Photolabeling of the human erythrocyte glucose carrier with androgenic steroids.

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.

High affinity estrogen binding by rabbit liver.

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.

The role of asparagine-linked oligosaccharides in the subunit structure, steroid binding, and secretion of androgen-binding protein.

Testicular androgen-binding protein (ABP) and liver sex hormone-binding globulin are encoded by the same gene. These proteins have the same primary amino acid sequences, but they differ in attached oligosaccharides; the differences are presumably due to cell-specific glycosylation mechanisms. To investigate the role of oligosaccharides in ABP/sex hormone-binding globulin subunit structure, secretion, and steroid binding, mutant rat ABP proteins were constructed that eliminated one or both of the two potential sites of asparagine (Asn)-linked glycosylation. Immunoblot analysis of wild type recombinant ABP yielded the typical heterogeneous banding pattern. Secreted ABP was composed of two protomers of M(r) 46,000 and M(r) 43,000, while cellular ABP yielded three mol wt species (M(r) 43,000, 41,000, and 39,000). Substitution of the Asn residue in either consensus sequence for Asn-linked glycosylation with an Ile residue resulted in increased mobility of the immunoreactive ABP species. These changes are consistent with the loss of an Asn-linked oligosaccharide. Substitution of both Asn residues yielded a single immunoreactive species in the medium and cell extracts that migrated as a M(r) 39,000 protein. These results demonstrate that the mol wt heterogeneity of ABP is due to differential Asn-linked glycosylation of both potential sites. All three mutant forms of ABP were secreted by the COS cells. However, the amount of immunoreactive ABP and [3H]5 alpha-dihydrotestosterone binding in the medium was lower than wild type (100%) in one of the single mutants (65%) and in the double mutant (29%). Unlike the glycosylation mutants, alteration of other residues, not involved in glycosylation, yielded cellular ABP and no detectable medium ABP.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of a gonadotropin-releasing hormone antagonist on androgen-binding protein distribution and other parameters in the adult male rat.

The effects of gonadotropins and gonadal steroids on androgen-binding protein (ABP) production and its distribution among the epididymis, seminiferous tubule fluid (STF), testicular interstitial fluid (TIF), and blood were studied in 300-g adult male Sprague-Dawley rats. The rats either received no treatment or their pituitary function was suppressed by administration of the GnRH antagonist [AcD2Nal,D4ClDPhe2,D3Pal3,Arg5,DGlu6 (AA),D-Ala10]LHRH (antagonist). Other groups of rats were treated with hCG, FSH, FSH plus hCG, testosterone, or estradiol, alone or together with antagonist. Treatment was conducted for 30 days, after which time, ABP was detected by its ability to bind [3H]5 alpha-dihydrotestosterone. Transport of ABP from the testis to the epididymis was inhibited by antagonist administration. Simultaneous treatment with antagonist and hCG, or antagonist and hCG plus FSH prevented antagonist-induced inhibition of ABP transport. Neither FSH, testosterone, nor estradiol alone was effective in this process. Inhibition of ABP transport to the epididymis was accompanied by its accumulation within the testis. Treatment with antagonist and FSH resulted in a 4.5-fold increase in the concentration of ABP in TIF, but had little effect on the amount of ABP in STF, indicating selective secretion of ABP from the basal surface of the Sertoli cells. Treatment with antagonist alone, antagonist together with testosterone or estradiol, or estradiol alone resulted in increased concentrations of ABP in both TIF and STF, but the increase in TIF was proportionately greater. Treatment with hCG or FSH plus hCG alone or with antagonist not only facilitated ABP transport to the epididymis, but also increased TIF levels of ABP above control values. The former treatment resulted in increased concentrations of testosterone in TIF, but not in STF. Both treatments resulted in testosterone levels in both compartments that were higher than those in animals treated with antagonist alone. No treatment had a statistically significant effect on blood levels of ABP. About 50% of ABP synthesis appears to be constitutive, i.e. is not regulated by hormones. Although ABP production continues in the presence of antagonist, its transport to the epididymis is halted, indicating that epididymal transport of ABP is a hormone-dependent process. It is likely that elevated intratesticular levels of testosterone or FSH and testosterone acting in concert regulate epididymal transport of ABP.

Structure-function relationships of rat androgen--binding protein/human sex-hormone binding globulin: the effect of mutagenesis on steroid-binding parameters.

