Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.     

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig

Summary Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1–2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.     

Characterization of glycoconjugates of guinea pig seminal vesicle by lectin histochemistry

In the present study, the expression of glycoconjugates in the guinea pig seminal vesicle was localized and partially characterized by lectin histochemistry using a battery of 30 different lectins specific for different carbohydrate residues. The results indicate that the glandular epithelium of the guinea pig seminal vesicle exhibits complex glycoconjugates rich in Man, -GlcNAc, -Gal, /-GalNAc, Fuc and complex NeuAc(2,6)Gal/GalNAc residues, as shown by its positive reactions to most lectins used. The Golgi region of the luminal secretory epithelial cells expresses a complex glycoconjugate pattern, as shown by its strong reactions to Man-(PSA, GNA), -GlcNAc-(S-WGA, PWA, DSA, UDA), -Gal- (RCA-I and -II), /-GalNAc-(SBA, Jac, VVA, BPA) and complex NeuAc-(SNA) specific lectins, indicating that the secretory epithelial cells are active in glycosylation and secretion process. It was also shown in the present study that the basal and luminal epithelial cells are different in their glycoconjugates. The basal epithelial cells are rich in NeuAc(2,3)Gal residues as they are stained specifically by MAA. The fibroblasts in the epithelial-smooth muscle interface and the smooth muscle cells close to the glandular epithelium are shown to express more glycoconjugates as they are stained intensely by GS-I-B4, GS-II and SBA. However, their role in the epithelial-stromal interaction in the seminal vesicle remains to be elucidated. In summary, the present study reports for the first time on the lectin binding patterns of the guinea pig seminal vesicle, and the results show that the seminal vesicle epithelium elaborates and secretes glycoconjugates in a complex pattern. Some of the lectins might be useful as histochemical markers for the secretory activity and specific structural components in the guinea pig seminal vesicle. © 1998 Chapman & Hall     

Electron microscopic immunolocalization of seminal vesicle-specific antigen in human seminal vesicle

Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.     

Processing of two protein precursors yields four mature guinea pig seminal vesicle secretory proteins

The translation of the two most abundant guinea pig seminal vesicle epithelium mRNAs (1800 nucleotides and 950 nucleotides) and the subsequent processing of their protein products were studied in an effort to elucidate the mechanism by which the four mature guinea pig seminal vesicle epithelium (GPSVE) secretory proteins are produced. The primary translation products of the 1800 nt and 950 nt mRNAs are two secretory protein precursors of 45 kDa and 20 kDa, respectively. Removal of signal peptides from these two precursors produces proteins of 43 kDa and 18.5 kDa, which are recognized by polyclonal antisera directed against the four mature secretory proteins. The existence of further processing intermediates in the production of the secretory proteins is suggested by the appearance of other immunoreactive polypeptides following incubation of GPSVE in nutrient medium containing [3H] leucine. Immunological and pulse-chase analysis strongly suggests that the 43-kDa protein gives rise to SVP-1, -3, and -4 and that SVP-2 is derived from the 18.5-kDa protein.     

Complex carbohydrate histochemistry of the lateral prostate and seminal vesicle of the guinea pig.

A systematic histochemical study of the complex carbohydrates of the lateral prostate and seminal vesicle of the guinea pig has been made. The complex carbohydrates of the guinea pig male accessory sex glands were partially characterized by various conventional carbohydrate histochemical methods including periodic acid-Schiff, selective periodate oxidation-Schiff reaction, Alcian blue staining at pH 2.5 and 1.0, and high iron diamine. The results indicated that neutral glycoconjugates with 1,2-glycol groups and sialic acids were present in the luminal border and apical cytoplasm of the glandular cells, basement membrane and connective tissue in the lamina propria of the lateral prostate. Similar patterns were demonstrated in the seminal vesicle except that there were relatively fewer or no neutral carbohydrates in the apical cytoplasm of the vesicular epithelial cells. The epithelial basement membrane and connective tissue at the epithelial-stromal interface of both glands were rich in acidic and sulphated glycosaminoglycans. Partial characterization by bovine testicular hyaluronidase indicated the presence of chondroitin sulphates in the lamina propria of the glands.     

Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.     

Identification, evolution, and regulation of expression of Guinea pig trappin with an unusually long transglutaminase substrate domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.     

