Isolation and culture of rat seminal vesicle epithelial cells : The use of the secretory protein SVS IV as a functional probe

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.     

Analysis of the major large polypeptides of rat seminal vesicle secretion: SVS I, II, and III

Rat seminal vesicle secretion (SVS) contains a variety of protein complexes that seem to be linked by interchain disulfide bonds. Upon reduction and analysis by sodium dodecyl sulfate (SDS) gel electrophoresis, this pattern resolves to 3 major high molecular weight (SVS I-100,000, SVS II-50,000, SVS III-37,000) and 3 major low molecular weight protein bands (SVS IV, V, and VI). A two-dimensional SDS gel (1st dimension unreduced, 2nd dimension reduced) permitted identification of the components of the cross-linked species. In the native secretion, SVS I forms a series of oligomers that include both SVS II and III. Essentially all of SVS III is involved in these complexes, while the bulk of SVS II occurs instead as an apparent homodimer. The smaller proteins (SVS IV-VI) are not involved in covalently crosslinked complexes. The reduced forms of the larger polypeptides were isolated by a variety of procedures involving agarose gel filtration in 6M guanidine hydrochloride, reversed-phase high pressure liquid chromatography, ammonium sulfate fractionation, and preparative polyacrylamide gel electrophoresis. Based on its size, solubility, and amino acid composition, SVS II was identified as the major clottable protein of the secretion.     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Neonatal seminal vesicle mesenchyme induces a new morphological and functional phenotype in the epithelia of adult ureter and ductus deferens

Mesenchyme from neonatal mouse and rat seminal vesicles (SVM) was grown in association with postnatal (adult) epithelial cells from the ureter (URE) and ductus deferens (DDE) in chimeric tissue recombinants composed of mouse mesenchyme and rat epithelium or vice versa. Functional cytodifferentiation was examined in these SVM + URE and SVM + DDE tissue recombinants with antibodies against major androgen-dependent seminal-vesicle-specific secretory proteins. Adult DDE and URE were induced to express seminal cytodifferentiation and produced the complete spectrum of major seminal vesicle secretory (SVS) proteins. The SVS proteins produced were specific for the species that provided the epithelium. In the case of SVM + URE recombinants, the URE, which normally lacks androgen receptors (AR), expressed AR. These results demonstrate that adult epithelial cells retain a developmental plasticity equivalent to their undifferentiated fetal counterparts and are capable of being reprogrammed to express a completely new morphological, biochemical and functional phenotype.     

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.     

cDNA cloning of androgen-stimulated mRNAs in rat seminal vesicles: partial characterization of newly isolated cDNA clones, pSv-1 and pSv-2

As the first step in surveying the molecular mechanism of androgen-responsive gene expression in rat seminal vesicles, the effect of androgen on the mRNAs was examined by in vitro translation assay. When the in vitro translation products of mRNAs from castrated animals (48 h) were compared with those from castrated and testosterone-treated animals (48 h) by SDS-PAGE, several discrete bands which were stimulated or repressed in response to androgen were observed in addition to major peptide bands of SVS IV and SVS V. From these findings, we constructed a partial cDNA library from the seminal vesicle poly(A +)RNAs of androgen-treated rats and screened by differential colony hybridization. Two distinct cDNA clones, pSv-1 and pSv-2, whose mRNAs were differentially stimulated in response to androgen and seemed to be expressed specifically in the seminal vesicles, were isolated. pSv-1 and pSv-2 hybridized to mRNAs of 1,600 and 3,500 nucleotides in length, respectively. These cDNA sequences, newly isolated in the present study, may provide useful probes for the study of molecular mechanism of androgen-responsive gene expression.     

Isolation of recombinant plasmids containing structural gene sequences for rat seminal vesicle secretory proteins IV and V

Double stranded cDNA was made to partially purified mRNA for the small seminal vesicle secretory proteins IV and V. This ds cDNA was then inserted into the Pst 1 site of pBR322 by the (G-C) homopolymer tailing technique. Bacterial transformants harboring plasmids with specific inserts were identified by translation of mRNA that was hybridized to plasmid DNA immobilized on nitrocellulose. Separate plasmids were obtained with cDNA inserts for both SVS IV and V. Neither hybridization results nor preliminary restriction analysis gave any indication for homology between them.     

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.     

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.     

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.     

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.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland

Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.     

Phospholipases A2 in the reproductive system of the bull

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.     

Neonatal exposure to oestrogens alters the protein profiles and gene expression in the genital tract of adult male mice.

After neonatal administration of supraphysiological doses of oestradiol, the concentration of tissue proteins, in adult mice, was significantly reduced by 39, 45 and 56% in epididymis, vas deferens and seminal vesicle respectively. The protein profiles showed persistent alterations. In epididymis, 4 protein bands were differentially increased (14.4, 43 and 67 kDa) or reduced (24 kDa) in oestrogenized males. In vas deferens, 4 proteins were increased (14.4, 49,67 and 76 kDa) and one (34 kDa) virtually absent. In seminal vesicle, about 20 proteins of varying molecular weights (12-140 kDa) were differentially increased or decreased. Testosterone substitution, at adulthood, was unable to reverse these effects. Treatments with oestradiol during adult life induced persistent alterations in the protein profiles of the 3 organs but, in contrast to neonatal treatment, these alterations could be reversed by androgen therapy. A cDNA library has been constructed with RNA prepared from adult seminal vesicle and screened by differential hybridization. Neonatal oestrogenization strongly reduced the abundance of some mRNA species. Eleven recombinants containing putative oestrogen-sensitive sequences were isolated. Two of them, having an insert of about 500 base pairs, were used for dot-blot hybridization. Results showed that the two clones contained sequences which were differently regulated by androgens.