The use of protamine to study [6,7-3H]-oestradiol-17β binding in rat uterus

1. The binding of [6,7-(3)H]oestradiol-17beta to uteri has been studied by using sucrose-gradient analysis and also the property of oestradiol receptors to form insoluble complexes with protamine. 2. Protamine precipitates the 8S and part of the 4S oestradiol-binding proteins in uterine cytoplasm from mature rats. It does not precipitate the oestradiol-17beta-binding proteins present in cytoplasm from non-target tissues or serum. No tritium-labelled material was precipitated by protamine after equilibration of [6,7-(3)H]oestradiol-17beta with either serum albumin or phosvitin. 3. Protamine precipitated a small amount of progesterone but not testosterone or cortisol that had been equilibrated with uterine cytoplasm. It did not precipitate any tritium radioactivity from muscle cytoplasm that had been equilibrated with either [1,2-(3)H]testosterone sulphate or [1,2-(3)H]dehydroepiandrosterone. 4. A simple method has been devised for measuring binding constants of tissue extracts for [6,7-(3)H]oestradiol-17beta, based on precipitation with protamine. Reasonable agreement was obtained between the values obtained by this method and those obtained by sucrose-gradient analysis. 5. This method has been used to study the effect of maturity, ovariectomy, adrenalectomy and hypophysectomy on the cytoplasmic binding of [6,7-(3)H]oestradiol-17beta. None of these procedures affected the dissociation constant K(d) or the number of binding sites/mg of cytoplasmic protein. When measured per uterus or per mg of DNA, ovariectomy and hypophysectomy decreased the number of binding sites. Adrenalectomy had no effect. 6. The properties of the 4S oestradiol-binding protein present in cytoplasm from mature uteri have been studied. It is not present in uteri from immature, ovariectomized, or hypophysectomized rats and it does not bind testosterone or cortisol. Unlabelled oestradiol-17beta, U-11,100A, N-ethylmaleimide and N-bromosuccinimide all decrease the binding of [6,7-(3)H]oestradiol-17beta to both 8S and 4S receptors. Binding to both 8S and 4S receptors decreases when oestradiol is transported to the nucleus. The 4S receptor is not the same as the 4S binding component formed by salt dissociation of the 8S receptor.     

Nuclear binding of oestradiol-17β and induction of protein synthesis in the rat uterus during postnatal development

1. The uterine response to a single injection of oestradiol-17beta during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17beta; (ii) induction of the synthesis of a specific cytoplasmic protein (;induced protein' of Gorski); (iii) rate of incorporation of (3)H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [(3)H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17beta could be demonstrated even at birth. Administration of oestradiol-17beta in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ;induced protein', based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of (3)H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3-4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.     

Histo-autoradiographic study of a testosterone target organ, the epididymis of the lizard (Lacerta vivipara), after administration of 3H-17 beta-estradiol]

[6,7 3H] oestradiol-17 beta was injected into castrated male viviparous lizards in order to compare retention of this steroid to that of testosterone during the period of sexual activity. Retention of the isotope in epididymis was greater than in blood, lung and stomach. Radioautographs of epididymis indicated that oestradiol or a metabolite was concentrated in cell nuclei and over discharged secretory granules as was observed with testosterone. Reason of such binding is not yet known.     

In-vitro zona-lytic activity in uterine fluid from ovariectomized mice treated with oestradiol-17 beta and progesterone

Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17 beta + progesterone, oestradiol-17 beta + progesterone, or oestradiol-17 beta alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.     

Demonstration of 8 S-cytoplasmic oestrogen receptor in rat mullerian duct.

1. High affinity macromolecular binding of the non-steroidal synthetic oestrogen [3H]diethylstilboestrol and of [3H] oestradiol-17beta in cytosol of Müllerian duct and uterus, and in blood plasma of perinatal rats, was investigated by sucrose density gradient sedimentation. 2. While [3H] oestradiol was bound to both the characteristic 8 S uterine cytoplasmic receptor and a 4 S component of uterine cytosol and plasma of 11-day-old rats, [3H] diethylstilboestrol was bound almost exclusively by the 8 S cytoplasmic receptor. 3. The greatly reduced binding of [3H] diethylstilboestrol to the 4 S plasma plasmic receptor in the Müllerian duct (precursor of the uterus) of 20-day-old foetuses.     

