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The genes for seminal vesicle secretory protein IV and prostate steroid binding protein component 3 of the rat share upstream homologies of which the most striking is a 30 nucleotide sequence located between position -190 and -330 relative to the major transcriptional initiation sites. This sequence does not appear to be a common repetitive element and deserves consideration as a potential site involved in the androgen regulated expression of these genes.

The amino acid sequence of the COOH-terminal cyanogen bromide fragment (residues 12 to 54) of the testis-specific basic protein of the rat has been determined. This analysis completes the primary structure of the whole protein by over-lapping the sequence of the 23 residues from the NH-2 terminus previously published (Kistler, W. S., Noyes, C., and Heinrikson, R.L. (1974) Biochem. Biophys. Res. Commun. 57, 341-347). The complete sequence of this small, highly basic protein is: (see article for formular).

The entire coding region for rat seminal vesicle secretory protein IV was obtained on a 3.5 kb Eco RI fragment isolated from a genomic library in lambda Charon 4A. The coding sequence for SVS IV message is interrupted twice by introns. The first lies just downstream from the juncture of the 21 amino acid secretory signal peptide with the start of the mature protein, and the second lies in the 3'-nontranslated region. The major transcriptional start site was mapped by primer extention and is 22 nucleotides upstream from the translational initiation codon. S1 protection experiments indicated additional minor transcriptional starts about 27 and 50 nucleotides further upstream from the major cap site. The entire transcriptional unit comprises about 1740 nucleotides. The SVS IV gene does not belong to an obvious gene family, and it is conserved in mice and guinea pigs.

The effects of castration on the synthesis (accumulation) of a major seminal vesicle secretory protein (SVS IV) were examined in young adult rats. In vitro incorporation of labeled amino acids into SVS IV by minced tissue was monitored by immunological methods. Castration resulted in a large decrease in the differential synthesis of SVS IV. A significant decrease in the relative incorporation of isotope into SVS IV was evident within 3 days of castration, and by 4 weeks relative incorporation dropped some 30-fold. These changes took place in the presence of a large generalized decline in protein synthesis so that incorporation into SVS IV on an organ basis decreased by over 200-fold. SVS IV messenger RNA levels were estimated by RNA excess solution hybridization using a cloned cDNA probe. Relative message levels declined after castration in harmony with the declines in SVS IV synthesis. SVS IV mRNA was decreased by a relative factor of approximately 20 and an absolute factor of approximately 200 in long-term (40-day) castrates. Accordingly, the seminal vesicle conforms to the general pattern of steroid regulated systems in which hormone withdrawal leads to differential decreases in the steady-state pool size for specific mRNAs. The seminal vesicle is unusual, however, in that a prolonged period is required for maximum differential effects to occur.

Seminal vesicle secretory protein IV (SVS IV) is perhaps the most abundant protein made by the epithelial cells of the rat seminal vesicle. In this report, we have asked whether the methylation of its gene correlates with its tissue-specific pattern of expression. Using methylation-sensitive restriction endonucleases HpaII and AvaI, we could examine seven potential methylation sites in or near the gene. All seven sites were largely unmethylated in the seminal vesicle, while in most nonexpressing organs, all sites were heavily modified. The correlation of demethylation with expression was broken by finding that both ventral prostate and coagulating gland (anterior prostate) DNA showed the same demethylation pattern seen for the seminal vesicle. SVS IV protein has not been reported in either of these two organs, and we could not detect mature SVS IV message in prostate RNA or SVS IV transcription by in vitro incubation of prostate nuclei. We conclude that demethylation of the SVS IV gene accompanies the differentiation of several androgen-dependent sex accessory glands but confirm that demethylation per se is not a sufficient signal to bring about gene transcription.

A very basic and acid-soluble small protein was isolated in the pure form from specimens of human testes that displayed histologic evidence of normal spermatogenesis. The protein is virtually indistinguishable in its electrophoretic properties from a testis-specific basic protein that has been previously described in a number of other eutherian mammalian species. The homogeneous human testicular basic protein is rich in arginine, lysine, and serine, and is devoid of cyst(e)ine, phenylalanine, glutamate, glutamine, isoleucine, and tryptophan. Its amino acid composition is extremely close to that of a testis-specific basic protein previously described in the rat. The basic protein from human testis is electrophoretically distinct from the principal basic chromosomal protein of human spermatozoa and could not be detected in ejaculated human spermatozoa by the procedure used to extract the chromosomal basic protein.

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.