Levels of circulating and urinary oestrogens during pregnancy in the marmoset monkey (Callithrix jacchus)

The levels of immunoreactive oestrone, oestradiol-17 beta and oestriol in plasma and urine were measured during early, mid- and late pregnancy in the marmoset monkey. In plasma, unconjugated oestrone remained less than 2% of total (conjugated plus unconjugated) oestrone throughout gestation, whereas unconjugated oestradiol-17 beta increased from 3% of the total value in early and mid-pregnancy to 35% in late pregnancy. The reversal in the unconjugated oestrone: oestradiol-17 beta concentration ratio from early (12:1) to late (0 . 15:1) pregnancy occurred despite the continuing predominance of oestrone in terms of total hormone. Total oestriol was measurable but in relatively low concentrations. Oestradiol conjugate was the predominant urinary oestrogen metabolite measured at each stage of pregnancy. The pattern of urinary oestrone and oestradiol-17 beta reflected plasma levels of total hormone, rather than unconjugated hormone, showing no further increase after mid-pregnancy. In contrast, oestriol increased throughout pregnancy and to a proportionately greater extent than oestrone or oestradiol-17 beta, but at lower absolute levels. High-pressure liquid chromatography of urine extract indicated the presence of considerable amounts of oestrogen immunoreactivity not accounted for by oestrone, oestradiol-17 beta and oestriol and with a retention time similar to that of 16 alpha-hydroxyoestrone. Gas chromatography and mass spectroscopy provided further evidence to suggest that 16 alpha-hydroxyoestrone is an abundant urinary oestrogen metabolite during pregnancy in the marmoset monkey.     

Conjugated and unconjugated oestrogens in fetal and maternal fluids of the cow throughout pregnancy.

The changes in concentration of oestrone, oestradiol (-17alpha and -17beta), oestrone sulphate and the oestradiol sulphates have been measured in allantoic and amniotic fluids and in maternal peripheral plasma throughout gestation. Oestrone sulphate was the major oestrone present in all of the fluids. It was measurable in allantoic fluid before Day 52 and reached a peak concentration of 475 ng/ml around Day 133. A lower peak occurred in the amniotic fluid around Day 110. The changes in oestradiol sulphates in allantoic fluid were similar to those of oestrone sulphate but at a much lower level. Considerable fluctuation was observed in the oestradiol sulphate concentrations in amniotic fluid. The ratio of oestradiol-17alpha sulphate to oestradiol-17beta sulphate was considerably higher in amniotic fluid than in allantoic fluid. Consistent changes in the levels of oestrone and the oestradiols were found in amniotic fluid but not in allantoic fluid during the second half of pregnancy. In maternal peripheral plasma oestrone sulphate was measurable before Day 72. In the limited number of samples analysed no difference in oestrogen concentration due to the sex of the fetus was evident in any of the fetal or maternal fluids.     

Circulating oestrogen concentrations during pregnancy in the African elephant (Loxodonta africana)

Oestrone, oestradiol-17 beta and oestriol were measured in plasma samples from non-pregnant and pregnant African elephants shot in the wild. Enzymic hydrolysis of plasma showed that approximately 90 and 96% of the total (i.e. conjugated plus unconjugated) concentrations of oestrone and oestradiol-17 beta, respectively were represented by conjugated hormones. Unconjugated oestrogens remained low (less than 50 pg ml) in all samples, with no distinction between non-pregnant and pregnant animals. Levels of total oestrone during pregnancy varied between 160 and 594 pg/ml but were not significantly different from non-pregnant values. Total oestradiol-17 beta concentrations were significantly elevated during pregnancy (P less than 0 X 01) and, despite considerable individual variation (193-1428 pg/ml), were consistently higher than non-pregnant values after 6 months of gestation. The elevated levels of oestradiol-17 beta resulted in a reversal of the total oestradiol-17 beta: oestrone concentration ratio at about 6 months of pregnancy. Concentrations of total oestriol did not exceed 103 pg/ml. An indirect method of measurement indicated that oestradiol-17 beta sulphate was probably the most abundant circulating oestrogen during pregnancy in the African elephant.     

