Glucocorticoid effects on somatomedins and somatomedin inhibitors

Glucocorticoid excess may be associated with poor growth despite normal levels of GH and adequate nutrition. Steroid-induced growth failure could be mediated by defective generation and/or action of somatomedins. To probe potential mechanisms, we examined the effect of corticosteroid administration on net somatomedin activity, immunoreactive somatomedin-C, and separated biologically active somatomedins and somatomedin inhibitors. Twelve children receiving alternate day steroid therapy had circulating somatomedin activity measured by porcine cartilage bioassay. Somatomedin activity fell 6 h after steroids [from 1.02 +/- 0.09 (+/- SEM) to 0.35 +/- 0.07 U/ml; P less than 0.001] and then rose toward normal. No significant change in somatomedin activity occurred during the day off therapy. Further studies were conducted in normal subjects given a single 60-mg dose of prednisone. Six hours after prednisone, somatomedin activity (rat cartilage bioassay) decreased by 46% (P less than 0.01), yet somatomedin-C did not change. To pursue this discrepancy, serum was fractionated on Sephadex G-50, pH 2.4, and separated somatomedin and somatomedin inhibitory bioactivity was measured. Biologically active somatomedins (Kav, 0.50-0.63) were comparable before and after prednisone treatment, as was inhibitory activity found at Kav 0.13-0.25. In contrast, somatomedin inhibitory activity at Kav 0.25-0.38 doubled (111 +/- 8% inhibition of somatomedin action vs. 54 +/- 11%; P less than 0.005) after prednisone therapy. The somatomedin inhibitor in these fractions blunted serum stimulation of sulfate, thymidine, and uridine uptake by test cartilage. These inhibitory effects could not be attributed to direct steroid action, as levels were less than 2 micrograms/dl in inhibitory fractions and addition of cortisol and prednisolone to the bioassay system failed to decrease somatomedin activity. We conclude that glucocorticoid administration is followed by an increase in circulating somatomedin inhibitors. Such inhibitors may explain the steroid-induced fall in net somatomedin activity and contribute to impaired growth.     

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin‐1β

Objective

Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin‐1β (IL‐1β), and combinations thereof.

Methods

Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17β‐estradiol, and dehydroepiandrosterone (DHEA), with or without IL‐1β. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix.

Results

Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 μg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration‐dependent stimulation of integration (with maximum values observed with DHEA at 3 × 10⁻⁵M, testosterone at 10⁻⁸M, and 17β‐estradiol at 10⁻¹¹M). For testosterone and 17β‐estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL‐1β at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL‐1β and sex hormones, integration of articular cartilage was reduced.

Conclusion

This is the first study to demonstrate that steroid hormones such as 17β‐estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL‐1β. In the absence of IL‐1β or after neutralization of IL‐1β, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.

     

Regulation of lactogenic hormone binding in rat liver by steroid hormones.

We have measured the effects of testosterone propionate, medroxy-progesterone acetate and cortisol on the binding of ovine [125I]iodoprolactin to 100,000 X g particulate fractions from liver of normal and estrogen-treated female rats. In untreated animals 6.7 +/- 1.1% (SD) of the radioactivity added to 0.5 mg of membrane protein was specifically bound to the hormone receptor. Specific binding was significantly (P less than .05) decreased after 7 daily doses of testosterone (1.0 mg) to 2.8 +/- 1.4%, medroxyprogesterone (0.25 mg) to 2.7 +/- 0.2% and cortisol (5.0 mg) to 3.1 +/- 1.3%. The serum prolactin concentration, 4.2 +/- 3.4 ng/ml in normal animals, was not affected by the hormone treatment. Ethinyl estradiol, 10 mug/day for 7 days, increasing the binding of [125I]iodo-prolactin to 16.6 +/- 6.0% and increased serum prolactin to 50.6 +/- 11.5 ng/ml. Simultaneous administration of testosterone, medroxyprogesterone or cortisol with estradiol did not diminish the estradiol-induced increase in serum prolactin, but completely prevented the increase in prolactin binding. Testosterone or cortisol given to animals pretreated with estradiol suppressed prolactin binding from 16.4 +/- 4.2% to less than 2.5% after 48-72 h. Parellel results were obtained with 125I labeled human growth hormone whereas 125I labeled-insulin binding was not affected by these treatments. Scatchard analysis showed that the decrease in lactogenic hormone binding was due to a reduced concentration of receptors with no significant change in affinity. Since serum levels of prolactin were not changed, we conclude that treatment with testosterone, medroxyprogesterone, and cortisol decreased lactogenic hormone binding by a direct action on the liver.     

