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Germ cells contain androgen receptor (AR). Germ cells do not contain androgen receptor. These diametrically opposed views are at the center of a long-running dispute regarding the cell site of androgen action within the testis (reviewed in 1, 2). In this chapter we examine AR distribution in the rat and mouse testis using an affinity-purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR (3–5). Results reveal the presence of AR in testicular somatic cells, as well as in elongated spermatids.

The peripheral-type benzodiazepine receptor (PBR) was identified and characterized by its high affinity for two distinct classes of compounds, the benzodiazepines (BZs) and the isoquinolines (IQs). An M(r) 18,000 IQ-binding protein has been identified as the PBR. In this report we isolated and sequenced a 626-base pair cDNA, specifying an open reading frame of 169 amino acid residues with a predicted molecular weight of 18,843, from MA-10 mouse tumor Leydig cells [i.e., mouse peripheral-type benzodiazepine receptor (mPBR)]. Expression of mPBR cDNA in simian virus 40-transformed 3T3 fibroblasts resulted in an increase in the density of both BZ and IQ binding sites. To examine whether the increased drug binding was due to the M(r) 18,000 PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant mPBR protein. The mPBR cDNA was inserted in the pMAL-c2 vector downstream from the malE gene, which encodes maltose-binding protein (MBP). Transfection of the recombinant pMAL-c2 in Escherichia coli provided high levels of expression of the MBP-mPBR fusion protein. Purified MBP-mPBR recombinant fusion protein incorporated into liposomes, but not MBP alone, was able to bind IQs but not BZs. Addition of MA-10 mitochondrial extracts to the liposomes resulted in the restoration of BZ binding. The protein responsible for this effect was then purified and identified as the M(r) 34,000 voltage-dependent anion channel protein, which by itself does not express any BZ and IQ binding. These results provide strong evidence that PBR is not a single protein receptor but a multimeric complex in which the IQ binding site is on the M(r) 18,000 subunit and expression of the BZ binding site requires both the M(r) 18,000 and 34,000 voltage-dependent anion channel subunits.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.

Steroid binding proteins bind steroid hormones with high affinity and their function is to carry those hormones in the extracellular compartment. Since their discovery more than fifty years ago, many reports concerning their physicochemical structures and functions have contributed to the better understanding of those proteins. Recent advances in recombinant DNA technology have led to the availability of molecular probes for these proteins, and new approaches have been used to analyse their gene structures as well as the regulation of their synthesis. In the present report, we will review the new findings of the last five years which include the cloning and sequencing of the cDNAs and genes for corticosteroid binding globulin, testosterone estradiol binding globulin and androgen binding protein, as well as the tissue distribution and regulation of their mRNAs in normal tissues and cancer cell lines.

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen-binding protein (ABP) isolated from rat epididymides by androgen affinity chromatography is a dimer with a native molecular weight of 85,000. When fractionated on sodium dodecyl sulfate gels two components were identified which were present in a 3:1 ratio; these were designated heavy (H) and light (L) (Mr of 45,000 and 41,000), respectively. The fact that H and L both have steroid binding sites and that they can be cross-linked suggests that both are protomers of native ABP. NH2-terminal analysis, amino acid analysis, and peptide maps suggest an extensive homology between H and L. In addition, peptide maps of H and L photolabeled with delta 6-[3H]testosterone suggest that the binding sites on these protomers are identical. ABP was also purified 33,800-fold from testes. The H and L protomers from this preparation were identical with those from the epididymides as judged by peptide maps. In addition, [35S]methionine was incorporated into the H and L synthesized by Sertoli cells in the same 3:1 ratio as in highly purified ABP from testis and epididymis. These observations suggest that L is not derived from H following secretion. Selective incorporation of [3H] fucose into the H protomer suggests that differences in carbohydrate composition account for at least part of the difference between H and L. These results support the conclusion that native ABP is composed of two kinds of protomers which exist in a ratio of 3:1. These protomers are similar polypeptides that differ partly in carbohydrate composition.

