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160. Androgen “priming” increases the FSH induced production of testicular androgen binding protein (ABP)
334
Abstracts
other hand, we have found at the tnne of the initiation of the meiotic division in the ZO- to X-day-old rat that there is an increase in the testicular concentration of testosterone {T) and particularly of 5r-androstandiol (DIOL) as well as a rise in Sr-reductase activity. The purpose of this work was to correlate the latter events with the presence of ABP in the testis. Testicular tissue from IO-. JO- and ho-day-old rats was homogenized in Tris-HCI buffer. IOOmM, pH 7.4 at 0 C. The cytosol fraction was obtained and endogenous androgens were removed with charcoal (1 mg;mg protein) during 18h at 0 C. Incubations were carried out at 17 c‘. 0 C and 50 C during 30’ with JH-T. ‘H-DIOL and “H-dihydrotestosterone(DHT). Binding to macromolecules was analyzed by 5- ?O”,, sucrose gradient centrifugation. it was found that ABP is not present in IO-day-old rats but it is present in 20- and 60-day-old animals. Some characteristics of the ?O-day-old rat testis ABP were studied. It can bind tritiated T. DIOL or DHT in a similar manner. No difference was detected incubating at 0 C. 27 C or 50 C. A 46 x IO “M concentration of non-radioactive DIOL was necessary to displace 3H-DIOl_. from ABP showing a fairly high binding capacity for this protein. The presence of ABP in the testis at the time of meiosis suggests that it might be playing a role, along with androgcns. ;~t thib atage of developmen t 160. Androgen “priming” increases the FSH induced produetion of testicular androgen binding protein (ABP) ‘HANSSON. V.. ‘FRENCH, F. S.. 3R~~z~~, E. M.. ~WEDDI~GTO~, S. C. and ‘NAYFEH, S. N.. ‘Rikshospitalet. Oslo, Norway, ‘University of North Carolina at Chapel Hill. U.S.A. and ‘Karolinska sjukhuset, Stockholm, Sweden Testicular androgen binding protein (ABP) is produced by the testis. secreted into the testicular fluid and carried into the epididymis by the way of efferent ducts. ABP is completely dependent on FSH stimulation ; it disappears rapidly after hypophysectomy and is dramatically stimulated by FSH. but not by LH or androgens alone (Nature New Biology 246, 56. 1973). The sensitivity of the testis to FSH, as measured by the ABP response, decreases rapidly with the time after hypophysectomy. f-fowever, pretreatment with high doses of androgens (2 mg TP per day) greatly enhanced the sensitivity. The mechanism by which testosterone propionate increases the sensitivitv of the testis for FSH is not known, however. several possjbitities might be considered. (a) It might stimulate the formation of FSH receptors on Sertoli cell membranes. (b) It might increase the blood flow through peritubular capillaries, therby allowing more FSH to reach its target sites in the seminiferous tubules. (c) It might also diminish the degradation of FSH in the liver. kidneyorwithinthetestisitself. (d) Finallv, testosterone propionate- might decrease the destruction 01 ABP within the testis (decreased proteolyric activity or inactivation of specific inhibitors). 161. Steroid binding proteins in rat preputial giand cytosol FELDMAN, M., VOIGT. W. and HSIA, S. L.. Departments of Biochemistry. Phar~lacology and Dermdtology, University of Miami School of Medicine. Miami, Florida. U.S.A. The preputialgland of the rat is an ondrogen-sensitive organ present in both male and female animals. The similar histology and androgen responsiveness of human sebaceous
cells. make the preputial gland an interesting model tbr the study of sebaceous glands. incubation of the lOS.OOOg supernatant of preputial gland homogenates with [H’] dihydrotestosterone(DHT) or[HJ] testosterone showed two peaks of radioactivity on sucrose density gradient centrifugation. corresponding to 2S and 4S complexes. Both peaks were destroyed by heating cytosol at 55 C for IOmin or treating with pronase. but not ribonuclease. The 4S protein is androgen specific. and does not bind estrogens, whereas the ZS protein binds estrogens with greater affinity than androgens, exhibiting linear binding with up to 31)OnM concentration of steroid. The association constanr of the 4s protein for DWT as determined by bat&wise gel equilibration was lO”M -I. Binding of DHT to the 4s component is greater at 37 C than at either 25 c‘ or 4 f. whereas at the lower temperatures binding to the 1s cornp[~ncllt predominates. The 1s protein has a molecular weight in the order of’ 15,000 as determined by gel filtration.
162. Inhibition of uptake, metabolism and binding of androgen in epididymis of male rats irl viva: Comparison of estradiol and cyproterone acetate TINT)AI.L. D. J., FRENCH. F. S. and NAYFEN. S. N.. Departments of Pediatrics and Biochemistry and The Laboratories for Reproductive Biology. University of North Carolina School of Medicine. Chapel Hill, U.S.A. Effects of a single dose of estradiol (E) on uptake, binding and metabolism of androgen in the rat epididymis itt ldluo were compared with the effects of a stmilar dose of cyproterone acetate (CA). Adult rats were castrated 18 h before the experiment. E or CA was injected i.v. 5 min prior to ‘Htestosterone (3H-T) or 3H-5r-dihydrotestosterone (jHDHT). and the epididymides were removed 3 h later. Total androgen uptake into epididymal supernatant was inhibited 58”,, by E as compared to 38”,, by CA. %-Reduction of ‘H-T was inhibited by E resulting in diminished formation of 3H-DHT. CA did not affect the metabolism of JH-T. E reduced total bound radioactivity in cytosol fractions to a greater extent than CA when either 3H-T or 3H-DHT was injected. E inhibited binding of 3H-DHT to both the intracellular cytoplasmic receptor (CR) and the intraluminal androgen binding protein (ABP). CA inhibited binding to CR without affecting binding to ABP. Androgen uptake into epididymal nuclei was inhibited both by E (95”,,) and CA (83”,,). E abolished all nuclear binding of 3H-DHT, white some bound radioactivity (20”” of the control) remained after treatment with CA. Both E’and CA caused bound radioactivity in nuclear extracts to decrease in direct proportion to the inhibition of binding tn CR.
SE. Steroid receptors: Cortieoids 163. Glucocorticoid binding macromolecules in chicken liver cytosol COC~~ET. C. and CHAMBAZ, E. M., C.E.R.M.O.. Universitk Scientifique et Medicale, Grenoble. France Glucocorticoid binding proteins have been extensively studied in several target organs, especially in the rat. Three different binders have been characterized in rat liver cytosol, of which only one might be involved in the mechanism of steroid specific enzyme induction in this tissue. The chicken liver may offer an interesting experimental model for the
Abstracts
other hand, we have found at the tnne of the initiation of the meiotic division in the ZO- to X-day-old rat that there is an increase in the testicular concentration of testosterone {T) and particularly of 5r-androstandiol (DIOL) as well as a rise in Sr-reductase activity. The purpose of this work was to correlate the latter events with the presence of ABP in the testis. Testicular tissue from IO-. JO- and ho-day-old rats was homogenized in Tris-HCI buffer. IOOmM, pH 7.4 at 0 C. The cytosol fraction was obtained and endogenous androgens were removed with charcoal (1 mg;mg protein) during 18h at 0 C. Incubations were carried out at 17 c‘. 0 C and 50 C during 30’ with JH-T. ‘H-DIOL and “H-dihydrotestosterone(DHT). Binding to macromolecules was analyzed by 5- ?O”,, sucrose gradient centrifugation. it was found that ABP is not present in IO-day-old rats but it is present in 20- and 60-day-old animals. Some characteristics of the ?O-day-old rat testis ABP were studied. It can bind tritiated T. DIOL or DHT in a similar manner. No difference was detected incubating at 0 C. 27 C or 50 C. A 46 x IO “M concentration of non-radioactive DIOL was necessary to displace 3H-DIOl_. from ABP showing a fairly high binding capacity for this protein. The presence of ABP in the testis at the time of meiosis suggests that it might be playing a role, along with androgcns. ;~t thib atage of developmen t 160. Androgen “priming” increases the FSH induced produetion of testicular androgen binding protein (ABP) ‘HANSSON. V.. ‘FRENCH, F. S.. 3R~~z~~, E. M.. ~WEDDI~GTO~, S. C. and ‘NAYFEH, S. N.. ‘Rikshospitalet. Oslo, Norway, ‘University of North Carolina at Chapel Hill. U.S.A. and ‘Karolinska sjukhuset, Stockholm, Sweden Testicular androgen binding protein (ABP) is produced by the testis. secreted into the testicular fluid and carried into the epididymis by the way of efferent ducts. ABP is completely dependent on FSH stimulation ; it disappears rapidly after hypophysectomy and is dramatically stimulated by FSH. but not by LH or androgens alone (Nature New Biology 246, 56. 1973). The sensitivity of the testis to FSH, as measured by the ABP response, decreases rapidly with the time after hypophysectomy. f-fowever, pretreatment with high doses of androgens (2 mg TP per day) greatly enhanced the sensitivity. The mechanism by which testosterone propionate increases the sensitivitv of the testis for FSH is not known, however. several possjbitities might be considered. (a) It might stimulate the formation of FSH receptors on Sertoli cell membranes. (b) It might increase the blood flow through peritubular capillaries, therby allowing more FSH to reach its target sites in the seminiferous tubules. (c) It might also diminish the degradation of FSH in the liver. kidneyorwithinthetestisitself. (d) Finallv, testosterone propionate- might decrease the destruction 01 ABP within the testis (decreased proteolyric activity or inactivation of specific inhibitors). 161. Steroid binding proteins in rat preputial giand cytosol FELDMAN, M., VOIGT. W. and HSIA, S. L.. Departments of Biochemistry. Phar~lacology and Dermdtology, University of Miami School of Medicine. Miami, Florida. U.S.A. The preputialgland of the rat is an ondrogen-sensitive organ present in both male and female animals. The similar histology and androgen responsiveness of human sebaceous
cells. make the preputial gland an interesting model tbr the study of sebaceous glands. incubation of the lOS.OOOg supernatant of preputial gland homogenates with [H’] dihydrotestosterone(DHT) or[HJ] testosterone showed two peaks of radioactivity on sucrose density gradient centrifugation. corresponding to 2S and 4S complexes. Both peaks were destroyed by heating cytosol at 55 C for IOmin or treating with pronase. but not ribonuclease. The 4S protein is androgen specific. and does not bind estrogens, whereas the ZS protein binds estrogens with greater affinity than androgens, exhibiting linear binding with up to 31)OnM concentration of steroid. The association constanr of the 4s protein for DWT as determined by bat&wise gel equilibration was lO”M -I. Binding of DHT to the 4s component is greater at 37 C than at either 25 c‘ or 4 f. whereas at the lower temperatures binding to the 1s cornp[~ncllt predominates. The 1s protein has a molecular weight in the order of’ 15,000 as determined by gel filtration.
162. Inhibition of uptake, metabolism and binding of androgen in epididymis of male rats irl viva: Comparison of estradiol and cyproterone acetate TINT)AI.L. D. J., FRENCH. F. S. and NAYFEN. S. N.. Departments of Pediatrics and Biochemistry and The Laboratories for Reproductive Biology. University of North Carolina School of Medicine. Chapel Hill, U.S.A. Effects of a single dose of estradiol (E) on uptake, binding and metabolism of androgen in the rat epididymis itt ldluo were compared with the effects of a stmilar dose of cyproterone acetate (CA). Adult rats were castrated 18 h before the experiment. E or CA was injected i.v. 5 min prior to ‘Htestosterone (3H-T) or 3H-5r-dihydrotestosterone (jHDHT). and the epididymides were removed 3 h later. Total androgen uptake into epididymal supernatant was inhibited 58”,, by E as compared to 38”,, by CA. %-Reduction of ‘H-T was inhibited by E resulting in diminished formation of 3H-DHT. CA did not affect the metabolism of JH-T. E reduced total bound radioactivity in cytosol fractions to a greater extent than CA when either 3H-T or 3H-DHT was injected. E inhibited binding of 3H-DHT to both the intracellular cytoplasmic receptor (CR) and the intraluminal androgen binding protein (ABP). CA inhibited binding to CR without affecting binding to ABP. Androgen uptake into epididymal nuclei was inhibited both by E (95”,,) and CA (83”,,). E abolished all nuclear binding of 3H-DHT, white some bound radioactivity (20”” of the control) remained after treatment with CA. Both E’and CA caused bound radioactivity in nuclear extracts to decrease in direct proportion to the inhibition of binding tn CR.
SE. Steroid receptors: Cortieoids 163. Glucocorticoid binding macromolecules in chicken liver cytosol COC~~ET. C. and CHAMBAZ, E. M., C.E.R.M.O.. Universitk Scientifique et Medicale, Grenoble. France Glucocorticoid binding proteins have been extensively studied in several target organs, especially in the rat. Three different binders have been characterized in rat liver cytosol, of which only one might be involved in the mechanism of steroid specific enzyme induction in this tissue. The chicken liver may offer an interesting experimental model for the