Free fatty acids do not influence the concentrations of free steroid hormones in serum under physiological conditions

In several recent studies it has been suggested that FFA may influence the concentrations of unbound steroid hormones in serum, but the experimental design of these studies has been questioned. We have reexamined the effects of oleic acid on the unbound concentrations of several steroid hormones in serum, including cortisol, testosterone, and estradiol. The results demonstrate that under physiological conditions, oleic acid does not affect the unbound concentrations of these hormones when assays are carried out with whole serum.     

Polychlorinated biphenyls (PCB 153 and PCB 126) action on conversion of 20-hydroxylated cholesterol to progesterone, androstenedione to testosterone, and testosterone to estradiol 17beta.

To look for one of the possible mechanisms of action we investigated the effect of two congeners of polychlorinated biphenyls (PCB153 as one of the most prominent environmental contaminants and PCB 126 as one of the most toxic contaminants similar to dioxin) on the cellular conversion of steroid precursors as an indicator for enzyme activity (20-hydroxylated cholesterol to progesterone for P450 (scc,) androstendione to testosterone for 17-beta-HSD, and testosterone to estradiol for P450 (arom)). The net synthesis and secretion of particular steroids was used as the indicator of enzyme activity. Co-culture of pig granulosa and theca cells isolated from small (SF) and large (LF) follicles, was carried out in medium M199 supplemented with 100 ng/ml of PCB 153 or 100 pg/ml of PCB 126. The inhibitory action of both PCB 126 and PCB 153 on progesterone secretion by cells isolated from SF and LF follicles was reversed in the presence of 20-hydroxylated cholesterol. The addition of PCB 126 into the culture medium caused a decrease in testosterone secretion by cells isolated from both SF and LF and this effect was reversed in the presence of androstendione. The inhibitory action of PCB 153 on testosterone secretion was reversed by the addition of androstendione to the culture medium in SF, while it caused even additional stimulatory action on cells collected from LF. No effect of PCB 126 and statistically significant decrease in estradiol secretion by cells collected from SF under the influence of PCB153 was observed. The inhibitory effect of PCB 153 was reversed when the culture was supplemented with testosterone. The opposite effect of both tested congeners on estradiol secretion in both basal and testosterone supplemented culture was seen in LF. PCB 126 increased it while PCB 153 decreased both, the basal and testosterone-stimulated estradiol secretion. In conclusion, the presented results suggest that the effect of both PCBs on steroid secretion observed in an early stage of the follicular phase of the estrus cycle is due to the inhibition of cholesterol mobilisation and thus insufficient substrate availability for hormone synthesis. On the contrary, in large preovulatory follicles inhibition of testosterone secretion is due to their action on 17-beta-HSD while stimulatory or inhibitory action on estradiol secretion is the result of their action on P450 aromatase activity.     

Testicular adrenal rests: evidence for luteinizing hormone receptors and for distinct types of testicular nodules differing for their autonomization.