Testosterone estradiol-binding globulin (rbTeBG) of rabbit plasma was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5 alpha-dihydrotestosterone-Sepharose, reactive blue-Sepharose, and finally preparative polyacrylamide gel electrophoresis. An overall purification of greater than 3000-fold was achieved with a recovery of 32%. This purified material was shown to possess the following physical characteristics: an equilibrium dissociation constant at 4 degrees C for dihydrotestosterone estimated to be 0.59 X 10(-9) M and a half-time of dissociation of rbTeBG-dihydrotestosterone complex of approximately 9 min at 4 degrees C. The purified protein shows considerable microheterogeneity with regard to size on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and to charge on isoelectric focusing gels. Analytical gel electrophoresis in the presence of SDS revealed two molecular weight components of 41,000 and 45,000. Size analysis on native gels and cross-linking studies yields a molecular weight of 101,600 and 83,000, respectively. These size components were shown to be very closely related with regard to primary structure. rbTeBG was completely absorbed by concanavalin A Sepharose resin, demonstrating the glycoprotein nature of rbTeBG. Highly purified rbTeBG and unpurified rbTeBG from rabbit plasma could be photolabeled with [3H]delta 6-testosterone and when analyzed on SDS-polyacrylamide gel electrophoresis (PAGE), two major bands of radioactivity with molecular weights of 41,000 and 45,000 appeared in both samples.

Androgen binding protein (ABP) was purified from rat epididymides by sequential ammonium sulfate precipitation, affinity chromatography, gel filtration, and DEAE ion exchange chromatography. The column matrix formed by coupling dihydrotestosterone-17 alpha-(hexanoic acidY to agarose via diisopropylamine was stable during the extensive washing required following application of crude tissue extracts to the affinity matrix. In addition, when used under the optimal conditions, the column produced a 1600-fold purified in a single step. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis, sedimentation equilibrium, and constant specific activity across the peak of the final chromatograph. The molecular weight determined by sedimentation equilibrium at pH 7.4 was 85 000. By contrast, the molecular weight determined by sedimentation equilibrium in guanidine.HCl and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately one-half that of the native protein, suggesting that suggesting that ABP is comprised of subunits.

Sertoli cells synthesize and secrete a number of cell-specific products including androgen binding protein (ABP), as well as "serum proteins" such as transferrin. The secretion of these proteins is regulated by extra-testicular hormones such as FSH and insulin; Leydig cell-produced steroids and proopiomelano-cortin-derived peptides; and the presence of peritubular myoid cells and/or factors secreted by these cells. Many of the Sertoli cell proteins are secreted in a cyclic fashion during the different stages of the spermatogenic cycle suggesting communication between Sertoli cells and developing germ cells. The availability of quantitative measurements for Sertoli cell-specific proteins such as ABP make it feasible to follow Sertoli cell function in vivo by measuring these products in serum. A bidirectional secretion of proteins by Sertoli cells is proposed to explain the presence of specific peptides in the male reproductive tract and blood.

Male rats homozygous for the growth and reproduction complex (grc), which is closely linked to the major histocompatibility complex, are sterile due to a uniform arrest of spermatogenesis after formation of primary spermatocytes. The present study was conducted to determine whether grc influenced the secretion of the Sertoli cell product androgen-binding protein (rABP) in two F2 hybrid crosses. One was an F2 hybrid of the R10 and ACP strains and the other was an F2 hybrid population derived from the R16 and BI strains. These crosses were chosen because they allowed examination of the effects of grc on rABP secretion when this gene complex was associated with two different major histocompatibility complex backgrounds. In both crosses the rABP content of testes and epididymides from rats homozygous for grc were markedly reduced whereas heterozygotes were normal. By contrast, the concentration of rABP in plasma was normal or near normal at all ages. These observations are compatible with the hypothesis that grc influences the secretion of rABP from the apical portion of Sertoli cells into the tubular lumen and its subsequent transport to the epididymis, but has little or no effect on rABP secretion from the base of Sertoli cells into the blood. In other studies we have shown that infertile animals that have received prenatal x-ray or that are heterozygous for the Hre gene have patterns of rABP secretion similar to that of grc/grc homozygotes. It is therefore possible that the abnormal rABP secretion into tubular lumen in these animals is secondary to germ cell depletion, which is a factor common to grc/grc, Hre/+, and x-ray-treated rats.

