Testosterone (T) levels in the plasma of male laboratory rats and mice were measured by radioimmunoassay. There was a striking individual variation with values ranging from less than 1 ng/ml to over 30 ng/ml in mice of the same age and strain housed under identical conditions. Using chronic indwelling catheters inserted into a jugular vein, blood was collected from adult conscious male rats every 24 hr for 4 or 8 days and every 30 min for 2 l / 2 or 8 hr. Considerable differences in plasma T levels were observed between different animals, and 2-to S-fold fluctuations of T concentrations in the plasma were detected between samples collected from the same animal at different times. These large and irregular changes in plasma levels of T are unlike the fairly stable levels observed in the human but bear certain resemblance to the pulsatile release of T described in bulls and rams and perhaps also to the social dominance related differences in plasma T levels in Rhesus monkeys. {Endocrinology 92: 1223, 1973
We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) Corticosteroid binding globulin (CBG) is the major transport protein for glucocorticoids in the blood of almost all vertebrate species (1), and >90% of the cortisol in human plasma is bound by this protein (2). The remaining fraction is distributed more evenly between albumin and the pool of nonprotein-bound or "free" steroid that is generally assumed to be biologically active (2, 3). In humans, CBG is an acidic, -58-kDa glycoprotein (4-6) comprising five N-linked oligosaccharide chains (7) that collectively represent -23% of the molecule by mass (6, 7). The binding site for natural glucocorticoids appears to be a hydrophobic pocket containing one of two cysteine residues that have been identified by amino acid composition analyses (8)(9)(10)(11). Apart from this information, and the identification of eight residues at the NH2 terminus of human CBG (5, 11), there is virtually no information about its primary structure or the location of its steroid binding site.Like many other plasma transport proteins, CBG is produced and secreted by hepatocytes (12), but has also been identified in a number of glucocorticoid responsive cells (2, 13), and may even interact directly with the plasma membranes of some cells (14,15). The objectives of this study were, therefore, to predict the amino acid sequence of human CBG from a cDNA and to determine whether tissues other than the liver possess the capacity to produce this protein. § METHODS cDNA Cloning. A monospecific rabbit antiserum for human CBG (6) was initially used to screen a Xgtll human liver cDNA library that was kindly provided by S. L. C. Woo (Baylor College of Medicine, Houston). The screening method was based on the technique described by Young and Davis (16), with the exception that peroxidase-labeled protein A was used to detect antibody-antigen complexes in the presence of the chromogenic substrate 4-chloro-1-naphthol. The recombinant phage isolated in this way were used to prepare plate lysates using NZC top agar (GIBCO). The phage were harvested and purified, and the cDNA inserts were excised and inserted into the EcoRI site of pBR322 according to Maniatis et al. (17). Plasmids containing CBG cDNAs were used to transform competent Escherichia coli (strain MM 294), and transformants were propagated in Luria broth in the presence of ampicillin and chloramphenicol to amplify the plasmid (17). Plasmids were isolated by the alkaline lysis method and purified using benzoylated-naphthoylated-DEAE cellulose (Sigma) according to Gamper et al. (18). The cDNAs were routinely excised from the plasmid and purified by polyacrylamide gel electrophoresis, prior to nick-translation with 32P-labeled dCTP (17).In an attempt to isolate a full-length CBG cDNA, the radiolabeled cDNA was employed to rescreen the library. Nitrocellulose filters (Schleicher & Schuell; BA85, 0.45-,um pore size) were used to transfer DNA and were hybridized with 2 x 106 dpm of the CBG cDNA probe per ml, in the pr...
The distribution of the androgen receptor (AR) in the adult rat testis was determined by biotin-streptavidin immunoperoxidase, employing tissue embedded in polyester wax which preserves antigenicity without compromising tissue preservation. The antibody probe used, which has been characterized previously, was an affinity purified, rabbit polyclonal antibody raised to the amino terminus peptide of the rat AR. Within the interstitial compartment, AR immunostaining was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells of blood vessels were negative. Furthermore, in those Leydig cells that were clearly identified as exhibiting AR immunostaining, the intensity of the reaction varied. In the seminiferous tubules AR immunostaining was observed in all peritubular myoid cell nuclei, but not in the distal layer of lymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific. Moderate AR immunostaining first became evident at late stage IV or early stage V of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, the spermatids in which nuclear elongation is apparent but chromatin condensation has not yet begun. Next, with onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm of the germ cells. These results are interpreted in the following manner: 1) The presence of AR in Leydig cells is consistent with the hypothesis that androgens modify Leydig cell activity in an autocrine fashion. Further, that not all Leydig cells exhibited AR immunostaining at steady state suggests a differential, functional activity of these cells within the population. 2) The intense AR immunostaining of smooth muscle cells present in the interstitium indicates that these cells are targets for androgens. 3) AR immunoreactivity in both Sertoli and peritubular myoid cells suggests their involvement in the androgenic control of spermatogenesis. The stage specific AR immunoreactivity in Sertoli cells, however, may be more indicative of a specific androgen response during these stages, whereas peritubular cells may participate in the tonal maintenance of spermatogenesis. 4) The specific presence of AR in step 11 elongated spermatids may suggest that androgens can act directly on germ cells to regulate spermatogenesis.
We have sequenced a cDNA for sex hormone-binding globulin (SHBG) isolated from a phage 2gtl 1 human liver cDNA library. The library was screened with a radiolabeled rat androgen-binding protein (ABP) cDNA, and the abundance of SHBG cDNAs was 1 in 750 000 plaques examined. The largest human SHBG cDNA (1194 base-pairs) contained a reading frame for 381 amino acids. This comprised 8 amino acids of a signal peptide followed by 373 residues starting with the known NH2-terminal sequence of human SHBG, and ending with a termination codon. The predicted polypeptide Mr of SHBG is 40 509, and sites of attachment of one O-linked (residue 7) and two N-linked oligosaccharide (residues 351 and 367) chains were identified. Purified SHBG was photoaffinity-labeled with zt6-[3H]testosterone and cleaved with trypsin. The labeled tryptic fragment was isolated by reverse-phase HPLC, and its NH2-terminal sequence was determined. The results suggest that a portion of the steroid-binding domain of SHBG is located between residue 296 and the 35 predominantly hydrophilic residues at the C-terminus of the protein.cDNA cloning; Amino acid sequence; Sex hormone-binding globulin; Steroid-binding domain
The localization of androgen-binding protein (ABP) in the reproductive tract of young adult male rats was studied with the peroxidase-antiperoxidase technique using frozen sections and light microscopy. Within the seminiferous tubules, a positive reaction was noted in the apical portion of the epithelium, apparently in spermatids and/or Sertoli cells. ABP was localized in granules in the apical cytoplasm of the principal epithelial cells of the proximal part of the caput epididymis and in the epithelial cells of the ductuli efferentes. The cells in the distal part of the caput as well as the corpus and cauda of the epididymis did not contain ABP. Numerous coated vesicles and multivesicular bodies were present in the supranuclear cytoplasm of the epididymal epithelium where ABP was taken up. The results indicate that ABP is taken up from the lumen by epithelial cells of the ductuli efferentes and proximal part of the caput epididymis.
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