We report one patient with 21-hydroxylase deficiency and associated bilateral macro-orchidism caused by nodular hyperplasia of testicular adrenal rests (TAR). The boy, referred to us when 10 years old, was born with bilateral cryptorchidism that was treated unsuccessfully with i.m. injections of human chorionic gonadotropin (hCG) and later on with orchidopexy. He was treated with oral dexamethasone (0.625 mg per day) for the following 13 years. After one year, there was a marked reduction in steroid hormone levels (17-hydroxyprogesterone (17-OH P) from 27.2 to 1.2 nmol/l, testosterone from >104 to 4.8 nmol/l, estradiol (E(2)) from 481 to 33 pmol/l). After the same period of time, both testicular volume and nodularity decreased: from 45 to 18 ml and from numerous to four nodules in the right testis, and from 40 to 13 ml and from numerous to three nodules in the left testis. At the third year, there were transient increases in serum gonadotropins, testicular volume (right testis = 25 ml, left testis = 20 ml) and steroid hormones, including cortisol (serum ACTH and dehydroepiandrosterone sulfate remained suppressed). At the fourth year of follow-up, there were still four nodules in the right testis and three in the left testis. The LH-dependency (which implies possession of LH/hCG receptors) of these nodules was also substantiated by their steroidogenic response to an acute i.m. hCG test. An exogenous ACTH stimulation test increased serum 17-OH P and cortisol. Since these nodules, unlike the majority of those present initially, were not suppressed by the corticosteroid therapy and since they were not detected when the patient returned for control at 23 years of age, they had partial autonomy from ACTH. At 23 years of age, the patient had a single nodule in the right testis (right testis volume = 13 ml, left testis volume = 10 ml), which should have accounted for the consistent difference in size between the two gonads. Serum LH was about 7 mU/l and FSH about 23 mU/l. The responsiveness of plasma steroid hormones to hCG had changed quantitatively and qualitatively. Secretion of cortisol was absent, secretion of 17-OH P and testosterone was reduced, and secretion of E(2) was much increased. The ACTH stimulation test showed that serum cortisol did not respond, while the other steroids responded in the order of 17-OH P>E(2)> testosterone. We conclude that there were three different groups of TAR when the patient was already 10 years old: (i) ACTH-sensitive (the majority), (ii) partially ACTH-insensitive but LH/hCG-sensitive (three nodules in the left testis and three in the right testis), (iii) almost entirely ACTH-insensitive and partially hCG-insensitive (a single nodule in the right testis). Probably, the never suppressed gonadotropin levels (presumably due to the bilateral testicular damage subsequent to the cryptorchid state) and the hCG therapy were major etiological factors for the appearance of the second and third population of TAR.     

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta

OBJECTIVE: Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1beta (IL-1beta), and combinations thereof. METHODS: Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17beta-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1beta. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix. RESULTS: Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 microg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 x 10(-5)M, testosterone at 10(-8)M, and 17beta-estradiol at 10(-11)M). For testosterone and 17beta-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1beta at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1beta and sex hormones, integration of articular cartilage was reduced. CONCLUSION: This is the first study to demonstrate that steroid hormones such as 17beta-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1beta. In the absence of IL-1beta or after neutralization of IL-1beta, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.     

Serum cortisol binding capacity and cortisol concentration in the pregnant baboon and its fetus during gestation.

The cortisol binding capacity of serum from 11 pregnant baboons (38 samples) and from 7 baboon fetuses delivered prematurely or at term was measured after removal of endogenous steroids. Values for maternal serum collected between 60 and 120 days after mating (59.0 +/- 6.4 mug/100 ml, mean +/- SD) were greater than those for serum collected at term (42.3 +/- 4.9 mug/100 ml). The cortisol-binding capacity of fetal serum collected between 100 and 132 days' gestation was similar to that of the corresponding maternal sample, but at term was only 50% of the maternal value. The rate of clearance of cortisol from both fetal and maternal serum may therefore increase progressively during the last trimester of pregnancy. This effect is likely to be more marked in the fetus. The cortisol binding capacity of 15 serum samples from 9 non-pregnant baboons was 33.4 +/- 5.5 mug/100 ml. Mestranol2 (administered 200 mug/day im for 15 days) significantly increased the serum cortisol binding capacity. The concentration of cortisol in maternal serum from 7 pregnant baboons (10 samples) was 44.0 +/- 8.4 mug/100 ml and was independent of the state of gestation. In fetal serum the cortisol concentration was 4 mug/100 ml before 168 days' gestation and reached 49 mug/100 ml after normal delivery at term. These findings suggest that the mechanisms for production of cortisol by the fetus mature as gestation progresses. The physiological significance of the marked difference between the cortisol concentration and the cortisol binding capacity of fetal serum awaits elucidation.     

