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.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

Polymorphism of rat seminal vesicle secretory proteins: Characterization of svp-1 and svp-2 and their identification with the major secretory proteins IV and V

The proteins secreted by the rat seminal vesicle can be separated into five major fractions (namely, RSV-I through V) by gel electrophoresis in denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and RSV-V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant of RSV-IV that can be expressed only in rats homozygous for a given allele at the svp-2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology of RSV-IV and RSV-V and of their corresponding gene.This work was supported by a CNR grant from Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie genetiche.     

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.     

Alanyl aminopeptidase of bovine seminal vesicle secretion

Multiple forms of an aminopeptidase hydrolysing L-alanine- and various other amino acid-beta-naphthylamides in bovine seminal vesicle secretion were studied after fractionation on gel filtration, anion exchange chromatography and chromatofocusing. Two forms of the enzyme were found in all these fractionations: one with a high molecular weight was aggregated or particle-bound and the other had a molecular weight of about 237,000. The high-molecular-weight form dissociated with Triton X-100 via an intermediate into the basic enzyme form with concurrent change in the pI and anionic sites. The basic form of the enzyme differed from the high-molecular-weight forms in substrate preference, response to some modifiers, thermal stability and kinetic constants.     

Phosphorylation of sperm-binding proteins from seminal vesicle secretion by sperm surface protein kinase

Rat seminal vesicle secretion does not demonstrate protein kinase activity, either towards endogenous or exogenous proteins. When epididymal sperm were incubated in vitro with seminal vesicle secretion, three prominent secretory proteins were bound to the sperm. Two of these proteins were highly phosphorylated. Thus, selected sperm-binding proteins from accessory gland secretions are phosphorylated by sperm surface protein kinases.     

Effect of antibodies against seminal vesicle secretion on fertility in the rat.

Fertile female Wistar rats were immunised against rat and mouse seminal vesicle secretion (SVS) to test the production of allo-antibodies and the effect of the antibodies elicited on fertility. Twenty-six per cent of the rat and mouse SVS-immunised females were infertile after the treatment. The sera were titrated by ELISA and used in Western blots to detect the proteins recognised. Although neither the antibody titres nor the proteins recognised by the sera showed a close relation with the degree of fertility, in all females the highest antibody titre in the fluids from the genital tract was found in the oviductal fluid and during the night of oestrus. This fact suggested that the site of action of the antibody could be the oviduct. Similar results were obtained using mouse SVS as immunogen--a fact that can be related to the antigenic similarity between the SVS of the two species. The antibodies react with the spermatozoa but not with eggs or embryos. Analyses performed on embryos collected from sterile females showed that there was a delay in fertilisation and normal embryogenesis. Our results suggest that SVS proteins are antigenic and that these antigens are bound to the spermatozoa and could take part in early pre-fertilisation events such as capacitation or sperm transport.     

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.     

A flavoprotein responsible for the intense sulfhydryl oxidase activity of rat seminal vesicle secretion.

A flavoprotein isolated in substantial yields from rat seminal vesicle secretion accounts for most if not all of the capacity of this fluid to catalyze the aerobic oxidation of a number of low molecular weight thiol compounds. The nature and possible physiological significance of this enzyme are discussed.     

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.     

Constitutive protein secretion by guinea-pig seminal vesicle epithelial cells.

1. Secretion of pulse-labelled protein by the isolated epithelium of guinea-pig seminal vesicle epithelium was rapid, unaffected by cholinergic and adrenergic drugs, cyclic nucleotides or changes in the sodium, potassium and calcium concentrations of the "chase" medium. 2. Low temperature, NH4Cl, hyper- and hypo-osmolarity and membrane-stabilizing agents inhibited secretion which was also dependent on aerobic metabolism. 3. Monensin reduced secretion of the six labelled, relatively low molecular weight proteins recovered from the medium in a concentration-dependent, apparently non-specific manner.     

Purification and characterization of a trypsin inhibitor from mouse seminal vesicle secretion

A Kazal-type trypsin inhibitor in mouse seminal vesicle secretion was purified to homogeneity via a series of purification steps including ammonium sulfate fractionation, affinity chromatography on a trypsin Affi-Gel 10 column, and HPLC on a reverse phase C4 column. It was shown to be a weak basic protein with an isoelectric point of 8.7 and to contain no carbohydrate. The protein had a specific activity of 184 U/mg protein in the inhibitory effect on the trypsin digestion of N-benzoyl-Pro-Phe-Arg-p-nitroanilide. Analysis of the kinetic data for the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin revealed that the protein was a competitive inhibitor with an inhibitory constant (Ki) of 0.15 nM. The molecular mass of the protein was determined to be 7 kDa by both gel chromatography and electrophoresis. Results of direct amino acid determinations indicated that this protein corresponded to the reading frame of MP12 cDNA identified from mouse prostate. We found that cleavage only at the reactive site of this protein (Arg19-Ile20) resulted in its denaturation.     

Seminal vesicle production and secretion of growth hormone into seminal fluid.

Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.