Oestrone, oestradiol and oestriol glucosiduronates and sulphates in human puerperal plasma and milk

Conjugated and unconjugated oestrone, oestradiol and oestriol were measured in simultaneous milk and plasma samples obtained from 21 women in the early post-partum period. Conjugated oestrogens comprised more than 90% of the total oestrogen content of both milk and plasma. Oestrone glucosiduronate was the major oestrogen metabolite in milk (33%), the levels being significantly higher (P less than 0.01) than in plasma. Oestriol glucosiduronates were the predominant oestrogen metabolites (63%) in plasma.     

Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.     

On the significance of in situ production of oestrogens in human breast cancer tissue.

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-17 beta on the epithelial cells

Epithelial and stromal cells of guinea-pig endometrium were separated by enzymic digestion, isolated by successive centrifugation, and maintained in culture as pure cell types for 5 days on growth medium. On Day 5, ultrastructural studies were performed on the two cell types, demonstrating that epithelial cells can grow as a monolayer composed of cohesive groups of polygonal cells (1.3 X 10(5) cells/cm2), while stromal cells were mostly fibroblastic. The effect of hormones was studied on the epithelial cells in culture. The monolayer was cultured into harvest medium for 3 days to ensure the complete removal of endogenous steroids, then these cells were incubated with 2 X 10(-9) M-oestradiol-17 beta for 3 days. There was a rise in the progesterone receptor level, varying from 1.3 to 10.8 times. The three enzymes known to interfere with oestradiol-17 beta metabolism were present in the epithelial cells grown in our culture conditions. By incubation with oestrone sulphate for 3 days it was demonstrated that, in cultured epithelial cells, oestrone sulphate is converted into oestradiol-17 beta sulphate, and oestrogen sulphates are hydrolysed to active oestrogens.     

On the significance of in situ production of oestrogens in human breast cancer tissue

We have previously shown that human breast cancer is autonomous in the regulation of its intra-tissue oestradiol concentration. Breast fatty tissue does not have this capacity, but rather reflects changes in the peripheral oestradiol concentration. To further evaluate the relative contribution of breast cancer and fatty tissue to the maintenance of tumour oestradiol we investigated whether a tumour-directed gradient in aromatase activity and oestrogen levels existed in mastectomy specimens. No such gradient was found, however, for aromatase, oestrone, oestradiol and their sulphates. Aromatase activity (expressed per gram of tissue) and the concentrations of oestradiol, oestradiol sulphate and oestrone sulphate were higher in tumour than in breast fatty tissue. Fatty tissue had a higher oestrone concentration. It is tentatively concluded that breast tumour aromatase activity is more important for the maintenance of tumour oestradiol levels than aromatase in breast fatty tissue.     

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: Relative importance of ring hydroxylation and aromatization in control and induced rat liver

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and β-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, β-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1).The principal aromatized products were oestriol and 2-hydroxyoestradiol-17β, with oestradiol-17β and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17β whereas hydroxylated metabolites were only minor products. Oestradiol-17β, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17β-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17β which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C₁₉-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.     

Comparison of serum oestrogen concentrations in post-menopausal women taking oestrone sulphate and oestradiol.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.     

Influence of the fetal pituitary-adrenal axis on fetal and maternal progesterone and unconjugated oestrogen concentrations in the pig

Fetal and maternal plasma progesterone and unconjugated oestrone and oestradiol-17 beta concentrations were measured in intact pig fetuses and those in which the pituitary had been destroyed. Progesterone concentrations were significantly higher (P less than 0.05) and oestrogen concentrations significantly lower (P less than 0.01) in hypophysectomized fetuses than in intact fetuses. When fetuses in one uterine horn only were hypophysectomized, oestrogen concentrations in the uterine vein draining this horn were lower than those from the contralateral vein. The results indicate that both fetal and maternal oestrogen concentrations are influenced by the fetal pituitary. When dexamethasone was infused (at 27 micrograms/h for 96 h) into 5 chronically-catheterized hypophysectomized fetuses no changes in peripheral fetal progesterone or oestrone were observed.     

