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Nuclear envelope of the seminal-vesicle epithelium.

ABSTRACT: The nuclear envelope of seminal-vesicle epithelium was isolated by a procedure involving enzymic digestion with deoxyribonuclease I, sonication in the presence of 0.34 M-sodium citrate, and centrifugation through sucrose density gradients. The mass of envelope DNA was only 0.8% of that of envelope protein, and by transmission electron microscopy the envelope was 98-99% pure. We showed that the envelope possess a protein kinase activity which is uninfluenced by cyclic nucleotides. Both lysine-rich histone and dephosphophosvitin as substrates gave a greater specific activity than did envelope protein itself. Optimum requirements with respect to Na+, Mg2+, pH and ATP were established for each substrate, and the influence of other factors on enzyme activity was investigated. Data, obtained mainly with the use of lysine-rich histone, are presented which indicate that nuclear envelope from intact and 96 h-castrated guinea pigs may have equal protein kinase activities and, in separate experiments, equal phosphoprotein phosphatase activities. Clarification of these initial observations must await identification of the natural substrates or the envelope's phosphorylation-dephosphorylation reactions.

SUBMITTER: Veneziale CM

PROVIDER: S-EPMC1163243 | biostudies-other | 1981 Aug

REPOSITORIES: biostudies-other

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Project description:Seminal vesicle autoantigen (SVA) is a 19 kDa glycoprotein purified from mouse seminal vesicle secretion. It was quantified to be 0.9% (w/v) in the seminal vesicle fluid. We examined its distribution in the accessory sexual gland, characterized its binding sites on the sperm surface and assessed its effect on sperm motility. It was immunolocalized on the epithelium of the primary and secondary folds in the tissue. Mouse spermatozoa collected from caudal epididymis were devoid of SVA. A cytochemical study illustrated the presence of SVA-binding region on the entire cells. The cytochemical staining intensity for the binding of SVA to spermatozoa remained even when the cells were pretreated with protease digestion, acid or heat at 100 degrees C for 10 min. Moreover, the SVA-sperm binding could be inhibited by the dispersed sperm lipid. The specificity of interaction between (125)I-SVA and phospholipids was studied by TLC overlay techniques. The radiolabelled protein showed strong binding to purified phosphatidylcholine and phosphatidylserine and weak binding to purified sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine, but did not interact with phosphatidic acid, lysophosphatidic acid or phosphatidylinositol. Among the lipids extracted from spermatozoa, SVA showed strong binding to phosphatidylcholine and weak binding to sphingomyelin and neutral lipids. The assay for SVA-sperm binding with (125)I-SVA determined the IC(50) as being (3.89+/-0.65)x10(-5) M(-1), which is compatible with an apparent dissociation constant of (9.10+/-0.02)x10(-5) M(-1) estimated by fitting the data of phosphatidylcholine-perturbed SVA fluorescence to a modified Scatchard plot. SVA showed an ability to suppress sperm motility. The average path velocity, straight-line velocity and curvilinear velocity of sperm were not detectable by computer-assisted sperm assay after incubation of the cells in the presence of 0.3% SVA at 37 degrees C for more than 40 min.

Project description:Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [(14)C]glycine and [(14)C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99-100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3alpha-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. ;non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.

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Nuclear envelope of the seminal-vesicle epithelium.

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