A nuclear binding assay for measurement of biologically active androgen receptors in animal tissues and human prostate cancer

A nuclear binding (NB) assay has been developed for the measurement in intact viable cells of biologically active (functional) estrogen and progesterone receptors, i.e. those capable of binding to nuclear acceptor sites [Spelsberg et al., Endocrinology 121: 631 (1987)]. This paper describes the application of this assay to analyses of androgen receptors in the guinea pig seminal vesicle and in human prostatic carcinoma. Cells from fresh animal seminal vesicles or human prostate carcinoma are isolated using collagenase and are incubated with [3H]R1881 for 1 h at 22 degrees C, after which nuclei are isolated at 4 degrees C and assayed for DNA and radioactivity. This NB assay demonstrates a saturable, temperature dependent, steroid and tissue specific nuclear binding of [3H]R1881 for the guinea pig-seminal vesicle system. The nuclear binding is of high affinity and low capacity. The NB assay reveals several important aspects of the androgen and estrogen receptors in target tissues: (1) the nuclear acceptor sites for androgen receptor (AR) are steroid receptor specific; (2) there are different concentrations of the androgen and estrogen receptors between the epithelium and the fibromuscular components of the guinea pig seminal vesicle; and finally (3) some biopsies of human prostate cancer appear to contain biologically inactive AR. This assay may be useful in the analyses of functional receptors in biopsies of human cancer cells.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.     

Immunocytochemical localization of insulin-like immunoreactivity in mouse seminal vesicle

Insulin-like immunoreactivity was localized in tissue sections and cell cultures of mouse seminal vesicle using the indirect technique of immunocytochemistry. Seminal vesicles were cut into fragments, fixed in 2.5% glutaraldehyde, embedded in epoxy resin, sectioned at 1 micron, and transferred to glass slides. Epithelial cell cultures of seminal vesicle were grown on coverslips in Dulbecco's Minimal Essential Medium for 4-6 days and fixed in 2.5% glutaraldehyde. Sections (etched with sodium ethanolate) or coverslips were incubated in guinea pig antiporcine insulin antiserum, in antiserum immunoabsorbed with porcine insulin, or in normal guinea pig serum. For indirect immunocytochemistry, incubation with primary antiserum was followed by treatment with rabbit anti-guinea pig immunoglobulin (Ig) G conjugated to peroxidase, or with protein A and then rabbit peroxidase anti-peroxidase (PAP). Finally, treated samples were incubated in phenylenediamine-pyrocatechol-H2O2 substrate mixture for 6-8 min at room temperature. Specific immunoreactivity to insulin antisera was confined to the epithelium of the seminal vesicle in tissue sections. No staining occurred in subepithelial connective tissue. Specific immunoreactivity was also observed in the cytoplasm of cultured seminal vesicle epithelial cells.     

Androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin II, a WAP-motif protein

We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.     

Polarized epithelial cells of the hamster seminal vesicle in a serum-free bicameral culture system: evidence of secretory and endocytic activities

Primary cultures of epithelial cells from the hamster seminal vesicle were established in a chemically defined medium supplemented with hormones and growth factors. Epithelial cell clusters were prepared combining enzymatic dissociation and mechanical disaggregation and then seeded in bicameral systems equipped with collagen-membrane inserts. A growth curve was generated and the cells were characterized morphologically and morphometrically by light and electron microscopy. The immunocytochemical detection of cytokeratins and the measurement of transepithelial electrical resistance were also performed. The secretory activity was studied by fluorography using L-[³⁵S]methionine as a precursor, and endocytosis was approached using horseradish peroxidase as a tracer. Our results show that epithelial cells of the seminal vesicle can be grown as a monolayer of morphological and functionally polarized cells which retain secretory and endocytic activities. These cell cultures might therefore prove useful to investigate further the regulation of secretion and endocytosis in the seminal vesicle and are a promising model to approach, in a broader scope, cell polarity and protein sorting and targeting.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

Nuclear protein matrix of seminal vesicle epithelium

Nuclear protein matrix was isolated from guinea pig seminal vesicle epithelium and liver. The two matrices were similar in fine structure as seen by transmission electron microscopy, in protein electropherograms, and in percent composition relative to protein, DNA, and RNA. Scanning electron microscopy was used to examine intact seminal vesicle nuclei, nuclei after treatment with Triton X-100 and DNAse I, and purified nuclear matrix. The matrix surface presented a 'porous' appearance by both scanning and transmission electron microscopy. The matrices of liver and seminal vesicle epithelium (SVE) and the intact nuclei of SVE were assayed for specific binding of free synthetic androgen, 17 alpha-methyltrienolone (R1881). Saturable specific binding was demonstrable for seminal vesicle matrix but not for liver matrix. Maximal binding of androgen occurred at a concentration of approximately 12 nM and was demonstrated to be 1.34 +/- 0.22 pmol of R1881 per mg of seminal vesicle matrix protein; the Kd was approximately 8 nM. The binding of labeled R1881 to matrix could be inhibited with low concentrations of unlabeled androgens, but not with estrogens or other steroids. Our data indicate that the binding of androgen to matrix could account for at least 21% of the binding to intact nuclei.     