Androgen-binding protein (ABP) and plasma sex hormone-binding globulin (SHBG) are encoded by the same gene and have the identical amino acid sequence within a species. Mammalian ABPs and SHBGs bind sex steroids with high affinity, but some binding properties differ among species. Human SHBG has a higher affinity for steroids and forms a more stable 5 alpha-dihydrotestosterone (DHT) complex (t1/2 = 40 min) than rat epididymal ABP (t1/2 = 5 min) at 0 C. In this study it was found that recombinant wild-type rat ABP/SHBG bound DHT and estradiol with nearly the same affinities as human SHBG, rather than the affinities of rat epididymal ABP. This finding is reminiscent of our previously published data demonstrating that ABP secreted by cultured Sertoli cells had a higher affinity for DHT than did epididymal ABP. Recombinant wild-type ABP had DHT dissociation properties similar to those of rat epididymal ABP. The differences in binding properties of epididymal ABP and recombinant ABP could in part be caused by intrinsic differences in the properties of the proteins due to posttranslational modifications or allelic variations in sequence. To aid in identification of the steroid-binding domain of ABP and SHBG, we developed recombinant rat ABP/SHBG chimeras containing human sequences in and flanking the putative steroid-binding region (residues 134-150). Four regions were mutated: 1) residues 129-132; 2) residues 133-138; 3) residues 148-155; and 4) 165-169; residues between these regions are identical in rat and human ABP/SHBG. Wild-type (ABPwt) and mutant proteins were expressed in COS cells, and their steroid-binding properties were determined. Conversion of rat amino acid residues 133-169 (ABP2,3,4) to human ABP/SHBG sequence resulted in a 2-fold increase in affinity for estradiol compared to ABPwt. Another mutant ABP2,3,4; Leu 137, which contained the rat Leu137 residue, had a 5-fold increase in estradiol affinity. Conversion of residues 129-132 to human sequence in ABP2,3,4 to form ABP1,2,3,4 resulted in a dramatic decrease in estradiol affinity. Conversion of each region separately also resulted in some changes in steroid-binding properties. The largest change was observed with ABP1, which had a 3-fold reduction in estradiol affinity compared to ABPwt. There was a 14-fold difference in estradiol affinity between ABP1 and ABP2,3,4; Leu 137. Alterations of some individual amino acid residues in region 2, which is the least conserved region between rat and human, caused subtle or major changes in the estradiol-binding properties; none affected DHT binding.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence of a cytoplasmic estrogen receptor in sexually mature rabbit epididymides: comparison with the estrogen receptor in immature rabbit epididymal cytosol.

Cytosol prepared from epididymides of castrated adult rabbits contains an estrogen-specific binding component that sediments in the 4--5S region on 0.01 M KCl sucrose gradients. The estrogen-specific binding component is not detectable on sucrose gradients unless the cytosol-[3H]estradiol mixture is first extracted with charcoal to remove uncomplexed and rapidly exchangeable ligand from the sample. The macromolecular bound [3H]estradiol that remains after charcoal extraction dissociates from the receptor with a dissociation half-time of greater than 24 h and is of high affinity (Kd = 6.5 +/- 0.9 X 10(-9) M). The estrogen receptor in the adult rabbit epididymis can also be demonstrated when charcoal-extracted samples are chromatographed on Sephadex G-200. Using this system, the major estrogen-specific binder has an apparent molecular weight of less than 200,000. In contrast, the epididymal estrogen receptor in sexually immature rabbits sediments as an 8S species on low salt gradients and has an apparent molecular weight of 200,000 or greater when assessed by chromatography on Sephadex G-200. The estrogen receptor in both age groups is distributed along the length of the epididymal duct. The highest concentration of the cytoplasmic receptor in both mature and immature rabbits is in the cauda epididymidis. The age-dependent changes in the amount of the receptor suggest that it may be involved in maturational changes in the epididymis. The alterations in the physicochemical properties may reflect physiological changes in the epididymis or in the endocrine status of the animal.

Estrogen and androgen regulation of protein synthesis by the immature rabbit epididymis.