Nutrition and somatomedin. V. Action and measurement of somatomedin inhibitor(s) in serum from diabetic rats.

Serum from normal rats stimulates growth cartilage in vitro; this stimulation is attributed to somatomedin activity. In contrast, serum from diabetic rats may produce dose-response lines with negative slopes and, in combination studies, suppress the stimulatory activity of serum from normal rats; this is attributed to somatomedin inhibitory factor(s). Somatomedin inhibitory activity in serum from diabetic rats cannot be attributed to recognized catabolic factors, such as glucocorticoids or free fatty acids. The inhibitory activity is resistant to 100 C heat at neutral pH but is partially removed by 100 C heat at pH 5.5. Somatomedin inhibitory activity can be estimated as the ability to decrease the stimulation of rat cartilage incubated for 2 days with serum from normal rats. With this method, serum from diabetic rats provides linear inhibition of both sulfate and thymidine uptake. This procedure is simple, reproducible, and allows detection of inhibitory activity in as little as 5 microliter whole serum. It allows identification of individual samples enriched in inhibitory activity and should be useful in further studies of somatomedin inhibitory factor(s).     

Somatomedin, growth and nutritional status

Serum somatomedins, or insulin-like growth factor(s) (IGF), originally characterized as primarily GH-dependent peptides, were found to also be dependent on insulin levels and nutritional status. Four properties characterize somatomedin peptides: their concentrations in serum are growth hormone dependent; they possess insulin actions in extraskeletal tissues; they promote the incorporation of sulfate into proteoglycans of cartilage; and they stimulate DNA synthesis and cell multiplication in certain types of cultured cells. Reduced somatomedin C levels are found in children with severe protein-energy malnutrition. Plasma concentration of growth hormone and cortisol are both elevated and there are low levels of insulin and somatomedin C. There is evidence that the ability of somatomedin C to stimulate cartilage is modulated by somatomedin inhibitor, factor that may act to limit growth in conditions of hormonal and/or nutritional deficiency. Dietary energy and protein appears to be particularly important for both generation of somatomedins and their action on growing cartilage. Measurement of somatomedins C concentration shows promise as a means for monitoring the response of malnourished patients and rats to nutrition repletion.     

Exogenous estrogen and endogenous sex hormones.

Estrogen replacement therapy is widely used to treat menopausal symptoms and prevent osteoporosis. The mechanism of these and other estrogen effects is currently under investigation. We studied the plasma steroid hormone and sex hormone binding globulin levels in frozen plasma obtained from 977 women aged 50 to 79 years from 1972 to 1974. Almost all of the 301 women who reported current use of noncontraceptive estrogen were taking conjugated estrogen by mouth; none reported use of a progestin. Women taking estrogen were significantly younger, thinner, and more likely to smoke cigarettes than women not taking estrogen. Sex hormone binding globulin and all endogenous hormones except testosterone were negatively correlated with age; estradiol was positively and cortisol and sex hormone binding globulin were negatively associated with obesity. After adjusting for age and obesity, dehydroepiandrosterone sulfate, androstenedione, and free testosterone were significantly lower in women currently taking estrogen than in women not using estrogen. These differences were independent of cigarette smoking. As expected, estrogens (including free estradiol), sex hormone binding globulin, and cortisol levels were higher in treated than untreated women. The possibility that some of the benefits and risks of replacement estrogen are secondary to altered adrenal steroid metabolism and androgen levels needs further evaluation.     

Does statin therapy influence steroid hormone synthesis?