Androgens are necessary for the establishment of secretory protein expression in the guinea pig seminal vesicle epithelium.

The guinea pig seminal vesicle epithelium (GPSVE) synthesizes and secretes milligram quantities of four related secretory proteins in an androgen-dependent manner. To investigate the role of androgens in the establishment of secretory protein synthesis during the development of the GPSVE, animals were castrated at Day 5, approximately 10 days before secretory protein accumulation begins in intact animals. Castration did not eliminate secretory protein mRNA from the SVE, but it did indefinitely postpone the developmentally programmed increase in secretory protein mRNA. Injection of neonatally castrated guinea pigs with either estradiol or dexamethasone did not alter levels of secretory protein mRNAs. However, treatment of castrated neonates with either testosterone propionate or dihydrotestosterone (DHT) led to specific increases in secretory protein mRNAs within 4 days. Although neonatally castrated animals accumulated and translated significant amounts of secretory protein mRNA, the newly synthesized secretory proteins failed to accumulate until exogenous androgens were provided. This observation suggests that androgens regulate both the accumulation of secretory protein mRNA and the accumulation of secretory proteins in the GPSVE.     

Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse.

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.     

Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig

The presence of caltrin-like proteins in reproductive tract fluid (RTF) and seminal vesicle content from male guinea pigs has been determined. Two fractions with electrophoretic mobility corresponding to Mr = 6200 (main band) and 5100 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Isoelectric focusing in thin-layer agarose gels revealed three bands with acidic pIs of 5.3, 6.0, and 6.2, respectively. RTF prevented the enhancement of calcium permeability induced by incubating guinea pig epididymal spermatozoa in medium for capacitation. Spermatozoa incubated for 2 h in minimal culture medium plus pyruvate and lactate containing RTF accumulated less than 30% of the 45Ca2+ accumulated by cells maintained in absence of this fluid. Calcium uptake by preincubated spermatozoa was also inhibited by RTF. Inhibition of calcium transport activity by RTF and seminal vesicle proteins was not decreased by heating the dialyzed preparations at 60 degrees C for 5 min. After this treatment, the inhibitory activity and the protein pattern were stable for 3 wk when stored at 4 degrees C. Unheated extracts lost calcium transport inhibitory activity after 2 or 3 days at 4 degrees C. In spite of the differences in pIs among the proteins from the guinea pig reproductive tract and bovine caltrin, several features indicate they may play a similar role in both species by controlling Ca2+ movement across the plasma membrane. By this mechanism, these proteins could regulate physiologic events essential for the fertilization process.     

Thiamine pyrophosphatase activity in secretory cells of the lateral prostate and seminal vesicle of normal and castrated guinea pigs and castrates treated with oestradiol

Summary Ultrastructural localization of thiamine pyrophosphatase (TPPase) activity was studied in secretory cells of the lateral prostate and seminal vesicle of normal and castrated guinea pigs and castrates treated with 17-oestradiol benzoate. The present study has demonstrated that TPPase reaction product is consistently localized in the three to four trans cisternae of Golgi complexes in both the lateral prostate and the seminal vesicle. The reaction was intense and the reaction product often filled the cisternae completely.After castration there was a decrease in TPPase activity in both glands as revealed by the reduction in the amount of the reaction product which was found mainly in one to two trans cisternae of the regressed Golgi complex. The reaction product changed from a dense to a more particulate or granular pattern or to discrete deposits of high electron-density.Administration of 17-oestradiol benzoate to the castrates caused changes in the localization and patterns of distribution of TPPase. In the lateral prostate there was an apparent increase in TPPase activity. The reaction product was found in two to four trans cisternae and occasionally in the trans-most cisternae of the dilated Golgi complex. The reaction product appeared as discrete, dense coarse precipitates. In the seminal vesicle TPPase reaction product was consistently found in one to two trans cisternae in cells with larger Golgi complexes. However, almost all cisternae of the smaller Golgi complexes were TPPase-positive. The cytochemicl results of the present study suggest that TPPase activity and possibly the process of glycosylation in secretory cells of the lateral prostate and seminal vesicle may have been affected after castration and after oestradiol administration.