The effects of serum oestrogen-binding components on the unbound oestradiol-17 beta fraction in the serum of developing female rats and on inhibition of [3H]oestradiol uptake by uterine tissue in vitro

Total oestradiol concentrations in the serum of young female rats were high and decreased after about 21 days of age. High affinity serum oestradiol-binding components (EBP), however, fell steadily from 5 to 23 days of age while the unbound oestradiol-17 beta fraction, which was low early in development, increased between 21 and 28 days of age. Injection (i.v.) of immature (EBP-rich) oestradiol-free serum into 21-day-old female rats led to a decrease in the unbound oestradiol fraction and an increase in serum FSH concentrations. Incubation of uterine tissue with [3H]oestradiol, with or without the addition of diethylstilboestrol (DES), showed the rate and degree of total and DES-suppressible uptake of [3H]oestradiol to be greatest from buffer, less from adult (EBP-poor) serum and negligible from immature (EBP-rich) serum; moreover, there was a positive correlation between the degree of uptake of [3H]oestradiol and the unbound fraction of [3H]oestradiol in the incubate. It is concluded that, at least in young rats, oestradiol activity depends more on the availability of free oestradiol than on its total plasma concentrations.     

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.     

In vivo oestrogenicity and binding characteristics of alpha-zearalanol (P-1496) to different classes of oestrogen binding proteins in rat liver

It is now well established that the mycotoxin zearalenone and some of its derivatives possess oestrogenic activity. In the present study, the binding characteristics of [3H]zearalanol (P-1496) to different classes of sites including [1] the oestrogen receptor, [2] the higher capacity lower affinity (HCLA) sites, [3] the antioestrogen sites and [4] a new class of binding sites apparently specific for P-1496 were examined in rat liver. Analysis of the binding by sucrose density gradient centrifugation confirmed that P-1496 binds to the oestrogen receptor but not to the higher capacity lower affinity sites for oestradiol-17 beta. Furthermore, saturation experiments using partially-purified fractions showed that P-1496 binds to the oestrogen receptor with an affinity very similar to that of oestradiol-17 beta (apparent dissociation constants ranged from 0.1-0.3 nM). Competition studies using partially purified cytosolic oestrogen receptor suggested that P-1496 binds to a second high affinity site distinct from the oestrogen receptor. This binding site was further characterized as selective for P-1496 by saturation analysis following the complete occupancy of oestrogen receptor by oestradiol-17 beta. The in vitro binding characteristics of P-1496 were then compared with in vivo effects on concentrations of serum triglycerides. Treatment of ovariectomized female rats daily with 1.5 or 2 mg P-1496/kg body weight resulted in marked increases in the concentrations of serum triglycerides associated with the very low density lipoprotein (VLDL) fraction. Dose-response studies indicated that there was no sex difference with respect to the dose necessary to produce significant increases in serum triglycerides. The present study shows striking similarities between the binding of P-1496 and oestradiol-17 beta to liver oestrogen receptor in vitro. However, differences are observed with respect to their binding to other cytoplasmic components of liver. In addition, although P-1496 is capable of eliciting in vivo oestrogenic effects in liver, it is much less potent than oestradiol-17 beta.     

Prolonged antioestrogenic activity of ICI 46, 474 in the ovariectomized mouse.

Subcutaneous administration of ICI 46,474 (3 mg) to ovariectomized mice was found to produce vaginal refractoriness to subcutaneously administered oestradiol for up to 6 weeks. Tritiated oestradiol-17 beta accumulated in the uterus and vagina of the ovariectomized mouse, with maximum accumulation at 3 to 4 hr. This property of the target tissues was used to investigate the binding of tritiated oestradiol after the administration of 3 mg ICI 46,474 to ovariectomized mice. Radioactivity in the vagina was found to be comparable to control values 6 weeks after ICI 46,474 administration; uterine levels of radioactivity returned to control values by 10 weeks. Administration of ICI 46,474 had an oestrogenic effect upon the mouse uterus whereas the vagina appeared to be initially stimulated and was then unable to respond to or to bind oestradiol.     