Statins reduce cholesterol and isoprenoid de novo biosynthesis as well as receptor mediated uptake of cholesterol for steroidogenesis. The present randomized placebo-controlled trial investigated whether pravastatin (40 mg/day) reduces the plasma concentrations of steroid hormones as well as of gonadotropins. Patients (n = 22; 15 males, 7 females) were treated with pravastatin (40 mg/day) or placebo. Levels of total and LDL cholesterol, the steroid hormones estradiol, testosterone, cortisol and dehydroepiandrosterone sulphate (DHEAS) as well as FSH and LH were studied. Pravastatin led to a significant reduction of total cholesterol and LDL cholesterol. There was no significant change in estradiol, testosterone, cortisol or DHEAS plasma concentrations. There was no compensatory change in FSH or LH. It is concluded that pravastatin does not alter steroid hormones or gonadotropins in a clinically applicable dose, which significantly reduces total and LDL cholesterol.     

The effects of insulin and growth hormone on the release of somatomedin by the isolated rat liver.

Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 muU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 muU/ml of insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itsel to stimulate somatomedin release. When added to a medium containing 1000 muU/ml of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 muU/ml insulin, a significant stimulation of somatomedin release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels.     

Catabolic/Anabolic Imbalance Is Accompanied by Changes of Left Ventricular Steroid Nuclear Receptor Expression in Tachycardia-Induced Systolic Heart Failure in Male Pigs

BackgroundSteroid hormones play an important role in heart failure (HF) pathogenesis, and clinical data have revealed disordered steroidogenesis in male patients with HF. However, there is still a lack of studies on steroid hormones and their receptors during HF progression. Therefore, a porcine model of tachycardia-induced cardiomyopathy corresponding to HF was used to assess steroid hormone concentrations in serum and their nuclear receptor levels in heart tissue during the consecutive stages of HF.Methods and ResultsMale pigs underwent right ventricular pacing and developed a clinical picture of mild, moderate, or severe HF. Serum concentrations of dehydroepiandrosterone, testosterone, dihydrotestosterone, estradiol, aldosterone, and cortisol were assessed by enzyme-linked immunosorbent assay. Androgen receptor, estrogen receptor alpha, mineralocorticoid receptor, and glucocorticoid receptor messenger RNA levels in the left ventricle were determined by qPCR.The androgen level decreased in moderate and severe HF animals, while the corticosteroid level increased. The estradiol concentration remained stable. The quantitative real-time polymerase chain reaction revealed the downregulation of androgen receptor in consecutive stages of HF and increased expression of mineralocorticoid receptor messenger RNA under these conditions.ConclusionsIn the HF pig model, deteriorated catabolic/anabolic balance, manifested by upregulation of aldosterone and cortisol and downregulation of androgen signaling on the ligand level, was augmented by changes in steroid hormone receptor expression in the heart tissue.     

Age, body mass index, race and other determinants of steroid hormone variability: the HERITAGE Family Study.

OBJECTIVE AND METHODS: To investigate from the HERITAGE Family Study database, 13 steroid hormones (androstane-3alpha, 17beta-diol glucuronide, androsterone glucuronide, cortisol, dehydroepiandrosterone (DHEA), DHEA ester (DHEAE), DHEA sulfate (DHEAS), dihydrotestosterone (DHT), estradiol, 17-hydroxyprogesterone, progesterone, pregnenolone ester, sex hormone binding globulin (SHBG) and testosterone in each sex for their relationships with age, body mass index (BMI), race and key lifestyle variables. Sample sizes varied from 676 to 750 per hormone. Incremental regression methods were used to examine the contributions of the variables to steroid hormone variability. RESULTS: Age was a major predictor for most steroid hormones. The greatest contribution of age was a negative relationship with DHEAS (R(2)=0.39). BMI was also associated with the variability of several steroid hormones, being the most important predictor of SHBG (R(2)=0.20) and of testosterone (R(2)=0.12) concentrations. When age and BMI were included, race still contributed significantly to the variations in cortisol (R(2)=0.02 for men and 0.04 for women), DHT (R(2)=0.02 for men and 0.03 for women), and progesterone (R(2)=0.03 for women). Nevertheless, race appeared to be less important than age and BMI. In addition, lifestyle indicators (food and nutrient intakes, smoking and physical activity) influenced steroid hormone variability. Their contributions, however, were minor in most cases once age, BMI and race had been taken into account. CONCLUSIONS: We conclude that age was the most important factor, followed by BMI, race and lifestyle factors in explaining steroid hormone variability.     

