal Vesicle Secretory Protein II by Protein Sequence Analysis-To
identify the 100-kDa LRP-2-binding protein, amino acid sequence analysis was performed. Initial attempts to sequence the N terminus of the LRP-2-binding protein using Edman degradation did not yield a sequence. Assuming that the protein might be N-terminal blocked, it was subjected to limited proteolysis, and the resulting fragments were evaluated by liquid capillary mass spectrometry and high performance liquid chromatography-tandem mass spectrometry. As a result, sequences were obtained from 13 peptides (Table II). Data base analysis of these sequences revealed that eleven corresponded to sequences contained within rat seminal vesicle secretory protein (SVS-II) (32). SVS-II is a 48-kDa seminal fluid protein that forms a 100-kDa disulfidelinked homodimer (16,17,32). Sequences from two of the peptides were unidentifiable. The results indicate that the 100-kDa-LRP-2-binding protein most likely corresponds to SVS-II.

SVS-II Binds with High Affinity to LRP-2 in Solid Phase
Assay-To confirm that LRP-2 binds to SVS-II, solid phase binding assays were performed using purified components. SVS-II was purified from rat seminal vesicle fluid according to previously published methods (16). As shown in Fig. 3A, 125 I-LRP-2 bound to purified SVS-II immobilized on microtiter well plastic but not to ovalbumin-coated wells. The binding to SVS-II could be inhibited by incubation with unlabeled LRP-2. A dissociation constant (K d ) of 10.4 Ϯ 4.9 nM (n ϭ 6) was determined for the binding of radiolabeled LRP-2 to SVS-II from the best fit of the data to a single class of binding sites model. Similarly, 125 I-SVS-II was shown to bind to wells coated with LRP-2, but not to ovalbumin-coated wells (Fig. 3B). A K d of 5.8 Ϯ 2.5 nM (n ϭ 5) was determined for the binding of SVS-II to immobilized LRP-2. The differences in the observed K d values derived from the two types of assays may reflect conformational differences between the solution phase and plastic-absorbed proteins. In addition, the LRP-2 antagonist RAP was shown to competitively inhibit 125 I-SVS-II binding to LRP-2coated wells (Fig. 3C), and an inhibition constant (K i ) of 9.0 Ϯ 2.5 nM (n ϭ 4) was determined. The findings indicate that LRP-2 can bind to SVS-II with high affinity and that the interaction can be blocked by RAP.
Given that LRP-2 shares a number of ligands in common with LRP-1, we were interested in evaluating the ability of LRP-1 to bind to SVS-II. As shown in Fig. 4A, 125 I-LRP-1 bound to purified SVS-II immobilized on microtiter well plastic, and the binding could be inhibited by incubation with solution phase SVS-II. Additionally, 125 I-SVS-II was found to bind to wells coated with LRP-1 but not to ovalbumin-coated wells (Fig. 4B). A K d of 1.4 Ϯ 1.0 nM (n ϭ 3) was determined for the binding of SVS-II to immobilized LRP-1. RAP was found to also inhibit the binding of SVS-II to immobilized LRP-1 (K i ϭ 4.4 Ϯ 0.6 nM) (Fig. 4C). A K i of 16.5 Ϯ 3.5 nM (n ϭ 2) was determined for SVS-II inhibition of binding of radiolabeled LRP-1 to immobilized SVS-II. The results indicate that LRP-1, like LRP-2 can bind to SVS-II in vitro; however, given that LRP-1 was not immunologically detected in the seminal vesicle (data not shown), the significance of this interaction remains uncertain.