To obtain evidence of a physiological role for androgens and estrogens in the regulation of the epididymis of sexually immature rabbits, the effects of these hormones on [35S] methionine incorporation into epididymal proteins in vitro were examined. Two-dimensional polyacrylamide gel electrophoresis revealed that short term incubation with estradiol changed the patterns of radiolabeled proteins detected in tissue homogenates of epididymal segments from castrated rabbits compared to those in segments from castrated rabbits that were not exposed to exogenous estradiol. Most of the changes seen in corpus tissue affected proteins with a wide range of pI values and relatively high mol wt (greater than 40K). The effects on caput and cauda tissue proteins were seen over a wide pH and mol wt range. Castration abolished many of the regional differences in protein synthesis; these were restored by incubation with estradiol. Testosterone had little effect on the synthesis of tissue proteins, except for stimulation of the synthesis of a single protein (17K; pI 5.1) in all three segments and stimulation of a small group of proteins (less than 14K; pI 7.0-7.2) in the corpus. Estradiol had little effect on proteins secreted by epididymal segments. Testosterone, however, stimulated the synthesis of a number of unique proteins secreted by the caput and corpus and resulted in a pattern of radiolabeled proteins similar to that obtained with intact animals. Additional secretory proteins could be stimulated in caput, but not corpus, tissue minces from intact rabbits by exogenous testosterone. No androgen-specific synthesis of secretory proteins was detected in the cauda of either castrated or intact rabbits. Estradiol affected the synthesis of both secreted and tissue proteins in terms of influencing which epididymal segment was most active at incorporating [35S]methionine into radiolabeled proteins and which was least active. Testosterone had a similar influence on secreted proteins, but did not have any analogous effect on tissue proteins. These results indicate that testosterone and estradiol influence the synthesis of proteins by the immature rabbit epididymis and that both may, therefore, be important physiological regulators of epididymal development and/or function.

Androgen and estrogen effects on protein synthesis by the adult rabbit epididymis.

The effects of castration and hormone replacement on [35S]methionine incorporation into newly synthesized proteins by the adult rabbit epididymis were studied in vitro. The proteins were analyzed using two-dimensional polyacrylamide gel electrophoresis. Short term (4-day) castration resulted in a few changes in the pattern of radiolabeled proteins observed in the caput, but no effect was seen in the corpus or cauda. The changes in the caput could be reversed if the samples were incubated with testosterone. The epididymis of short term castrates failed to respond to exogenous estradiol. Long term castration (4-6 weeks) resulted in changes in protein synthesis among all three epididymal segments. Short term (4-h) incubation with testosterone restored the pattern of proteins secreted by the caput and cauda to that in intact rabbits. Short term incubation with estradiol did not restore the pattern of radiolabeled secreted proteins, but it did slightly intensify a 28K protein (pI 5.2) that was present in the caput and cauda of castrated animals. No clear-cut effect of the hormones on proteins secreted by the corpus was observed. Short term incubation with testosterone or estradiol restored the patterns of tissue proteins synthesized by the caput and corpus of castrated rabbits to that in intact animals. In the cauda, estradiol also enhanced the presence of a small group of high mol wt proteins present in the control castrate sample, while testosterone inhibited these proteins. This group of proteins was absent in cauda tissue samples from intact rabbits.

The ontogeny of biologically active androgen-binding protein in rat plasma, testis, and epididymis.

Androgen-binding protein (ABP) can be detected in the blood of sexually immature male rats by its ability to specifically bind [3H]5 alpha-dihydrotestosterone (5 alpha-DHT). Since the androgen-binding site is functional, we consider this ABP to be biologically active. ABP can be detected (641 +/- 107 ng/ml; n = 5) in plasma by the 15th day of postnatal life, it reaches a maximum concentration (1631 +/- 323 ng/ml; n = 5) on day 20 of age, and is no longer detectable after day 40. ABP can be detected in the testes of all age groups studied (15 days to adult). However, no ABP is detectable in the epididymis until the animals are 25 days old. Plasma ABP comigrates on nondenaturing gels with photolabeled ABP from the adult or immature rat epididymis. Serum that had been treated with Affigel blue to remove albumin and with hydroxylapatite to decolorize it was photolabeled using [3H]17 beta-hydroxy-4,6-androstadien-3-one. Photolabeled serum ABP migrated on polyacrylamide gels containing sodium dodecyl sulfate as 60,000 and 48,000 dalton androgen-specific peaks. In contrast, photolabeled adult epididymal ABP exhibited the 47,000 and 41,000 dalton peaks characteristic of ABP subunits. When photolabeled plasma and epididymal ABP were combined and electrophoresed on the same gel under denaturing conditions, prominent 60,000 and 47,000 dalton peaks were obtained, indicating that the two species of ABP retained their identities when combined. Photolabeled epididymal ABP from 25-day-old rats exhibited similar subunit mol wt in the same ratios as ABP from the adult. When epididymal ABP from the two age groups was combined and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the resulting pattern was identical to that obtained when the samples were run individually, except that there was an increase in peak height. These data indicated that there is no significant difference in the subunit mol wt of epididymal ABP from the two age groups.