Effect of luteinizing hormone and cyclic adenosine 3',5'-monophosphate on steroidogenesis in the ovarian follicle of the rabbit.

Mature ovarian follicles have been isolated from estrus rabbits and the effects of gonadotropin and cyclic AMP on steroidogenesis in this tissue determined. Gonadotropins used include LH, FSH, and prolactin; follicular levels of progesterone, 17-hydroxyprogesterone, testosterone and 17beta-estradiol in all experiments were quantitated by radioimmunoassay. Incubation of follicles with LH in concentrations ranging from 0.05 to 50 mug/ml medium yielded increases in the total ng steroid/follicle. Five mug LH/ml gave a maximal response with no further increase in steroid concentration when the LH was raised to 50 mug/ml. Prolactin had no effect on follicular steroidogenesis while the stimulatory action of a FSH preparation could only be partially attributed to LH contamination. Cyclic AMP also proved to be a potent stimulatory agent in follicular steroidogenesis with maximal increases at 20 mumoles/ml and a decline in the ng steroid/follicle when cyclic AMP was raised to 30 and 40 mumoles/ml. The effects of LH and cyclic AMP proved to be nonadditive; incubation of follicles simultaneously with 5 mug LH/ml and 20 mumoles cyclic AMP/ml yielded steroid concentrations which were no different than levels following incubation with either agent alone. Taken together, these results demonstrate that both LH and cyclic AMP cause increases in radioimmunoassayable steroid in the rabbit follicle in vitro and that LH probably acts by way of a cyclic AMP intermediate.     

Effects of estrogens on adrenal 3 beta-hydroxysteroid dehydrogenase in ovariectomized women.

The acute and chronic effects of estradiol (E2) on the serum levels of four delta5,3-beta hydroxysteroids and their four delta4, 3-keto products were studied in four ovariectomized women with and without adrenal stimulation by ACTH. Six hour infusions of saline and of synthetic 1-24 ACTH were administered and later repeated with a two hour infusion of E2 50 mug/h. The patients were then given 50 mug of ethinyl estradiol (EE2) p.o. for 4 to 6 weeks and the control and ACTH infusions were again repeated. Levels of pregnenolone3 (Pe), 17alpha-hydroxypregnenolone (17 Pe), progesterone (Po), 17alpha-hydroxyprogesterone (17 Po), dehydroepiandrosterone (DHEA), androstenedione (Adione), androstenediol (Adiol), and testosterone (T), as well as cortisol and DHEA-sulfate were measured by radioimmunoassay on serum samples taken at 1200 and 1300 h. There was no significant effect of E2 or EE2 in the doses administered with or without exogenous ACTH on 3 betaOHSD activity as reflected in absolute steroid levels or in the ratio of concentrations of each delta5:delta4 steroid pair. During the 4th and 5th hour of ACTH infusion, the plasma level of 17 Pe (mean 22.5-fold stimulation) was most elevated, followed by 17 Po (12.5-fold), Pe (10-fold), cortisol (5.9-fold) and Po (4.5-fold), with smaller increases for the other steroids. These results, as well as the pattern of change in plasma levels in one of the subjects in whom fifteen minute samples were measured, provide further evidence suggesting that the major pathway for cortisol biosynthesis in vivo proceeds from Pe via 17 Pe, and not via Po.     

Measurement of cortisol, cortisone, 11-deoxycortisol and corticosterone in foetal sheep plasma during the perinatal period.