LRP-2 Binds to a Site within the 13-Residue Repeat Region of SVS-II-
To determine the binding site within the SVS-II protein, a series of fragments of SVS-II were generated recombinantly and evaluated for their ability to promote LRP-2 binding in gel-blot overlay assays. The recombinant fragments corresponding to the 85-residue N-terminal domain, the 103-residue C-terminal domain, or the 204-residue 13-residue repeat domain of SVS-II (Fig. 5) were expressed as fusions with GST. As shown in Fig. 6, LRP-2 bound the 204-residue 13-residue-repeat domain-containing fragment but not the N-and C-terminal domain fragments. As controls, LRP-2 bound filter-immobilized SVS-II and RAP, but not GST. LRP-2 binding to SVS-II, the 204-residue 13-residue-repeat domain-containing fragment and RAP was inhibited by RAP (Fig. 6, C).
LRP-2 Mediates Cellular Uptake and Lysosomal Degradation of SVS-II-To determine whether LRP-2 functions to mediate endocytosis leading to lysosomal degradation of SVS-II, we evaluated the ability of LRP-2-expressing cells to internalize and degrade radioiodinated SVS-II. As shown in Fig. 7, cultured F9 cells, treated to augment expression of LRP-2 (24, 27) mediated internalization and degradation of 125 I-SVS-II. Both processes could be inhibited by polyclonal LRP-2 antibodies that have been previously shown to block LRP-2 function (24,27,33). Control IgGs had no effect on 125 I-SVS-II internalization and degradation. The magnitude of the inhibition by LRP-2 antibodies (200 g/ml) was nearly as great as that obtained using either 1000-fold molar excess of unlabeled SVS-II or RAP as competitors. The degradation of radiolabeled SVS-II was inhibited by chloroquine (Fig. 7A), a drug that blocks lysosomal proteinase activity through its ability to increase the pH of lysosomal vesicles. As expected, the chloroquine treatment resulted in an intracellular accumulation of 125 I-SVS-II (Fig. 7B). The findings indicate that LRP-2 can function to mediate endocytosis and lysosomal degradation of 125 I-SVS-II. DISCUSSION Evidence is presented in this study to indicate that LRP-2, a multi-ligand endocytic receptor and a member of the LDL receptor family, is expressed by seminal vesicle epithelial cells and is potentially a component of seminal vesicle-secretory vesicles. In addition, it was discovered that LRP-2 binds with high affinity (K d ϭ 5.6 nM) to SVS-II, a prominent seminal vesicle-derived coagulatory protein. The LRP-2 binding site was mapped to a 204-amino acid region of the protein contain-ing a series of 13-residue repeats. As evidence that LRP-2 is also capable of mediating cellular clearance of SVS-II, LRP-2expressing cells were shown to endocytose and degrade SVS-II. Both processes can be inhibited by anti-LRP-2 IgG or RAP, a known antagonist of LRP-2 function (23, 24, 33, 34).

TABLE II
Mass spectroscopic sequence analysis of trypsin-generated proteolytic fragments of 100-kDa seminal vesicle fluid protein X designates isoleucine or leucine which cannot be distinguished by low energy collisionally activated dissociation; M(o) designates oxidized methionine; lowercase letters designate tentative assignments; _ designates an unknown amino acid; ---designates an unknown number of unknown amino acids; b2 ϭ refers to the mass (in Da) of the first two amino acid residues; UI indicates that the peptide sequence obtained did not correspond to any sequence in the database. The physiological significance of the LRP-2 interaction with SVS-II is not yet known. LRP-2-mediated endocytosis of SVS-II may function in vivo to clear multimerized and/or transglutaminase-cross-linked SVS-II, thereby preventing coagulation within the male reproductive tract and the urinary tract which might lead to an obstruction. Proteinaceous plugs have been reported in the accessory sex glands and urinary bladders of male rats (35,36). The expression of LRP-2 in epithelial cells proximal to the ductal region of the gland might be important to clear SVS-II that has become cross-linked by retrograde flow of transglutaminase-containing fluids derived from the coagulatory and prostate glands. LRP-2 that is expressed by epithelial cells lining the female reproductive tract (1,4,37) might also mediate clearance of proteolytic fragments of SVS-II generated during dissolution of the coagulatory plug. Indeed, following copulation, seminal vesicle antigens have been detected within uterine epithelial cells, indicative of endocytosis as a mechanism of their elimination (38).
Immunohistological staining of the seminal vesicle not only showed LRP-2 to be localized within the epithelial cells lining the ductal region of the gland, but possibly in membrane bound vesicular bodies found in the lumen of the gland. While LRP-2-expressing epithelial cells lacking apparent vesicular budding were observed, numerous regions of epithelium in the ductal area appeared to be active in secretion of LRP-2-containing vesicular bodies. The pronounced LRP-2 staining of the budding vesicles in these areas may indicate that LRP-2 serves a role in vesicle secretion. Vesicular bodies have been observed by electron microscopy in the lumen of the seminal vesicle and other reproductive organs including the epididymis, vas deferens and ampulla (39). In addition, such vesicular bodies have been isolated from seminal vesicle (39 -41), epididymal (42,  Table I  43), and prostatic fluids (44,45). Following our previous finding of LRP-2 expression by epididymal epithelial cells (2), we isolated vesicular bodies from rat cauda epididymal fluid and found that they contained LRP-2 (unpublished observations, Ranganathan and Argraves). Vesicular bodies in seminal and epididymal fluids have been speculated to play a role in sperm maturation, perhaps involving proteolytic and glycolytic modification of spermatozoa membrane glycoproteins (46 -48). It remains to be determined what role is served by LRP-2 associated with seminal and epididymal vesicular bodies.