Analysis of the steroid binding domain of rat androgen-binding protein.

The site-directed photoaffinity ligand [3H]17 beta-hydroxy-4,6-androstadien-3-one (delta 6-testosterone) was used to label the steroid binding domain of rat androgen-binding protein (rABP). After digestion with trypsin, the major radiolabeled peptide was isolated by reverse phase chromatography. The peptide was found to have the following amino acid sequence: Ile Ala Leu Gly Gly Leu Leu Leu Pro Thr Ser. Gaps in the sequence that one would anticipate if delta 6-testosterone formed an adduct with a single amino acid were not encountered. Several different amino acids appear to have been labeled as expected given the free radical nature of photoactivated delta 6-testosterone. The sequence obtained corresponded to a tryptic peptide (amino acids 171-181) of the rABP precursor. The only other protein having this amino acid sequence was human sex hormone binding globulin. The binding domain lies in a hydrophobic pocket that contains a predicted beta-sheet and turn secondary structure, as would be anticipated given the hydrophobic nature of the steroid molecule. A hydropathy and secondary structure analysis of rABP was performed as a basis for discussing the results of the current study in relation to previous studies on the steroid binding domain on human sex hormone binding globulin.

Analysis of the disruptive action of an epididymal protease and the stabilizing influence of molybdate on nondenatured and denatured glucocorticoid receptor.

The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.

Human testosterone-binding globulin is a dimer composed of two identical protomers that are differentially glycosylated.

Affinity-purified human testosterone-binding globulin (hTeBG) is composed of two subunits [mol wt (Mr), 52,200 and 48,600], as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, and immunochemical localization with a monoclonal antibody raised against rat androgen-binding protein. Fluorography of SDS-PAGE gels on which photoaffinity-labeled hTeBG was analyzed yielded essentially identical results. Enzymatic deglycosylation of hTeBG with neuraminidase to remove sialic acid led to the production of two subunits of 50,800 and 47,300 Mr when assessed by SDS-PAGE. Treatment of hTeBG with an optimal concentration of N-glycanase to remove Asn-linked oligosaccharides produced a single subunit of 44,100 Mr. When hTeBG was treated with neuraminidase and O-glycanase to remove O-linked oligosaccharides, three subunits were seen, two of which had Mr not clearly different from those obtained with neuraminidase treatment alone plus a subunit of 40,900 Mr. Treatment of hTeBG with a combination of all three enzymes produced a single subunit of 42,900 Mr. Chemical deglycosylation with trifluoromethane-sulfonic acid produced a single subunit with a Mr identical to that produced by treatment with all three enzymes. We concluded that this is the Mr of completely deglycosylated hTeBG. Based on this Mr, carbohydrates contribute 18% and 12% to the apparent Mr of the heavy and light subunits of hTeBG, respectively. Two-dimensional PAGE analysis of hTeBG with its oligosaccharides intact indicated that the heavy subunit was composed of seven isoelectric variants with pI values of 5.87-6.55, while the light subunit was composed of four charge variants with pI values of 6.14-6.55. Treatment of hTeBG with the enzymes resulted in a shift in the pH values to a more basic pH range, indicating that carbohydrate removal also removed charged species from the protein. The greatest cathodal shift occurred when hTeBG was treated with a combination of the three enzymes (pI 7.33-7.77) or when it was chemically deglycosylated (pI 6.37-7.02). Despite the apparent removal of all carbohydrates, the single subunit was still composed of multiple isoforms. This finding suggests that other charged species remain on the hTeBG molecule.

The effects of estradiol, tamoxifen, and testosterone on the weights and histology of the epididymis and accessory sex organs of sexually immature rabbits.

