The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

The androgen-dependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning

Seminal vesicle secretory protein IV of a mouse has been isolated, and the cDNA coding for its mRNA has been cloned and sequenced. The 556-nucleotides encode 16 amino acid signal peptides and 92 residues of mature protein. Considerable homology between mouse and rat SVS IV cDNA was found. In the leader peptide and 3'-noncoding region there is 92% and 85% homology, respectively. The other regional homologies are 86% for the first 12, 68.5% for the last 35, and 40% for the middle 44 amino acids. The expression of mouse SVS IV mRNA is under the control of androgen. Administration of testosterone to castrated mice resulted in induction of the mRNA level to 50% of the mature male in 96 h of hormone treatment. Secretion of the protein after testosterone injection follows a similar pattern.     

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.     

Isolation and characterization of gelatin-binding bison seminal vesicle secretory proteins

Bovine seminal plasma (BSP) contains a family of major proteins designated BSP-A1/A2, BSP-A3, and BSP-30kDa (collectively called BSP proteins) that bind to sperm at ejaculation and potentiate sperm capacitation. Homologous proteins have been identified in stallion, boar, goat, and ram seminal plasma. We report here the isolation and characterization of homologous proteins from bison seminal vesicle secretions. Seminal vesicle secretory proteins were precipitated by adding cold ethanol and recovered by centrifugation. The precipitates were resuspended in ammonium bicarbonate, dialyzed, and lyophilized. Lyophilized proteins were dissolved in 0.05 M phosphate buffer (PB) and loaded onto a gelatin-agarose column. The unadsorbed proteins and adsorbed proteins were eluted with PB and 5 M urea in PB, respectively. The gelatin-adsorbed fraction was analyzed by SDS-PAGE and revealed the presence of four major proteins designated BiSV-16kDa, BiSV-17kDa, BiSV-18kDa, and BiSV-28kDa (BiSV: bison seminal vesicle proteins). Heparin-Sepharose chromatography allowed the separation of BiSV-16kDa, which did not bind heparin from other BiSV proteins, which bound heparin. Immunoblotting revealed that BiSV-16kDa cross-reacted with BSP-A3 antibodies, BiSV-17kDa and BiSV-18kDa cross-reacted with BSP-A1/-A2 antibodies, and BiSV-28kDa cross-reacted with BSP-30kDa antibodies. Radioimmunoassays indicated that approximately 25% of bison seminal vesicle total proteins are related to BSP proteins. The amino-terminal sequencing indicated that BiSV proteins share almost 100% sequence identity with BSP proteins. In addition, BiSV proteins bind to low-density lipoproteins isolated from hen's egg yolk. These results confirm that BSP protein homologs are present in mammalian seminal plasma and they may share the same biological role.     

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.     

Immunofluorescent localization of a murine seminal vesicle proteinase inhibitor

The indirect immunofluorescent technique was used to localize a proteinase inhibitor isolated from murine seminal vesicles. The inhibitor was found in the lumen and in the apical epithelium of the seminal vesicle but not in the testes, epididymides, ductus deferens or Cowper's glands. It was also associated with the anterior acrosomal region of ejaculated sperm and sperm recovered from the female tract within 5 min of coitus. The inhibitor is removed from uterine sperm between 2 to 4 h postcoitus, however sperm recovered from the uterus 2 h postcoitus will rebind inhibitor. The inhibitor is not normally associated with epididymal or ductus sperm although these sperm will bind purified inhibitor in vitro.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Autophagy in the epithelial cells of murine seminal vesicle in vitro

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.     

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 (Kd = 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.     

A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa

The rat seminal vesicle produces large amounts of a protein-rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm-coating antigen. We report in this paper that high-molecular-weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm-bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high-molecular-weight, sperm-coating antigen.     

Abundance of intrinsic disorder in SV-IV, a multifunctional androgen-dependent protein secreted from rat seminal vesicle

The potent immunomodulatory, anti-inflammatory and procoagulant properties of protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have previously been found to be modulated by a supramolecular monomer-trimer equilibrium. More structural details that integrate experimental data into a predictive framework have recently been reported. Unfortunately, homology modelling and fold-recognition strategies were not successful in creating a theoretical model of the structural organization of SV-IV. It was inferred that the global structure of SV-IV is not similar to that of any protein of known three-dimensional structure. Reversing the classical approach to the sequence-structure-function paradigm, in this paper we report novel information obtained by comparing the physicochemical parameters of SV-IV with two datasets composed of intrinsically unfolded and ideally globular proteins. In addition, we analyse the SV-IV sequence by several publicly available disorder-oriented predictors. Overall, disorder predictions and a re-examination of existing experimental data strongly suggest that SV-IV needs large plasticity to efficiently interact with the different targets that characterize its multifaceted biological function, and should therefore be better classified as an intrinsically disordered protein.     

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.     

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.     

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.     

Calcium-binding protein in bull seminal vesicle secretion and seminal plasma.

A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.