Placental and luteal steroidogenesis in the pregnant rhesus monkey.

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.     

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3β-hydroxysteroid oxidoreductase (3β-HSD) from human placental homogenates were studied invitro. The apparent Michaelis constants for 3β-HSD with the substrates pregnenolone (Δ⁵P) and dehydroepiandrosterone (DHA) were 170 nM and 40 nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for pregnenolone was 20 μM and for DHA, 17 μM. The activity of 3β-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17β (Ki=46 nM), cholesterol (Ki=0.68 μM) and 16α-hydroxydehydroepiandrosterone (16αOHDHA) (Ki=2.2 μM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17β (Ki=69 nM), pregnenolone (Ki=91 μM), cholesterol (Ki=1.3 μM) and 16αOHDHA (Ki=1.9 μM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (Δ⁴P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Cholesterol side chain cleavage and aromatase activities in the corpus luteum of the pregnant rhesus monkey.

Cholesterol side-chain cleavage (CSCC) and aromatase activities were measured in luteal mitochondria and tissue pieces, respectively, from rhesus monkeys on days 22, 49, 128 and 160 of gestation. CSCC activity did not vary significantly during gestation and thus probably does not respond to chorionic gonadotropin which is elevated on day 22 of pregnancy. It is not known, however, whether CSCC can be stimulated prior to day 22 when the corpus luteum is steroidogenically more active. Both 3H-pregnenolone and 3H-progesterone were synthesized from [1,2-3/]cholesterol. Aromatase activity declined from high levels on days 22 and 49 to a nadir on day 128 of pregnancy. Utilizing either [1beta-3H]androstenedione or [1beta-3H]testosterone as substrate yielded comparable results throughout gestation.     

Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate.

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2 degrees C) for the synthetic androgen R1881 (17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17alpha,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 +/- 1.4 and 14.3 +/- 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 +/- 0.8 and 1.6 +/- 0.4 x 10(8) M-1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20-24 hr incubation at 15 degrees C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 +/- 3.3 fmoles per mg cytosol protein and 2.7 +/- 0.6 x 10(7) M-1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17β (E₂) on the netabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated $\text{in vitro}$ was studied. Doses considered as being physiological of 0.09 and 0.9 μM had a stimulatory effect on the conversion (p < 0.008 to 0.0l6). However, a supraphysiological dose of 45 μM showed an inhibitory activity related to the maximal stimulation (p < 0.03). A dose of 0.9 μM E₂ favoured the accumulation of (³H)-progesterone in the tissue (p < 0.05). These results suggest that E₂ may regulate the synthesis of progesterone in human term placenta.     

Solubilization and partial purification of 4,16-androstadien-3-one synthesizing enzyme from boar testis microsomes and requirements for the enzymatic activity

The enzyme related to the synthesis of 4,16- androstadien-3-one from progesterone was investigated using boar testis. To elucidate the activity, in the soluble form, the lyophilized powder of 12,000 g supernatant from homogenate was first treated with n-butanol after which the enzyme could be extracted with 1 mM ethylenediaminete-traacetic acid(EDTA), 1 mM dithiothreitol(DTT) and 20 % glycerol. The enzyme was stable in this medium.The enzyme filtered through a column of Sephacryl S-200 super fine gel exhibited requirements of NADPH-cytochrome C reductase and phosphatidylcholine for maximum enzymatic activity. The requirements of reductase and phosphatidylcholine were not observed in the crude extract fraction. The enzyme separated by column chromatography with DEAE-cellulose required phosphatidylcholine for the synthesis but the reductase had no effect. These lines of evidence suggest that the activity of the enzyme, as related to synthesis of 4,16-androstadien-3-one from progesterone might be regulated by phosphatidylcholine and reductase, $\text{in situ}$.     

Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH).

Methyltrienolone (R 1881 - 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of [3H] R 1881 binding is different from that measured in rat prostate: progesterone and R 5020 (17, 21-dimethyl-19-nor-4, 9-pregnadiene-3, 20-dione) being more potent while 19-nortestosterone is less potent competitor. Moreover, the synthetic progestin [3H] R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol.     

Estrogen metabolism in neural tissues of rabbits: 17 beta - hydroxysteroid oxidoreductase activity.

Incubation of adult rabbit neural tissue homogenates with (3H)-estrone and (3H)-estradiol revealed that the conversion of estrone to estradiol is higher in both male and female animals than estradiol to estrone. Both 17 oxidation and reduction are higher in male animals than in females. However, it is observed that the quotient of estrone leads to estradiol/estradiol leads to estrone for pituitary tissue and hypothalamus are higher in females than in males. There is no such dimorphism in cerebral cortex. The overall metabolism in pituitary is higher than in hypothalamus and cortex in both sexes. These results suggest that 17 beta hydroxysteroid oxidoreductase activity may play an important role in the regulation of estrogen function in neuroendocrine tissues.     

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Failure to detect delta 5-3 beta-hydroxysteroid oxidoreductase activity in the preimplantation rabbit embryo

Preimplantation rabbit embryos were incubated with pregnenolone and dehydroisoandrosterone under conditions which gave formazan precipitation by the histochemical technique. The metabolic fate of the labeled steroids was assessed simultaneously. There was no concomitant transformation of pregnenolone to progesterone and dehydroisoandrosterone was not transformed to androstenedione. It is concluded that the formazan precipitation is coupled with an "activity" other than delta 5-3 beta-hydroxysteroid oxidoreductase.     