Hormonal and physical changes associated with bovine conceptus development.

Conceptus (placental membranes, fetal fluids and fetus) development was characterized between Days 27 and 111 of gestation. Progestagens, oestrone, oestradiol, oestrone sulphate and prostaglandins (PG) F were measured in maternal plasma and allantoic and amniotic fluids. Protein concentrations are described for fetal fluids. The early increase in placental membrane weight from 1.12 g (27 days) to 58.45 g (50 days) was associated with oestrogen production presumably of conceptus origin. Oestrogens increased significantly in allantoic and amniotic fluids throughout the period studied with oestrone being the primary free oestrogen, rising from 2 pg/ml (Day 33) to 144 ng/ml by 111 days in allantoic fluid. Changes in plasma oestrogens of the maternal circulation were not detected until after Day 70 at which time oestrone concentration was greater than that of oestradiol. Fetal fluid concentrations of progestagens, oestrone sulphate and PGF were not related to maternal plasma levels and a sequestration of these hormones by the allantois is postulated.     

Modulation of phospholipase A2 activity in human endometrium and amniotic membrane by steroid hormones

Phospholipase A2 (PLA2) activity was measured in endometrium and amnion by a double isotope ratio technique using 1-palmitoyl-2-oleoyl phosphatidylcholine as substrate in the presence and absence of a range of unconjugated steroids and steroid sulphates (0.2–6.4 × 10⁻⁴ M). In the presence of 0.1% Triton, PLA2 activity was inhibited by the majority of steroids tested, pregnenolone sulphate being the most effective (12.9 ± 3.0% control activity) while oestradiol sulphate, oestrone and testosterone had only a minimal or no effect (99.1 ± 19.0, 85.4 ± 4.4 and 104.2 ± 16.3% control respectively). In the absence of Triton, the inhibitory effect of the free steroids was reduced or absent but oestradiol sulphate and testosterone sulphate stimulated activity by 2–13 and 1.5–3 times respectively. The effect was dose related, linear with time and independent of the stage of the menstrual cycle. Inhibition by pregnenolone sulphate, dehydroepiandrosterone (DMA sulphate and oestrone sulphate was maintained in the absence of Triton (24.9 ± 3.8, 67.1 ± 10.1 and 87.2 ± 13.8% control respectively). In amnion all 5 steroid sulphates caused a marked stimulation of PLA2 activity in both the presence and absence of Triton. The effect was greatest without Triton and at 6.4 × 10⁻⁴ M, testosterone, pregnenolone, oestrone, DHA and oestradiol sulphates increased PLA2 activity 20, 15, 12, 10 and 6-fold respectively. These findings indicate a direct action of steroid sulphates on PLA2 activity in endometrium and amnion.     

Effect of intrauterine application of oestradiol-17 beta and prostaglandin E-2 on the porcine oestrous cycle and uterine endocrinology.

Silastic beads were inserted into the uterine lumen on Day 10 after oestrus. Gilts received beads containing oestradiol-17 beta only, oestradiol benzoate, or oestradiol-17 beta+prostaglandin (PG) E-2. Oestrous cycles were slightly longer in treated than in untreated pigs (20.2 +/- 0.4 days), and durations were 22.6 +/- 1.3, 26.2 +/- 1.7 and 23.2 +/- 1.8 days for oestradiol-17 beta, oestradiol benzoate and oestradiol-17 beta+PGE-2 treatments, respectively (P greater than 0.05). Thus, PGE-2 and an oestrogen such as oestradiol benzoate that persist for a longer period cannot prolong the cycle more than oestradiol-17 beta alone. Additional cyclic gilts underwent similar treatments with beads containing oestradiol-17 beta, oestradiol-17 beta+PGE-2 or cholesterol, and cannulation of one utero-ovarian vein on Day 10. Blood samples were collected from the catheter every 15 min from 08:00 until 11:00 h and from 20:00 until 23:00 h for 5 consecutive days starting the day after surgery and peripheral plasma samples were also collected daily. On Day 16, beads containing oestradiol-17 beta were surrounded by endometrial folds whereas cholesterol beads were free. Concentrations of plasma progesterone did not vary significantly from Days 11 to 16 in gilts treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2, but decreased in cholesterol-treated gilts. Concentrations of plasma oestrone and oestradiol-17 beta were more than ten times higher in gilts treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2 than in cholesterol-treated gilts on the day after bead insertion, but decreased rapidly to values comparable to those in cholesterol-treated gilts by Day 14. In contrast, concentrations of oestrone sulphate remained high until Day 16. Concentrations of PGE-2 in the utero-ovarian vein plasma did not differ (P greater than 0.05) between treatments but those of PGF-2 alpha were higher (P less than 0.004) in gilts treated with cholesterol than in those treated with oestradiol-17 beta or oestradiol-17 beta+PGE-2. It is postulated that insufficient oestradiol-17 beta is released by the beads toward the end of a 'recognition period' to prolong the cycle for more than 3-6 days.     

Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.     

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17β-oestradiol 3-sulphate, 16-oxo-17β-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.     

A direct assay for oestrone sulphate and its use to investigate the effect of ampicillin on plasma levels of oestrone sulphate

A direct radioimmunoassay for measuring plasma levels of oestrone sulphate has been developed using 8-anilino-2-naphthalene sulphonic acid to displace oestrone sulphate from plasma binding proteins. Oestrone sulphate was assayed by using an antiserum raised against glucuronide which cross-reacted 100% with oestrone sulphate. The direct assay gave a good analytical recovery of oestrone sulphate and there was a good correlation (r = 0.82, P less than 0.001) for plasma levels of oestrone sulphate measured by the direct assay and a method involving steroid conjugate extraction and enzyme hydrolysis. The mean (+/- S.D.) plasma level of oestrone sulphate in men was 1100 +/- 280 pg/ml. The effect of taking the antibiotic, Ampicillin, on plasma levels of oestrone sulphate was investigated in four men. Plasma levels of oestrone sulphate were significantly reduced after taking Ampicillin for 5 days. Ampicillin may act to lower plasma levels of oestrone sulphate by reducing the growth of bacteria in the gut or by inhibiting oestrogen sulphotransferase activity.     

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.     

Steroid hormones in uterine washings and in plasma of gilts between days 9 and 15 after oestrus and between days 9 and 15 after coitus

Between Days 9 and 15 after oestrus, concentrations of pregnenolone, pregnenolone sulphate, dehydroepiandrosterone (DHEA), DHEA sulphate, androstenedione, oestrone and oestrone sulphate in free uterine fluid collected from non-pregnant gilts were greater than respective values in plasma (P less than 0.05). The total contents of pregnenolone, progesterone, DHEA, testosterone, oestrone and oestradiol in washings from pregnant uteri exceeded (P less than 0.05) respective non-pregnancy levels during this same period. Concentrations of pregnenolone, pregnenolone sulphate, DHEA, DHEA sulphate, androstenedione, oestrone, oestrone sulphate and oestradiol in free uterine fluid recovered from gravid uteri were also higher (P less than 0.05) than respective plasma values. By contrast, the progesterone concentration in uterine fluid from pregnant animals was lower (P less than 0.001) than the plasma value. Concentrations of DHEA, DHEA sulphate, androstenedione and oestrone sulphate in plasma of pregnant gilts between Days 9 and 15 after mating exceeded (P less than 0.05) the respective concentrations in unmated gilts between Days 9 and 15 after oestrus. Plasma levels of pregnenolone sulphate were lower (P less than 0.05) in the pregnant animals. We therefore suggest that the endometrium of the pig can concentrate steroid hormones in uterine fluid and that increases in steroid levels in this milieu between Days 9 and 15 after coitus reflect steroidogenesis by embryonic tissues and modification of enzyme activities within uterine tissues under the influence of progestagens. The pool of steroid sulphoconjugates present in uterine fluid between Days 9 and 15 post coitum could serve as an important precursor source for progestagen, androgen and oestrogen synthesis by tissues of pig embryos before implantation.     

The direct radioimmunoassay of oestrogen glucuronides in human female urine.

Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.