The effects of estradiol benzoate (EB), testosterone propionate (TP), and Tamoxifen, alone or in combination, on the weight and morphology of the male accessory sex glands were studied in intact and in castrated immature rabbits. TP treatments (2 mg/kg) exerted a stimulatory effect on the glands, resulting in a significant increase in the weight of the epididymis, the proprostate, and the prostate of castrated rabbits, and of the bulbourethral glands of both intact and castrated rabbits, and in marked morphological changes in all the glands. the epithelium was stimulated but retained its pseudostratified columnar appearance and resembled more the epithelium of normal mature males than that of age matched controls. The bulbourethral glands were the most responsive and the vesicular gland the least responsive to androgen treatment. The response of the glands to EB (25 micrograms/kg) was characterized by significant weight increases in all the glands of both castrated and intact rabbits, hypertrophy of the musculo-fibrous components and proliferation of the basal layer of the epithelium leading to squamous metaplasia and leukocytic infiltration. Hyperplasia of the fibromuscular stroma was most evident in the cauda epididymidis and in the vesicular gland. Squamous metaplasia and leukocytic infiltration were most evident in the ejaculatory duct and in the structures adjacent to it. The antiestrogen, Tamoxifen (250 micrograms/kg), and TP (2 mg/kg) given in conjunction with EB (25 micrograms/kg) tended to reduce the weight increase caused by estrogen, but the decrease was significant in only a few instances. Tamoxifen (250 micrograms/kg) administered alone stimulated the epithelium of the accessory sex glands and induced squamous metaplasia, but did not induce hyperplasia of the fibromuscular stroma. The study demonstrates that accessory sex glands display a consistent pattern of differential sensitivity to both androgens and estrogens and that these hormones exert their action on different cell types within the organ.

Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the restricted rat.

The hypothesized relationship between androgen-binding protein (ABP) and sperm maturation was investigated using a mutant rodent: the restricted rat. The seminiferous epithelium of these animals undergoes a spontaneous degeneration, but changes are progressive. Restricted rats in the transition to infertility were used to determine if changes in ABP were related to the decreased fertility found in these animals. Fertilizing ability was determined by insemination of cauda epididymal spermatozoa into hormonally primed female rats and examination of ova for evidence of fertilization 48 h later. Epididymal and testicular tissues were analyzed for ABP using a charcoal assay. Androgen levels were determined by RIA. Testicular weights were significantly reduced compared to those of normal littermates in restricted rats at all ages; epididymal weights were significantly reduced in rats 140 days and older. Among restricted rats, sperm fertilizing ability was variable, but was significantly lower than that in normal littermates; it was consistently highest at 90 days of age. Epididymal ABP content (picomoles per organ) was significantly reduced in restricted rats at all ages; peak values occurred at 90 days. Testicular ABP content was significantly reduced only in the youngest and oldest animals. Plasma testosterone levels were not statistically lower than those found in normal littermates, and ventral prostate weights were maintained at normal levels in all four groups of animals. A significant positive correlation existed between sperm fertilizing ability and epididymal ABP, but not between sperm fertilizing ability and plasma testosterone. Since ABP is an index of Sertoli cell function, these data indicate that sperm fertilizing ability is closely related to Sertoli cell function and/or ABP.

Investigations on the relationship between sperm fertilizing ability and androgen-binding protein in the hypophysectomized, pregnenoloneenolone-injected rat.

Hypophysectomized rats maintained with 2 mg pregnenolone have low plasma testosterone levels, but rete testis levels are normal. This model system was used to examine the importance of androgen-binding protein (ABP) in maintaining high luminal androgen concentrations for the development and maintenance of sperm fertilizing ability in the epididymis. Hypophysectomized rats were injected with pregnenolone (1, 0.5, 0.2, or 0.02 mg/100 g BW) for 14 days, starting 1 day after surgery. Sham-operated rats and a group of hypophysectomized rats were injected with oils as controls. At the end of the experimental period, sperm fertilizing ability, tissue weights, ABP, and testosterone levels were determined. The 1- and 0.5-mg doses of pregnenolone resulted in rete testis testosterone levels that were 67% and 29%, respectively, of the levels found in sham-operated animals. Plasma testosterone levels were not different from levels found in hypophysectomized, oil-injected controls with any of the pregnenolone doses. The three highest doses of pregnenolone resulted in levels of testicular ABP that were not statistically different from sham-operated levels (2 pmol/organ). Epididymal ABP content was maintained at sham-operated control levels (30 pmol/organ) with the 1-mg dose; with the 0.5- and 0.2-mg doses, ABP levels were 76% and 73% of sham-operated levels, respectively. Epididymal ABP specific content (picomoles per 100 mg tissue) was maintained at sham-operated control levels with the 1-, 0.5-, and 0.2-mg doses. Sperm fertilizing ability was maintained at sham-operated control levels with the three highest doses of pregnenolone. The 0.02-mg dose of pregnenolone resulted in values that were not statistically different from hypophysectomized, oil-injected control values for all parameters tested. A significant positive correlation existed between ABP and sperm fertilizing ability. These data demonstrate that testicular and epididymal ABP levels can be maintained in hypophysectomized rats with pregnenolone treatment alone, and that sperm fertilizing ability can be maintained when intraluminal androgen levels are low and ABP levels are near normal. This suggests that ABP may be important in the maintenance of normal sperm fertilizing ability.

