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.     

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.     

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.     

Tissue-specific expression and androgen regulation of different genes encoding rat prostatic 22-kilodalton glycoproteins homologous to human and rat cystatin

22-Kilodalton (kDa) protein cDNA clones were isolated from a rat prostatic library. Nucleotide sequence analysis revealed three different cDNA sequences encoding two somewhat different open reading frames of 176 amino acids. The N-terminal 24 amino acids of these sequences show the typical characteristics of signal peptides of secretory proteins. The C-terminal end of the derived protein sequences displays sequence similarity to a number of cysteine proteinase inhibitors, called cystatins, suggesting a common physiological function. Upon Northern blotting with a labeled cDNA fragment, three different 22-kDa protein mRNAs, i.e. 950 nucleotides (nt), 920 nt and 860 nt, could be detected in the rat ventral prostate and the lacrymal gland. In both tissues these messengers were regulated by androgens showing the most rapid androgen response for the 950 nt mRNA form. Administration of cycloheximide nearly completely abolished the observed androgen effect suggesting that a short-living protein is required for the full induction of the 22-kDa protein genes. Hybridization experiments with specific oligonucleotides which distinguish between the mRNAs encoding both 22-kDa protein variants indicate that one protein form is less androgen dependent in the ventral prostate and not expressed in the lacrymal gland.     

Molecular cloning of the cDNA for two major androgen-dependent secretory proteins of 18.5 kilodaltons synthesized by the rat epididymis

cDNA clones representing two closely related androgen-dependent secretory proteins of 18.5 kDa were selected by screening a rat epididymal cDNA library constructed in lambda gt 11 with affinity-purified antibody directed against the 18.5-kDa proteins. The entire amino acid sequence of the 18.5-kDa secretory proteins and a putative signal sequence of 18 amino acids was derived from 682 base pairs of the nucleotide sequence of overlapping cDNA clones. Confirmation of the identity of the cDNA clones was obtained by matching a partial amino acid sequence obtained for the N terminus of the pure protein with that of the sequence derived from the nucleotide code of the cDNA. Evidence is presented that the difference between the two closely related proteins may be associated with differential post-translational modification of the N terminus of the protein following cleavage of the signal sequence. Northern blot analysis revealed that the mRNA for the proteins is approximately 850 nucleotides long and that the concentration of the mRNA in the tissue is androgen-dependent. The proteins and their mRNAs were restricted to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from the skin, brain, liver, kidney, heart, skeletal muscle, and testis. With the exception of a weak cross-reaction with mouse epididymis, the proteins were not detected by Western blots of extracts of guinea pig, rabbit, or bull epididymis. The two proteins account for a substantial proportion of the total protein in epididymal luminal fluid and become incorporated as components of the sperm plasma membrane where they may play a specific role in the post-testicular phase of sperm development.     

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.     

Morphometric and stereological study of the seminal vesicle of the guinea pig.

The seminal vesicle of the guinea pig has been widely used as a model for the study of hormonal action on the male accessory sex organ, but there have been few attempts to quantify their cellular and tissue components. In the present study, the seminal vesicle of the guinea pig was described in the form of a morphometric model. Tissue samples were taken from the distal, middle and proximal regions of the gland and processed for light microscopy. Using a combination of a stereological point-counting technique and direct measurement, the relative volumes of different components (lumen, epithelium, lamina propria and fibromuscular layer) were determined. The relative numbers of the secretory cells and basal cells were also estimated. Following the estimation of the average size of the seminal vesicle, the relative volume of different components and the relative number of secretory cells were transformed into absolute data on a per average seminal vesicle basis. Similarly, the average sizes of the secretory cells and nuclei were also determined. The quantitative data generated from the present study will serve as a baseline for further studies of the seminal vesicle of the guinea pig. The techniques used in the present study are easy to apply, and data generated were objective and reproducible.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.     

Immunoelectron microscopic evidence for different compartments in the secretory vacuoles of the rat seminal vesicles

Summary Immunoelectron microscopy of the rat seminal vesicle was performed using specific antibodies to secretory proteins. Proteins were precipitated from rat seminal vesicle secretion and were separated by SDS—polyacrylamide gel electrophoresis. Among the great number of bands the two most prominent bands were selected and designated SVS II and IV. Their apparent molecular weights were 48 kDa and 16.5 kDa respectively. The bands were excised from the gels and used for antibody production in rabbits. The respective antisera were used for immunohistochemical studies both at the light and electron microscopic levels in the rat seminal vesicle and the different prostatic lobes in infantile, adult and castrated animals. A positive immunoreaction was observed in seminal vesicle and lateral prostatic epithelium of the intact adult rat, while it was lacking in prepubertal and castrated animals. The subcellular distribution of both proteins was clearly different: SVS II was exclusively confined to the electron dense core of the secretory vacuoles, while SVS IV was detected only in the clear halo surrounding the central granule. It is suggested that the spatial arrangement of both proteins in the seminal vesicle secretion vacuole reflects a particular functional significance of each of these proteins. These proteins may serve as a tool in the study of regulation of androgendependent protein synthesis.     

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.     

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.     

Sequencing of the ovine interleukin-8-encoding cDNA using the polymerase chain reaction

The nucleotide (nt) sequence encoding the ovine homologue of interleukin-8 (IL-8) was determined. The mRNA is 1494-nt long with an ORF of 101 codons. The long 3' non-coding element contains several ATTTA repeats implicated in the swift turnover of other chemokine mRNAs. The encoded protein of 11 kDa before processing, and 9 kDa as mature protein, contains the Cys-Xaa-Cys motif common to -chemokines, and has conserved amino acids (aa) at positions identified as receptor contact sites for IL-8. Identities with other published IL-8 aa sequences are: dog, 91%; pig, 87%; rabbit, 84%; human, 78%; guinea pig, 69%. A 49% aa identity is also found with a chicken embryo fibroblast protein.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.     

Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.