Comparison of rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin--II. Immunological properties

Purified rabbit epididymal androgen binding protein and serum testosterone estradiol binding globulin have been immunologically compared. A comparison using the steady state gel method of Ritzen et al. indicated immunological cross-reactivity. In order to further compare their immunological properties we developed a radioimmunoassay for both rbABP and rbTeBG using specific antisera directed against each. When these assays were compared, the extent or completeness of displacement proved to be the only parameter that was significantly different. This data obtained with homologous and heterologous radioimmunoassays is consistent with the idea that these two proteins contain minor antigenic determinants which are distinct.     

An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α).The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellman's reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins.Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.     

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.     

There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin

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)     

Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.

Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.     

Serum estradiol, testosterone and dihydrotestosterone in male monkeys treated with testosterone propionate.

Serum estradiol (E2), testosterone (T) and dihydrotestosterone (DHT) were measured in juvenile (pre-pubertal) male rhesus monkeys injected with either 8 mg or 80 mg of testosterone propionate (TP). After one week, the three steroids were elevated and remained essentially unchanged for the duration of the study. There was little difference in serum E2 or DHT when comparing the two groups of steroid-treated monkeys. In contrast, T levels were consistently greater in the animals given the high dosage of TP.     

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.     

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.     

Demonstration of a specific binding protein for testosterone and 5alpha-dihydrotestosterone in rabbit serum. Separation from the corticosteroid binding globulin (CBG)

Rabbit serum contains a specific androgen binding protein which can be separated from the corticosteroid binding globulin (CBG) in rabbit serum by sucrose gradient ultracentrifugation and polyacrylamide gel electrophoresis. It has a sedimentation constant of 4–5 S (mean 4.4 S) and high binding affinities for 5α-dihydrotestosterone, 5α-androstan-3α, 17β-diol and testosterone but negligible affinity for androstenedione, progesterone or corticosterone. Concentrations of the androgen binding protein expressed as 5α-dihydrotestosterone (DHT) binding capacity at saturation are higher in adult female (4.0 ± 0.3 μg DHT bound/100 ml) than in adult male sera (1.4 ± 0.8 μg DHT bound/100 ml). Immature male sera contain slightly higher amounts than adult females.     

Relationship of testosterone pulses to androgen binding in the pituitary of rams

The effects of testosterone on cytosol and nuclear androgen receptors of ram pituitary were examined in two experiments. In Exp. I, 500 micrograms testosterone were injected intravenously and groups of 4 rams were slaughtered at 0, 15, 30, 45, 90 and 360 min after injection. Cytosolic receptor concentration decreased from 21 +/- 0.9 to 6 +/- 0.9 fmol/mg protein 30 min after the testosterone injection (P less than 0.001), and then returned towards the preinjection level after 90 min. The pattern of nuclear receptor concentration was the opposite; a maximal increase (12 +/- 3.5 to 32 +/- 5.7 fmol/mg protein) was observed 30 min after injection (P less than 0.001), followed by a progressive but incomplete decrease by 360 min. In Exp. II, blood was collected every 20 min for 17 h in three successive series, each of 12 rams, which were then slaughtered. Plasma LH and testosterone concentrations were measured by radioimmunoassay. No changes were observed in cytosol receptor concentration, but nuclear receptor concentration was negatively correlated with the interval elapsed since the beginning of the last testosterone pulse (r = -0.62; P less than 0.001). The highest values for nuclear receptor concentrations were observed at an interval equal to or less than 120 min. These results indicate that natural pulses are associated with androgen binding particularly in the pituitary nuclei.     

Diazepam binding inhibitor (DBI) reduces testosterone and estradiol levels in vivo

Diazepam binding inhibitor (DBI) is a putative endogenous ligand capable of binding to the central type benzodiazepine (BZD) receptor located on the GABAA receptor and the peripheral type BZD receptor on the mitochondrial outer membrane. We examined the effects of an intracerebroventricular injection of DBI on the serum levels of the gonadal hormones, testosterone and estradiol, respectively, in male and female mice. DBI (0.3-10 nmol/mouse, i.c.v.) significantly reduced the levels of both gonadal hormones in a dose-dependent manner. The decrease in the gonadal hormone levels became evident at 1 hr and lasted for at least 4 hrs after the DBI injection. The effects of DBI (3 nmol/mouse, i.c.v.) in male and female mice were completely attenuated by the coadministration of flumazenil (66 nmol/mouse), a selective antagonist for the central type BZD receptor. These results suggest that DBI acts as an endogenous modulator to regulate the levels of gonadal hormones in vivo, and that the DBI-induced decrease in gonadal hormone levels is mediated by down regulation of the GABAergic system, implicated in gonadotropin-releasing systems and/or the hypothalamic-pituitary-gonadal axis.     

Percentage binding of testosterone and dihydrotestosterone and unbound testosterone and dihydrotestosterone in rabbit maternal and fetal plasma during sexual organogenesis.

The percentages of bound testosterone (17 beta-hydroxy-4-androsten-3-one; T) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one; DHT) and their unbound concentrations were determined in pregnant rabbits and their fetuses from the 18th day of gestation to birth. T and DHT were also measured in fetal testes. In the testis, the total T/total DHT ratio, very high at 22 days (73.7 +/- 15.2), decreased until birth (6.7 +/- 0.8). In male fetuses the concentrations of total and unbound circulating T and DHT were always low and did not show any peak during sexual organogenesis. The percent binding of T (from 73.0 +/- 0.5 to 77.6 +/- 0.6) and DHT (from 76.5 to 83.7 +/- 1.1) in fetuses were similar in both sexes and significantly lower than those measured in mothers (T: from 87.2 +/- 0.6 to 91.6 +/- 0.9; DHT: from 87.3 +/- 0.9 to 93.8 +/- 0.9).