Some physicochemical characteristics of photoaffinity-labeled rabbit androgen-binding protein.

Photoaffinity-labeled androgen-binding protein (ABP), present in cytosol prepared from the caput epididymidis of intact sexually mature rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one (delta 6-testosterone). Photo-labeled ABP could not be distinguished from unlabeled ABP by gel filtration chromatography, electrophoresis on nondenaturing polyacrylamide gels, or sucrose gradient ultracentrifugation. Rabbit ABP was found to have a sedimentation coefficient of 4.6S and a Stokes radium of approximately 45A. Based on these parameters, its native molecular weight was calculated to be approximately 86,300. A model of ABP as a prolate ellipsoid having an axial ratio of 10 is consistent with its frictional ratio of 1.555. When photolabeled rabbit ABP was examined on polyacrylamide gels containing sodium dodecyl sulfate, two androgen-specific peaks of approximately 52,000 and approximately 48,000 daltons were detected. Both peaks contained approximately the same amount of radioactivity. When photolabeled ABP was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on gels containing sodium dodecyl sulfate, an additional androgen-specific peak corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent dimers of the lower molecular weight species. The 52,000- and 48,000-dalton subunits were observed regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.

Some physicochemical characteristics of photoaffinity-labeled rabbit testosterone-binding globulin.

Photolabeled testosterone-binding globulin (TeBG), obtained from the plasma of sexually immature male rabbits, was produced using 17 beta-hydroxy-[1,2-3H]4,6-androstadien-3-one. Photolabeled TeBG could not be distinguished from unlabeled TeBG by gel filtration chromatography, electrophoresis on nondenaturing gels, or sucrose gradient ultracentrifugation. Rabbit TeBG had a Stokes radius of approximately 45 A and a sedimentation coefficient of approximately 4.6S. Based on these parameters, its native molecular weight was calculated to be approximately 78,000. The frictional ratio of rabbit TeBG was found to be approximately 1.6. When photolabeled TeBG was examined on polyacrylamide gels containing sodium dodecyl sulfate, a single androgen-specific peak of approximately 40,000 daltons was obtained. When photolabeled TeBG was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on sodium dodecyl sulfate gels, an additional androgen-specific peak of radioactivity corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent oligomers of the TeBG subunits. The 40,000-dalton subunit was obtained regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.

Hormonal regulation of androgen-binding protein in the rat.

Effects of gonadotropins and gonadal steroids on androgen-binding protein (ABP) production by the testis and its secretion into the blood and transport into the epididymis were studied in 20- and 30-day-old rats. These animals had been treated with hCG, FSH, the Nal-Glu GnRH antagonist [Ac-D2Nal1,D4ClDPhe2,D3Pal3,Arg5,DGlu6(AA) ,DAla10]LHRH (antagonist), testosterone propionate, or estradiol benzoate, alone or in combination, for 10 days before assessment of ABP. Antagonist administration suppressed the testicular content (nanograms per organ) of ABP to below control (untreated) levels in both age groups. When hCG or testosterone was given along with the antagonist, they overcame the effect of the antagonist, and the resultant ABP values exceeded untreated control levels in both the 20- and 30-day-old rats. Treatment of rats with these hormones in the absence of the GnRH antagonist also elevated the ABP content of the testis above that of untreated controls. FSH administered with antagonist was able to prevent the antagonist-induced suppression of testicular ABP content. When rats were treated with FSH alone, the content of ABP in the testis was increased above untreated control levels in the 30-day-old group, but not in the 20-day-old group. The simultaneous administration of FSH and hCG did not result in an increase in testicular ABP content above that caused by hCG or testosterone alone. The increase in the ABP content of the testis caused by FSH administration was only about one sixth that caused by hCG or testosterone. Since testosterone or hCG, even in the presence of antagonist, was able to maximally stimulate ABP production by the testis of both age groups, we conclude that testosterone is the major in vivo regulator of its synthesis. Only combined treatment with hCG and FSH was able to increase transport of ABP into the epididymis of 20-day-old rats. All treatments that increased the testicular content of ABP in the 30-day-old rats also increased its transport into the epididymis. Treatments that drastically reduced the content of ABP in the testis of 20-day-old rats (antagonist, estradiol, estradiol plus antagonist) also reduced ABP secretion into the serum. Only treatment with estradiol reduced the secretion of ABP into the serum of 30-day-old rats. None of the treatments increased the ABP secretion into the bloodstream above untreated control levels.