Testosterone estradiol-binding globulin (rbTeBG) of rabbit plasma was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5 alpha-dihydrotestosterone-Sepharose, reactive blue-Sepharose, and finally preparative polyacrylamide gel electrophoresis. An overall purification of greater than 3000-fold was achieved with a recovery of 32%. This purified material was shown to possess the following physical characteristics: an equilibrium dissociation constant at 4 degrees C for dihydrotestosterone estimated to be 0.59 X 10(-9) M and a half-time of dissociation of rbTeBG-dihydrotestosterone complex of approximately 9 min at 4 degrees C. The purified protein shows considerable microheterogeneity with regard to size on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and to charge on isoelectric focusing gels. Analytical gel electrophoresis in the presence of SDS revealed two molecular weight components of 41,000 and 45,000. Size analysis on native gels and cross-linking studies yields a molecular weight of 101,600 and 83,000, respectively. These size components were shown to be very closely related with regard to primary structure. rbTeBG was completely absorbed by concanavalin A Sepharose resin, demonstrating the glycoprotein nature of rbTeBG. Highly purified rbTeBG and unpurified rbTeBG from rabbit plasma could be photolabeled with [3H]delta 6-testosterone and when analyzed on SDS-polyacrylamide gel electrophoresis (PAGE), two major bands of radioactivity with molecular weights of 41,000 and 45,000 appeared in both samples.

Androgen-binding protein (ABP) isolated from rat epididymides by androgen affinity chromatography is a dimer with a native molecular weight of 85,000. When fractionated on sodium dodecyl sulfate gels two components were identified which were present in a 3:1 ratio; these were designated heavy (H) and light (L) (Mr of 45,000 and 41,000), respectively. The fact that H and L both have steroid binding sites and that they can be cross-linked suggests that both are protomers of native ABP. NH2-terminal analysis, amino acid analysis, and peptide maps suggest an extensive homology between H and L. In addition, peptide maps of H and L photolabeled with delta 6-[3H]testosterone suggest that the binding sites on these protomers are identical. ABP was also purified 33,800-fold from testes. The H and L protomers from this preparation were identical with those from the epididymides as judged by peptide maps. In addition, [35S]methionine was incorporated into the H and L synthesized by Sertoli cells in the same 3:1 ratio as in highly purified ABP from testis and epididymis. These observations suggest that L is not derived from H following secretion. Selective incorporation of [3H] fucose into the H protomer suggests that differences in carbohydrate composition account for at least part of the difference between H and L. These results support the conclusion that native ABP is composed of two kinds of protomers which exist in a ratio of 3:1. These protomers are similar polypeptides that differ partly in carbohydrate composition.

In the rat, the effects of progestin and androgen administration on serum, testicular and epididymal androgen binding protein (rABP) concentrations were determined and related to the organ weight and morphology. Adult rats were treated with medroxyprogesterone acetate (MPA; 17 alpha-acetoxy-6 alpha-methylprogesterone), testosterone propionate (TP) and mibolerone (MB; 7 alpha, 17 alpha-dimethyl-19-nortestosterone). MPA reduced testicular and epididymal weights and the concentrations of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. During MPA treatment testicular and epididymal ABP content declined in parallel with organ weights and hormone concentrations, whereas serum ABP concentrations increased. Combinations of MPA and TP reduced testicular and epididymal ABP, but the reductions were less than with MPA alone; this combined treatment also elevated serum AMP. Both MB and TP reduced ABP in the male reproductive tract, but unlike MPA did not increase the concentration of this protein in serum. The results suggest that MPA acts directly on Sertoli cells resulting in increased ABP release into the blood. The comparison was made of steady state polyacrylamide gel electrophoresis (SS-PAGE) and radioimmunoassay (RIA) methods of estimating rABP. The potency ratio of testicular ABP estimated by the two methods (RIA:SS-PAGE) was three times higher than this ratio in the epididymis in normal and hormonally treated animals. Due to differences in end points, these observations imply that these assays do not quantify the molecules in the same way in one or both of these tissues. The results indicate, however, that both assays are suitable for following rABP concentration in animals with altered hormonal states.

Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time.

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)