Interaction of the anti-oestrogen, nafoxidine hydrochloride, with the soluble nuclear oestradiol-binding protein in chick liver.

Nafoxidine hydrochloride (Upjohn, 11100A)injected with oestradiol into immature chicks inhibits the hormone-induced increase in [3H]oestradiol-binding activity in salt extracts of liver nuclei as well as the subsequent production by liver of egg-yolk phosphoprotein. Substantial inhibition of both oestradiol-induced responses is seen when nafoxidine is given in a dose approximately equimolar with that of oestradiol. In vitro nafoxidine competitively inhibits binding of [3H]oestradiol in nuclear extracts. The Ki for the inhibition is 43 nM, which indicates an affinity of nafoxidine for the binding protein about 4% of that of oestradiol. The inhibitory action of nafoxidine in vivo thus is more potent than the relative binding affinity determined in vitro might indicate. One possible explanation is that the primary site of nafoxidine action is at a point proximal to nuclear receptor interaction. Nafoxidine injected alone into the chick does not induce phosphoprotein synthesis, but it does increase [3H]oestradiol-binding activity in extracts of liver nuclei to a limited extent. No differences in the properties of the oestradiol-binding activity in extracts from nafoxidine-treated chicks or from oestradiol-treated chicks were detected. Chick liver cytosol does not contain detectable high-affinity oestradiol-binding activity. A low-affinity oestradiol-binding component with a sedimentation coefficient of 3.5S was found, but it was unaffected by treatment of chicks with earlier nafoxidine or oestradiol. The results suggest a difference in the mechanism of oestradiol action in the chick liver and in the widely studied rat uterus, on which the usual model for oestradiol action is largely based.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Selective retention of oestradiol by cell nuclei in specific brain regions of the ovariectomized rat

—Cell nuclei were isolated from four regions of the brains of ovariectomized female rats 2 hr after the injection of [³H]oestradiol. By light microscopy, the nuclear pellets contained highly purified nuclei of neuronal and glial cells with little cytoplasmic contamination. Tritium was concentrated in cell nuclei from the preoptic-hypothalamic area, to a lesser extent in nuclei from the amygdaloid region and hippocampus, and least of all in cerebral cortical nuclei. In comparison with whole homogenates (= 1-0), the nuclear concentrations of radioactivity were 12·9, 4·7, 1·9 and 0·8, respectively. Approximately 40 per cent of the radioactivity in homogenates of the preoptic-hypothalamic area was present in cell nuclei, and upon TLC more than 85 per cent of the radioactive material in the nuclei exhibited the R_F of oestradiol-17β. Pretreatment of ovariectomized females with 1 mg of unlabelled oestradiol 30 min before the injection of labelled hormone abolished the nuclear uptake of [³H]oestradiol in all four regions of the brain. A concurrent injection of 10 μg of unlabelled oestradiol-17β significantly reduced nuclear uptake, while a similar injection of testosterone or oestradiol-17α had no significant effect. One mg of oestradiol-17α, but not testosterone, did reduce nuclear uptake. The retention of [³H]oestradiol by the preoptic-hypothalamic area decreased exponentially in the tissue from 30 min to 4 h after an intraperitoneal injection; however, nuclear binding reached a peak at 1-2 h and still showed high retention at 4 h. These results, together with observations in other laboratories of morphological changes induced by oestrogens, establish that certain regions of the brain are bona fide targets for the action of oestradiol.     

Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

The binding of [6,7-(3)H]oestradiol-17beta to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.     

Studies on the in vitro metabolism of [3H]cortisol and [3H]oestradiol by sheep skin and wool roots.

The in vitro metabolism of [3H]cortisol, [3H]cortisone and [3H]estradiol-17 beta by adult sheep skin and wool follicle tissue (wool roots) was examined. The main metabolic product of the incubation of [3H]cortisol with sheep skin was [3H]cortisone, and the conversion was reversible. Wool roots were unable to carry out detectable interconversion, nor did this tissue give rise to other significant metabolites. Sheep skin and wool roots both rapidly converted [3H]oestradiol-17 beta to [3H]oestrone and the conversion could be carried out by follicle and non-follicle skin structures. It is suggested that sheep skin contains both 11 beta- and 17 beta-hydroxysteroid dehydrogenases, but that wool follicles contain only the latter enzyme.     

Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life

Female mice of the NMRI strain were treated with the synthetic oestrogen diethylstilboestrol (DES) for the first 5 days after birth. Pools of ovaries were removed from groups of 6-, 12-, 21-, 28- and 56-day-old females. An homogenate of an ovarian pool was incubated for 1 h in the presence of [3H]pregnenolone. Synthesized steroids were extracted and separated in a two-dimensional thin-layer chromatography system. Homogeneity of tentative steroids was verified with recrystallization to constant specific activity. Synthesis of [3H]progesterone and [3H]testosterone was demonstrated at 6 days, [3H]androstenedione at 12 days, [3H]17 alpha-hydroxyprogesterone at 21 days, and [3H]oestradiol-17 beta at 28 days. Up to 28 days (21 days for progesterone), the synthetic activity was lower in homogenates of DES-exposed ovaries than in control homogenates. After 28 days, values for recovered [3H]progesterone, [3H]androstenedione and [3H]oestradiol-17 beta were higher in DES homogenates than in control homogenates while the reverse was true for [3H]17 alpha-hydroxyprogesterone and [3H]testosterone. The results are compatible with an early and direct DES inhibitory effect on ovarian steroidogenesis and, later in immature life, a DES-induced disruption of the normal FSH-LH stimulation of ovarian development.     

Pituitary uptake of 125I-D-Leu6, Des-Gly N2(10) LH-RH-ethylamide in ovariectomized rats pretreated with oestradiol-17 beta.

The objective of this study was to determine if pretreatment of ovariectomized rats with oestradiol-17 beta affects the anterior pituitary uptake of 125I-D-Leu6, Des-Gly NH2(10)-LH-RH-ethylamide (125I-D-Leu6-LH-RH). Oestradiol-17 beta (0.5 microgram/0.5 ml oil) or oil was administered to ovariectomized rats at 2, 4, 8, 12, 16, 20 or 24 h before death, and at 30 min before death, 5 ng 125I-D-Leu6-LH-RH were injected intravenously. The serum LH response to analogue administration in oil-treated rats did not change over time, but that in oestradiol-treated rats was depressed for 4 h and restored 8-24 h after oestradiol treatment, with the greatest response being at 16 h. However, the pituitary (adrenal, CNS cortex and thyroid) uptake of 125I-D-Leu6-LH-RH in oestradiol-treated and control rats did not change over the 24-h time period. These data suggest that oestradiol-17 beta does not affect pituitary responsiveness to 125I-D-Leu6-LH-RH by inhibiting or facilitating the uptake of this analogue by the anterior pituitary.     

The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

High-affinity binding of oestradiol-17β by cytosols from testis interstitial tissue, pituitary, adrenal, liver and accessory sex glands of the male rat

The specificity of the binding of oestradiol-17beta by cytoplasmic fractions of several tissues of the male rat was investigated. 1. Agar-gel electrophoresis, Sephadex chromatography, adsorption by dextran-coated charcoal and sucrose-gradient centrifugation were used to estimate the binding capacity and specificity. The four different methods all gave similar results for the capacity of the specific oestradiol-17beta-binding macromolecules in the testis. 2. The presence of a specific saturable binding protein with a sedimentation coefficient of 8S was demonstrated in liver, adrenal, pituitary, prostate, epididymis and testis interstitial tissue. The highest concentration of oestradiol-17beta-binding macromolecules was found in testis interstitial tissue (0.12pmol/mg of protein) and in the pituitary (0.075pmol/mg of protein). 3. The oestradiol-17beta receptor in the testis cytosol showed the characteristics of a protein with respect to Pronase treatment and temperature sensitivity. In competition experiments with different steroids the receptor showed a high affinity for oestradiol-17beta, a moderate affinity for diethylstilboestrol and oestradiol-17alpha and a low affinity for oestrone, oestriol, testosterone and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one). 4. The wide distribution of oestradiol-17beta receptors in the male rat is in apparent contradiction to the current concept of the specificity of steroid-hormone action. Further research is required to investigate a possible physiological meaning of the presence of specific receptors in the different tissues.     

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.