Monoclonal antibodies to rat androgen-binding protein recognize both of its subunits and cross-react with rabbit and human testosterone-binding globulin.

A series of four monoclonal antibodies to rat androgen-binding protein (ABP) has been developed. These antibodies recognize both the heavy (48,400-dalton) and light (43,000-dalton) subunits of the native ABP molecule. In addition, they recognize the subunits from which Asn-linked oligosaccharides have been removed by treatment with N-glycanase, indicating that these moieties do not form the immunological determinants recognized by the antibodies. Two of these antibodies are capable of recognizing both nondenatured ABP, as assessed by dot blot analysis, and denatured ABP, as determined by Western blot analysis of ABP after electrophoresis under denaturing conditions. The immunoreactivity of denatured ABP is decreased with two of the antibodies, suggesting that they more readily recognize the antigenic epitopes when the protein is in its native configuration. The antibodies were capable of immunoprecipitating covalently labeled ABP from solution. All four monoclonal antibodies produced were determined to be immunoglobulins M by both enzyme-linked immunosorbent assay and Ouchterlony immunodiffusion even though the initial serum response of the immunized animals indicated the presence of immunoglobulins G. All of the monoclonal antibodies raised against rat ABP cross-reacted with rabbit and human testosterone-binding globulin (TeBG). They were able to detect two subunits when Western blots of intact rabbit [mol wt (Mr, 43,000 and 40,500] or human (Mr, 47,600 and 44,500) TeBG were probed, but only a single subunit (Mr, 39,300) when deglycosylated samples of rabbit TeBG were analyzed.

Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins.

The disruption of the reproductive system of male and female animals in the wild has been attributed to environmental chemicals (xenobiotics). The effects seen mirror alterations one might anticipate if the steroid hormone-dependent processes that regulate these systems were impaired. To determine whether xenobiotics (present at a concentration of 100 microM) exert their action through steroid-mediated pathways, we examined their ability to inhibit the binding of [3H]physiological ligands (present at a concentration of 7 nM) to the androgen and estrogen receptors, rat androgen-binding protein (ABP), and human sex hormone-binding globulin (hSHBG). The gamma- and delta-isomers of hexachlorocyclohexane, congeners of dichlorodiphenyl-trichloroethane (DDT; p,p'-DDT; p,p'-DDE; o,p'-DDT), dieldrin, atrazine, and pentachlorophenol, caused a statistically significant inhibition of specific binding of [3H]5 alpha-DHT to the androgen receptor that ranged from 100% (p,p'-DDE) to 25% (dieldrin). Methoxychlor, o,p'-DDT1, pentachlorophenol, and nonylphenol significantly reduced [3H]17 beta-estradiol binding to the estrogen receptor by 10, 60, 20, and 75%, respectively. The binding of [3H]5 alpha-DHT to ABP was inhibited 70% by the delta-isomer of hexachlorocyclohexane, but the gamma-isomer did not reduce binding significantly. Methoxychlor, p,p'-DDT, atrazine, and nonylphenol reduced [3H]5 alpha-DHT binding to ABP by approximately 40%. Nonylphenol reduced the binding of [3H]5 alpha-DHT to hSHBG by 70%. Hexachlorocyclohexane reduced [3H]5 alpha-DHT binding to hSHBG by 20%, but the stereospecific effects observed with ABP did not occur. o,p'-DDT and pentachlorophenol resulted in a statistically significant 20% inhibition of [3H]5 alpha-DHT binding to hSHBG. Some xenobiotics resulted in dissociation of [3H]ligands from their binding proteins that was statistically identical to that caused by the unlabeled natural ligand, whereas others resulted in slower or more rapid dissociation rates.