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP)

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [³H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) for binding to SBP was assessed.Binding was enhanced by reduction of the Δ⁴ double bond to 5α-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14, 15. Affinity was always reduced by modifications of the C-17β hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17α-ethinyl had limited effects on binding; whereas, other modifications such as 7α-methyl or 17α-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5β steroids had reduced affinity.     

Androgen metabolism in male and female breast tissue

Incubation studies have been carried out using normal breast tissue and breast tissue from patients with gynecomastia, mammary dysplasia and breast carcinoma to determine the pattern of androstenedione metabolism. All tissues formed estrone (E₁) and testosterone (T) in all incubations. Estradiol (E₂) was isolated in incubations of tissue from 1 to 6 patients with mammary dysplasia, 5 of 6 patients with gynecomastia and in all incubations with normal and carcinoma tissue. Estrone formation was lowest in mammary dysplasia and gynecomastia, and higher in apparently normal breast tissue. The greatest E₁ formation was found in incubations with breast carcinoma tissue, although there was considerable variation within this tissue group. Estradiol formation was low in all tissues, with the highest conversion rates in carcinoma tissue. Testosterone formation in carcinoma tissue was greater than in mammary dysplasia or gynecomastia, but similar to apparently normal tissue. These results indicate that breast tissue from different pathological states varies in its capacity to aromatize androstenedione (A) to estrogenic products and to convert it to other androgens. They have also shown that the pattern of metabolism is distinctive for the nature of the pathological abnormality.     

In vitro effects of estrogens on rat prostatic 5 alpha-reduction of testosterone

The inhibitory effects of a variety of estrogens on rat prostate testosterone Δ4–5α-reductase activity were measured by a specific $\text{in vitro}$ assay. The conversion of ³H-testosterone (initial concentration 2.8 × 10^?9 M) to labelled 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol was used as a measure of Δ4?5a-reductase activity. At a concentration of 1.8 × 10^?6 M, estradiol was the most potent inhibitor (83.4%) of the estrogens tested. Various ester derivatives, e.g. 3-acetate, 3-phosphate, were effective inhibitors. The 17-glucuronide and 3-sulfate conjugates were less effective inhibitors. The estriol isomers exerted similar degrees of inhibition (40–60%). The 3-methoxy derivatives of estradiol and estriol were poor inhibitors. The introduction of certain groups into the steroid structure, e.g. 15α-hydroxy and 6-ketone, greatly decreased the inhibitory effect of estradiol. The nature of the oxygen function at carbon 17 did not greatly influence the inhibitory effects.     

Studies on the mechanism of action of 1 alpha,24-dihydroxyvitamin D3. III. The specific binding of 1 alpha,24-dihydroxyvitamin D3 to the receptor of chick parathyroid gland

Three protein fractions of the cytosol of the chick parathyroid glands, which had the sedimentation constants of 2.5 S, 3.7 S and 5.5 S, were found to bind with 1 alpha,25-dihydroxyvitamin D3. Among these proteins, the 3.7 S protein was assumed to be the specific receptor protein. The 3.7 S receptor protein was also capable of binding to 1 alpha,24-dihydroxyvitamin D3 but not 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor protein was estimated to be 1.2 times greater than that of 1 alpha,25-dihydroxyvitamin D3, while 1 alpha,25-dihydroxyvitamin D3 bound to the receptor protein about 10 times stronger than 1 alpha,24(S)-dihydroxyvitamin D3. The dissociation constant for the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 2.7 x 10(-11) M, the dissociation constants were calculated to be 2.2 x 10(-11) M and 2.6 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3.     

Evaluation of adrenal function in rats by the measurement of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone.

Methods für the determination of urinary free corticosterone, free aldosterone and free 11-deoxycorticosterone (DOC) in rats are described. The free corticosteroids were measured in urine samples of 0.1–0.5 (2.0) ml by radioimmunoassay after purification by column chromatography. The validity of the methods is demonstrated by the data of the free urinary corticoids under basal conditions and after adrenal suppression and various forms of adrenal stimulation. The basal excretion of free corticosterone, free aldosterone and free DOC was 123.71 ± 15.31 (x? ± SD), 3.87 ± 1.29 and 10.61 ± 2.24 ng/day, respectively, exhibiting a decrease to 26.20 ± 5.21, 1.05 ± 0.47 and 1.35 ± 1.20 ng/day after adrenal suppression by dexamethasone. Irrespective of the mode of adrenal stimulation i.e., synthetic ACTH and systemic (cold, hunger) or neurotrophic (ether, reserpine) stress stimuli free corticosterone increased to about 450 ng/day, while free aldosterone excretion decreased during hunger and cold and was strongly enhanced after the application of reserpine. Furthermore, determination of urinary free DOC, which increased by a factor of 4, may be applied in the metyrapone test. There was a good correlation between the excretion of free corticosterone and that of free aldosterone and free DOC under basal conditions and after ACTH application, demonstrating that ACTH is responsible for the secretion of all the 3 corticoids measured. It is concluded, that the measurement of the urinary excretion of corticosterone, aldosterone and DOC is a valuable parameter of adrenal function in rats. Furthermore, in small laboratory animals like rats steroid measurements in urine are often more advantageous than Measurements in plasma.