Previous studies demonstrated that the polypeptide diazepam binding inhibitor (DBI) and its receptor, the peripheral-type benzodiazepine receptor (PBR), are involved in the regulation of steroid biosynthesis and that one site of PBR action resides in mitochondria. In the present investigation, evidence is presented that a functional form of PBR is also present at the cell surface. First, PBR was immunolocalized in the rat testis using biotin-streptavidin peroxidase immunocytochemistry, and results revealed that PBR was present exclusively in the interstitial Leydig cells. Next, the distribution of PBR in MA-10 Leydig cells was further examined using confocal microscopy. MA-10 cells were either fixed and immunostained or fixed/permeabilized and immunostained for PBR, followed by generation of confocal microscope optical sections, three-dimensional reconstructions of these sections, and then generation of vertical confocal sections of the three-dimensional reconstruction. In the fixed/unpermeabilized cells, PBR immunostaining at the cell surface was clearly evident, whereas in the fixed/permeabilized cells, intracellular PBR distribution was more robust. These results suggest that the plasma membrane fraction of the receptor could mediate the action of extracellular PBR ligands on Leydig cell function. Next, we examined whether DBI, the naturally occurring PBR ligand, is secreted by testicular cells and whether it could activate the cell surface PBR. Immunolocalization of DBI demonstrated that it was present in both Leydig and Sertoli cells. Further, using an immunoblot assay, we demonstrated that DBI is present in rat testicular interstitial fluid. Metabolic labeling of cultured immature rat Sertoli cells and MA-10 mouse tumor Leydig cells, followed by immunoprecipitation of the secreted proteins with an anti-DBI antiserum, demonstrated that both Leydig and Sertoli cells secrete DBI and could serve as a cell source for the interstitial fluid DBI. Then, we partially purified the DBI present in conditioned medium and interstitial fluid by reverse phase chromatography and demonstrated it to be bioactive, based on displacement of a radiolabeled benzodiazepine (Ro5-4864)-specific ligand for PBR; pronase treatment of different preparations eliminated all bioactivity. We then examined the effects of DBI on Leydig cell function. DBI added to MA-10 cells affected DNA synthesis and cell growth in a biphasic manner; at low concentrations (1 nM), DBI was mitogenic, increasing [3H]thymidine incorporation and cell numbers by 30-40%, while at high concentrations (1 microM), DBI inhibited cell growth (30-40%). Similar effects on cell growth were obtained using the benzodiazepine Ro5-4864.

No abstract

No abstract

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38 degrees C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule.(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract