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Search for other papers by D. Y. WANG in
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SUMMARY
The binding by plasma from man, rabbit and rat of the sulphate esters of dehydroepiandrosterone, testosterone, 17-acetoxypregnenolone and pregnenolone has been studied using equilibrium dialysis.
The plasma of these species had a large capacity for binding dehydroepiandrosterone sulphate and testosterone sulphate. A large capacity for binding pregnenolone sulphate and 17-acetoxypregnenolone sulphate was observed with plasma from man and rabbit.
The percentage binding of the steroid sulphates studied was higher for human plasma than for rabbit plasma and, in the case of the sulphates of dehydroepiandrosterone and testosterone, for rat plasma. The higher percentage binding by human plasma was confirmed using multiple equilibrium dialysis.
Evidence is presented that suggests that the sulphate esters of dehydroepiandrosterone, testosterone and pregnenolone are bound to the same sites. Dehydroepiandrosterone sulphate was displaced by non-steroidal organic sulphates, suggesting that the binding sites involved are not specific for steroid sulphates.
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Search for other papers by D. Y. Wang in
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ABSTRACT
Mice were immunized with a mixture of four steroid antigens: dehydroepiandrosterone (DHA), oestradiol, progesterone and testosterone linked to bovine serum albumin through the 7, 6, 11α and 17β positions respectively. The response to immunization varied widely with no one mouse producing an optimal response to all four steroids. In the two fusion experiments performed, antibodies to all four antigens were developed. Data are presented which show that the immune response of the spleen donor is related to the relative numbers and quality of the antibodies produced to each steroid. Despite the structural identity of the progesterone and testosterone haptens, antibodies elicited in response to their respective antigens could readily be distinguished from each other.
From the large number of monoclonal antibodies obtained those most useful for radioimmunoassay were three high affinity antibodies to oestradiol and two antibodies raised against DHA but with high affinity for DHA sulphate.
J. Endocr. (1984) 100, 367–376
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Search for other papers by VICKY AMOR in
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SUMMARY
The rates of synthesis of DNA, RNA and protein of mouse mammary gland explants in organ culture have been determined. Stimulation with insulin resulted in maximal rates of synthesis of these components, all occurring between 18 and 22 h of culture.
The use of metabolic inhibitors of DNA, RNA or protein synthesis showed that after insulin stimulation, inhibition of any one of these processes was associated with a reduction in the synthesis of the other two components. Also the maximal rate of protein synthesis is governed by the net amount of RNA formed throughout the period of culture.
Evidence is presented that the stimulation of DNA, RNA or protein synthesis by insulin is not due to increased transport of amino acids and that insulin appears to act rapidly on processes which subsequently lead to enhanced synthetic activity.
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Search for other papers by D Wang in
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Search for other papers by DM Stocco in
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The rate-limiting step of steroidogenesis is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane which involves a cycloheximide-sensitive newly synthesized protein. A protein believed to carry out this function was recently cloned from MA-10 mouse Leydig tumor cells and named the steroidogenic acute regulatory protein (StAR). In the present study, we evaluated the expression and regulation of StAR in primary cultures of rat Leydig cells. StAR mRNA was expressed in Leydig cells as two major transcripts of 3.8 and 1.7 kb and one minor transcript of 1.2 kb. Induction of StAR mRNA transcripts could be detected as early as 30 min after the addition of human choriogonadotropin (hCG) with peak levels attained between 2 and 4 h. hCG in concentrations of 0.1-10 ng/ml caused a dose-dependent increase in StAR mRNA expression. hCG administered at a dose of 10 ng/ml increased the 3.8 kb StAR mRNA level about 14-fold and the 1.7 kb StAR mRNA level about 13.6-fold. hCG-stimulated StAR mRNA was associated with increased StAR protein levels as determined by immunoblot analysis (a 4.5-fold increase). Murine interleukin-1 alpha (mIL-1 alpha) at a concentration of 100 ng/ml inhibited hCG-induced cytochrome P450 side-chain cleavage (P450 scc) mRNA expression and testosterone formation almost completely. Interestingly, mIL-1 alpha had no effect on hCG-induced StAR mRNA or protein levels. Furthermore, mIL-1 alpha (10 ng/ml) decreased conversion of (22R)-hydroxycholesterol to testosterone while the conversion of pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone and androstenedione to testosterone were not affected. These results indicate that the major inhibitory effect of IL-1 on Leydig cell function occurs at the level of P450 scc.
Search for other papers by R. C. HALLOWES in
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Search for other papers by D. Y. WANG in
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Search for other papers by D. J. LEWIS in
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SUMMARY
Explants of mammary glands from mature virgin and pregnant Sprague—Dawley rats were maintained in organ culture for up to 96 h. The effects of insulin, corticosterone, ovine prolactin and bovine growth hormone on the synthesis of DNA, RNA and casein in the explants were studied.
DNA synthesis in explants from virgin rats was maintained by insulin but was not increased by the addition of the other hormones tested. DNA synthesis in explants from pregnant rats was increased by insulin, and the addition of corticosterone and either prolactin or growth hormone to the culture medium increased this synthesis. The rate of RNA synthesis in explants from virgin rats was similar in medium 199 with or without additional hormones. RNA synthesis in explants from pregnant rats was increased by the addition of insulin or insulin plus corticosterone to the medium. In explants from both virgin and pregnant rats the maximal rate of hormone-stimulated DNA or RNA synthesis occurred during the first 24 h of culture.
Casein synthesis, as measured by the uptake of 32P-labelled orthophosphate by explants from virgin and pregnant rats, was increased by insulin plus corticosterone plus either prolactin or growth hormone. The rate of casein synthesis was maximal between 48 and 72 h and was reduced by actinomycin D. In the pregnant rats no significant differences were demonstrated between the effects of the hormones on DNA, RNA or casein synthesis in explants from the 5th, 10th, 16th or 19th day of pregnancy.
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Search for other papers by R. D. BULBROOK in
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Search for other papers by A. SNEDDON in
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Search for other papers by T. HAMILTON in
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SUMMARY
The disappearance of isotopically labelled dehydroepiandrosterone, testosterone and their sulphates from the peripheral circulation of man, rabbit and rat has been investigated.
Metabolic clearance rates, distribution volumes and half-lives have been determined for these compounds in the above species.
In man, the steroid sulphates have a much lower metabolic clearance rate than the corresponding free steroids. This large difference stems from longer half-lives and lower distribution volumes of the former.
In the rabbit or rat the steroid sulphates and the appropriate free steroids do not show such marked differences in their metabolic clearance rates: the half-lives and distribution volumes are comparable.
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Search for other papers by STRETTON YOUNG in
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Search for other papers by R. D. BULBROOK in
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SUMMARY
(1) The incorporation of [1,2-3H]testosterone in vivo into various tissues of virgin, pregnant, post-partum and tumour-bearing female rats was studied.
(2) In virgin female rats the clearance of radioactivity from mesenteric fat, mammary gland, uterus, spleen, lung and blood was similar. This similarity in the rates of clearance of radioactivity for all the tissues examined was also found for the tissues of pregnant, post-partum, and tumour-bearing rats.
(3) After the administration of [1,2-3H]testosterone different amounts of radioactivity were found in each of the tissues examined. In virgin rats the levels of incorporation were fat > uterus ≥ mammary gland > lung > blood ≥ spleen. This pattern was also obtained in post-partum and tumour-bearing animals; the tumours in the latter behaved in a similar way to normal mammary glands. In the pregnant rat, the foetus incorporated the least amount of radioactivity.
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Search for other papers by VICKY AMOR in
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Search for other papers by R. D. BULBROOK in
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In experiments on the effect of insulin on the stimulation of incorporation of radioactive thymidine into the DNA of pregnant mouse mammary gland explants in culture, considerable variation between the results of successive experiments was noticed. Dependence of the extent of incorporation on the stage of pregnancy was investigated as a possible cause.
Cultures of explants of mammary gland were made (Trowell, 1959) from Schneider mice at various stages of their first pregnancy. The length of pregnancy was taken from the time of appearance of a vaginal plug. Insulin (Sigma Chemical Co., 24·1 i.u/mg.) was used at a concentration of 5 μg./ml. medium. After 24 hr. of incubation the cultures were pulsed for 4 hr. with [methyl-3H]thymidine (18·3 c/m-mole; Radiochemical Centre, Amersham) at a concentration of 0·5 μc/ml. medium (Stockdale & Topper, 1966). The tissue was removed and weighed, the acid-insoluble material hydrolysed (Mahin & Lofberg, 1966), and
Search for other papers by J.-F. Wang in
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Search for other papers by L. J. Fraher in
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Search for other papers by D. J. Hill in
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ABSTRACT
We have characterized an insulin-like growth factor (IGF)-binding protein present in ovine amniotic fluid. Using an activated charcoal-binding assay, whole amniotic fluid specifically bound approximately 20–30% of 125I-labelled human (h) IGF-II added, while the binding of 125I-labelled hIGF-I was minimal. Radioimmunoassay for IGF-I or -II in ovine biological fluids showed that values in amniotic fluid were 9- to 13-fold less than in fetal plasma, while gel filtration of amniotic fluid on Sephadex G-50 eluted with 1 mol acetic acid/l revealed no additional binding activity which had been complexed to IGFs at neutral pH. Together, these observations suggest that the binding activity in amniotic fluid is largely unsaturated. Competition studies for the displacement of 125I-labelled IGF-II binding to amniotic fluid by increasing amounts of unlabelled IGF-I or -II, using the charcoal assay, showed that IGF-II was 30-fold more potent than IGF-I. Scatchard analysis revealed a single class of binding site for IGF-II, with a binding affinity of 0·68 ±0·18 litres/nmol (mean ± s.d., n = 3). Ligand blot analysis of amniotic fluid by separation on 8% SDS-PAGE, transfer to nitrocellulose membranes, incubation with 125I-labelled IGF-II and autoradiography revealed a single band of IGF-binding protein with approximate molecular size of 38 kDa. Additional IGF-binding species of 20, 28, 48 and > 180 kDa were present in ovine fetal plasma. Separation of amniotic fluid on Concanavalin A–Sepharose revealed that it had little carbohydrate content. These results show that ovine amniotic fluid contains an unsaturated, non-glycosylated IGF-binding protein with high affinity for IGF-II. These characteristics differ from those of the IGF-binding proteins purified from human amniotic fluid.
Journal of Endocrinology (1990) 127, 325–333
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Search for other papers by G P Becks in
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Abstract
Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10−8–10−3 mol/l), dibutyryl cAMP (0·25–1 mmol/l), forskolin (5–20 μmol/l) or 12-0-tetradecanoylphorbol-13-acetate (TPA; 10−11–10−6 mol/l), with or without exposure to TSH (200 μU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [γ-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10−6–10−3 mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10−11–10−6 mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol. An 80 kDa species of immunoreactive PKC was recovered from the membranes of cells cultured in 3H medium, but its presence in membrane was decreased following incubation with TSH. The actions of TSH on intracellular PKC distribution were reversed by prior incubation with TPA, which itself stimulated the appearance of membrane PKC immunoreactivity. These results suggest that the ability of TSH to suppress IGFBP release does not depend primarily on cAMP stimulation, but may involve changes in the activation of PKC, possibly inhibition or down-regulation.
Journal of Endocrinology (1994) 141, 231–242
