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Run-on transcription in isolated nuclei has been used to study the effects of testosterone on gene expression in rat seminal vesicles. General transcriptional rates were increased by about 6-fold with an additional 2- to 3-fold differential stimulation of the genes for secretory proteins IV and V. These transcriptional changes are insufficient to explain overall changes in cellular mRNA levels, indicating that androgens must also have major effects on post-transcriptional processing of RNA transcripts or on mRNA stability. Analysis of nuclear RNA by Northern blotting with intron probes suggests substantial androgen effects on primary transcript processing.

Functional development of the ventral prostate and seminal vesicles in the male rat has been examined by incubating tissue with [35S] methionine and using specific antisera to detect the synthesis of individual marker proteins. 4 major secretory proteins of the seminal vesicles were chosen along with 4 major prostatic secretory peptides, including 3 which form the prostatic steroid-binding protein. None of the seminal vesicle proteins was synthesised before 25-30 days but all were then induced in parallel to reach adult values by 45-50 days. The 3 peptides of the prostatic steroid-binding protein were synthesised at the earliest age studied (10 days) and were also induced in parallel to maximal levels by 25-30 days. In contrast, the 4th prostatic marker, a glycopeptide, was not synthesised until about 25 days. Treatment of male rats from 3 days of age with testosterone induced precocious development of the seminal vesicles, causing substantial synthesis of marker proteins well before their synthesis would normally have been expected, and also advanced the synthesis of the prostatic 22 K glycopeptide. The results confirm that functional development in the ventral prostate precedes that of the seminal vesicles but that within the ventral prostate there is a considerable difference in the developmental profiles of its secretory proteins.

Epithelial cells from enzymically dispersed seminal vesicle tissue of castrated adult rats were grown on collagen-coated surfaces in serum-containing medium for up to 6 weeks. Androgens were neither required for growth nor were they mitogenic. They also failed to induce the synthesis of tissue-specific secretory proteins that serve as markers of androgen action in vivo. A variety of potential growth factors and culture procedures (such as feeder layers, extracellular matrices, collagen gels, and growth from tissue explants) were examined for their ability to support androgen action. Testosterone and 5 alpha-dihydrotestosterone were only able to induce seminal vesicle secretory protein synthesis in the presence of neurotransmitters (acetylcholine, noradrenaline, or serotonin). The mechanism of this permissive effect of neurotransmitters on androgenic induction has been examined. It is not related to their mitogenic activity since other mitogens (epidermal growth factor and corticosterone) did not facilitate androgen action. Agents that raise intracellular cAMP were also ineffective so cAMP is probably not involved, but the phosphatidyl inositol pathway may be important.

Proteins synthesised by seminal vesicles of normal rats were labelled with [35S]methionine and were then separated using two-dimensional gel electrophoresis. Isoelectric focusing or nonequilibrium pH gradient electrophoresis were used in the first dimension followed by polyacrylamide gel electrophoresis under denaturing conditions. Using antiserum to total seminal vesicle secretion and running the immuno-precipitated proteins on two-dimensional gels, secretory proteins were identified and shown to be much more complex than previously thought. Proteins synthesised by seminal vesicles from rats castrated 1-2 weeks before were also labelled with [35S]methionine and separated on two-dimensional gels. Comparison of the two-dimensional protein maps from normal and castrated animals showed that a substantial number of proteins were differentially induced or repressed by testosterone. Of the secretory proteins, some were clearly regulated in a highly differential manner but others appeared to be unaffected by castration. The results are discussed in relation to previous measurements of mRNA sequence complexity and show that previous conclusions derived from using less sophisticated methods are oversimplifications of the response.

Rat seminal vesicles serve as a model system for studying androgen action. We have sequenced and compared full length cDNAs for two major proteins (S and F) synthesised and secreted under hormonal control. Overall, mRNAS and mRNAF share 57% nucleotide sequence homology suggesting that their genes arose by duplication of a common ancestor. However, the mRNAs display a highly unusual regional distribution of sequence homology, with the untranslated regions (UTRs) being substantially more homologous than the protein-coding regions (PCRs). Detailed analysis of nucleotide substitutions at synonymous and replacement sites shows that the PCRs have evolved very rapidly. Evolutionary conservation of the UTRs is no higher than that of UTRs generally and thus provides no evidence of a specific regulatory role for the UTRs in androgen action. The primary sequences of proteins S and F have diverged so rapidly that they are the best examples of neutrally evolving proteins for which comparative nucleotide sequence data are available. However, despite their rapid divergence, the predicted higher order structures for both proteins consist largely of non-regular conformation. This is discussed in terms of their roles as structural components of the rodent copulatory plug.

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.

Androgenic steroids control the synthesis and secretion of several proteins by the seminal vesicles of the male rat. Prominent among them are 2 basic proteins, S and F, previously used as markers of androgen action. These proteins are not found among translation products of a wheat-germ protein-synthesising system supplied with mRNA from seminal vesicles of normal rats. Instead, higher molecular weight forms, S' and F', are formed which are nonetheless related antigenically to S and F respectively. Injected into Xenopus Laevis oocytes, seminal vesicle mRNA does direct synthesis and secretion of polypeptides S and F. Partial peptide analysis confirms that S' and F' have extensive amino acid sequence homology with S and F respectively. We conclude that S' and F' are precursor forms of the secreted proteins and thus at least 2 abundant proteins of this male accessory tissue are secreted by a mechanism common to a wide number of secreted proteins.

In a previous report [Higgins et al. (1976) Biochem. J.158, 271-282] we described the effects of alterations in androgen status on the synthesis of two basic secretory proteins of the rat seminal vesicle. In the present paper we examine the effects of testosterone on the activity of mRNA in the seminal vesicle. Total cellular poly(A)-rich RNA was isolated and translated in a cell-free system prepared from wheat germ. Translation products were separated on denaturing polyacrylamide gels and the protein bands corresponding to the two basic secretory proteins were identified immunologically. Incorporation of radioactive methionine into these bands was taken as a measure of the individual mRNA activities. Total mRNA activity was estimated by radioactivity in total acid-precipitable material. The results show that 1 to 2 weeks after castration the activities of mRNA molecules for the basic secretory proteins were decreased 10-20-fold on a tissue basis. Testosterone given in vivo rapidly and substantially restores mRNA activity to normal. Since these changes correlate closely with variations in the rates of synthesis of the secretory proteins in whole cells it suggests that androgenic steroids control protein synthesis chiefly via mRNA availability. In this respect their action resembles those of other steroid hormones acting in other systems. However, these effects of testosterone on the mRNA molecules for the major secretory proteins could not be distinguished from those on total mRNA. Thus the proportion of the total mRNA population accounted for by the two specific mRNA molecules showed less than a 2-fold variation with androgen status. Similarly the two secretory proteins always accounted for 25-33% of general protein synthesis. This is in sharp contrast with the markedly differential effects of other steroid hormones controlling synthesis of major proteins in other well-studied systems. We interpret our results as indicating that testosterone regulates the mRNA population of the seminal vesicle as a whole.