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.     

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.     

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.     

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.     

Localization,metabolism, and binding of estrogens in the male rat

Estrogen assimilation by male Wistar rats was examined in these studies in several accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) as well as in a variety of nonaccessory sex organs. When [³H]estradiol was injected into intact 3- to 4-month-old rats in a pulse dose, no selective accumulation of radioactivity recovered as estradiol was found in the accessory sex glands when compared to other organs. This was due at least in part to the metabolism of estradiol to estrone and to the relatively low concentration of high affinity estrophilic molecules in the accessory sex organs. The order for the rate of formation of estrone from estradiol in tissues obtained from intact animals was ventral prostate > lateral and dorsal prostate > anterior prostate and seminal vesicles. Steroid specificity studies for cytosol estradiol binding by the ventral prostate and seminal vesicles revealed that estrophilic molecules exist in these organs. Based on Scatchard plot analyses in 24-h castrates, the number of available estradiol binding sites was too low in the ventral prostate to quantify accurately, but the seminal vesicles contained distinctly more estrophilic activity than the ventral prostate. The affinity for the seminal vesicle cytosol estradiol-estrophile binding exceeded that quantified for the seminal vesicle dihydrotestosterone-androphile reaction while the number of estradiol binding sites was less than that quantified for dihydrotestosterone. In relation to the accessory sex organs of other species, the rat seminal vesicles have a relatively small amount of cytosol estrophile. The findings that the seminal vesicles catabolize less estradiol and contain significantly more estrophilic activity than the ventral prostate is consistent with and offers insight into the noted estrogenic sensitivity of the seminal vesicles and lack thereof in the rat ventral prostate. With aging of the rat from 3–4 months to 22–26 months, the affinity of the seminal vesicle estradiol-estrophile interaction was unchanged but the number of binding sites increased significantly.