A method is described for the resolution and individual quantitation of cortisol, cortisone, 11-deoxycortisol and corticosterone in foetal sheep plasma. The steroids were extracted by solvent partition and separated by LH-20 Sephadex column chromatography. Radioimmunoassay was used for the measurement of 11-deoxycortisol and cortisone and competitive protein-binding for corticosterone and cortisol. The relative levels of these steroids in the plasma of chronically catheterized sheep foetuses from 12 days before birth to term and then in the newborn lamb until 2 days of age are recorded. Cortisol gradually increased from a basal concentration of between 0 - 5 and 3 - 0 mug/100 ml plasma between days 12 and 5 pre partum, and then rose rapidly to 10 mug/100 ml plasma during the last 5 days of pregnancy to reach a maximum during or just after birth. Two days post partum the levels had fallen to approximately 3 mug/100 ml plasma. The mean value for 11-deoxycortisol between days 8 and 3 pre partum was 0 - 4 mug/100 ml plasma and increased in the final days before delivery to 1 - 0 mug/100 ml. Corticosterone initially showed slightly higher levels (approximately 1 - 5 mug/100 ml) in the earlier period of investigation but then fell during the immediate pre-partum period to 0 - 8 mug/100 ml. Cortisone was not detected at any stage of the investigations. The relationship between levels of cortisol and 11-deoxycortisol in foetal plasma and myometrial contractility is shown. An increase in uterine activity was seen to occur at the time that cortisol levels were at their maximum. The 11-deoxycortisol values throughout this particular study remained low. The results are discussed in relation to recorded levels in the adult and to previous studies in vitro with regard to changing steroid biosynthetic enzyme activity.     

Follicle-stimulating hormone stimulates estradiol-17beta synthesis in cultured Sertoli cells.

Sertoli cells isolated from testes of 20-day-old rats and maintained in primary culture synthesized estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol] (measured by specific radioimmunoassay) when testosterone (17beta-hydroxy-4-androsten-3-one) 0.5 muM, was added to the culture medium. No detectable estradiol synthesis occurred when cells were incubated in medium containing pregnenolone (3beta-hydroxypregn-5-en-20-one), 0.5 muM, or containing no added steroid substrate. Follicle-stimulating hormone (FSH) (NIH-FSH-S10, 5 mug/ml) stimulated estradiol synthesis 12- to 80-fold when added to medium containing testosterone, but not when added to medium containing pregnenolone or no exogenous steroid substrate. A highly purified FSH preparation, with FSH potency 50 times that of the NIH-FSH, caused a similar stimulation at a concentration of 0.25 mug/ml of medium, whereas luteinizing hormone (NIH-LH-S18, 5 MUG/ML) Caused only marginal stimulation. Dibutyryl-adenosine 3':5' cyclic monophosphate, 0.1 mM, caused a 30-fold increase in estradiol synthesis by Sertoli cells cultured in medium containing testosterone. These studies provide direct demonstration of estradiol-17beta production by Seroli cells from normal animals, and offer evidence that the synthesis of this steroid is regulated at the level of the aromatizing enzyme system by FSH and adenosine 3':5' cyclic monophosphate.     

Nonsuppressible insulin-like activity and thyroid hormones: major pituitary-dependent sulfation factors for chick embryo cartilage.

Serum from hypothyroid hypophysectomized rats did not stimulate sulfation or incorporation of amino acids into chick embryo sterna. When such rats were treated for a short time with growth hormone (somatotropin), their serum stimulated incorporation both of sulfate and of amino acids. The different actions of the two types of sera were not due to changes in thyroid state. The results support the existence in serum of a sulfation factor for chick embryo cartilage that is dependent upon growth hormone. Highly purified preparations of nonsuppressible insulin-like activity from human serum stimulated incorporation of amino acids, and of uridine into RNA, in chick embryo sterna in vitro; chondrocytes prepared from this tissue had specific high-affinity binding sites for this insulin-like activity. However, sulfate incorporation was stimulated very little, unless serum from hypothyroid hypophysectomized rats was also present. When L-3,5,3'-triiodothyronine was added as well, the stimulation was enhanced further. From these and other experiments, we conclude that (i) nonsuppressible insulin-like activity or a closely related peptide is the growth-hormone-dependent growth and sulfation factor for chick embryo cartilage: (ii) a second, unidentified factor must be present for the insulin-like activity to stimulate sulfation; and (iii) stimulation of sulfation by thyroid hormones in vitro is additive to that of nonsuppressible insulin-like activity.