Biomedical subjects

R Knuppen

Publications and source records attributed to R Knuppen.

At least 55 records · Page 3Linked to original sources

PubMed1989

Quantification of estrogen- and progesterone receptors from cryostat sectioned human tumor tissue: comparison of ligand binding and immunocytochemical assays.

We have recently described an assay procedure to measure estrogen and progesterone receptors in extracts from frozen sections by a ligand binding assay. With this methodological approach it is now possible to perform comparative experiments not only to DCC/Scatchard analyses from different tissue blocks, but also to immunocytochemical determinations in identical tissue blocks: (1) When receptor quantities measured by the two biochemical methods were compared, a high correlation of estrogen receptor content was found between determinations in supernatants from frozen sections and DCC/Scatchard analyses. A slightly poorer correlation in the comparison of the ligand binding assays was obtained for the progesterone receptor. (2) The percentage of tumor cells stainable by immunocytochemistry for estrogen and progesterone receptors could hardly be correlated to the receptor concentrations (fmol/mg) measured quantitatively by the two ligand binding assays. (3) As the final result tumor specimen could be grouped into classes of receptor status according to the presence or absence of a nuclear stain in immunocytochemical assays or according to receptor concentrations above or below distinct threshold which were fixed at 20 fmol/mg for Scatchard analyses of both receptor species, 20 fmol/mg for estrogen- and 40 fmol/mg for progestin receptors for the assay with sections. In this diagnostical consideration the concordance of both biochemical methods to the immunocytochemical assessment was high for estrogen and less pronounced for progesterone receptors. (4) In some breast cancer specimen analyzed biochemically an unspecific progestin binding component could be detected superimposed on the progesterone receptor peak after isoelectric focusing.

Breast Neoplasms↗

PubMed1989

Gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.

As a first step to investigate whether gonadotropin releasing hormone (GnRH) analogs might be able to modulate directly the proliferation of human epithelial ovarian carcinomata, we checked if binding sites for GnRH are present in these malignancies. Specific binding of [125I][D-Ala6-des Gly10]-GnRH-ethylamide (GnRH agonist = GnRH-A) could be demonstrated in plasma membranes from 32 out of 40 ovarian carcinomata tested. This binding was dependent on temperature, time and plasma membrane concentration. Mathematical analysis of the binding data showed that the interaction of GnRH-A with the binding sites was consistent with a single class of low affinity, high capacity binding sites (Ka = 1.42 +/- 0.14 X 10(5) M-1; range: 0.3-3.8 X 10(5) M-1; R = 209 +/- 69 X 10(-12) M/mg membrane protein; range 16-400 X 10(-12) M/mg MP; means +/- S.E., n = 32). Native GnRH and the GnRH antagonist [D-p-Glu1, D-Phe2, D-Trp3,6]-GnRH had Ka values comparable to those of the GnRH-A used. [125I]GnRH-A binding could not be displaced by oxytocin, thyrotropin releasing hormone and corticotropin releasing factor in concentrations up to 10(-4) M. Somatostatin cross-reacted with binding sites from some carcinomata, while it did not displace GnRH-A binding in membranes from others. Though the functional role of this specific binding site for GnRH in human epithelial ovarian carcinomata is still obscure, it might be part of an autocrine regulatory system and provide a possible point of attack for therapeutic approaches using GnRH analogs in this malignancy.

Adenocarcinoma↗

PubMed1989

Simultaneous quantification of oestrogen and progesterone receptors by a ligand binding assay in frozen sections.

We describe two modifications of a double-isotope assay for measuring the concentrations of oestrogen receptors and progesterone receptors in tumour cytosols and extracts of frozen tumour sections and endometrial sections. The concentrations of these receptors are derived from single-point/isoelectric focussing assays after incubation of cytosols or section supernatants either with ([125I]vinyl)-nortestosterone and [3H]oestradiol or [125I]oestradiol and [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor[6,7(3)H]pregn-4-ene-3,20-dione). The concentrations of the oestrogen and progesterone receptors found in cytosols (r greater than 0.93) and extracts from sections (r greater than 0.8) by dual label assays are highly correlated with those found by single label assays. The method described represents an approach to the determination of oestrogen and progesterone receptors biochemically in an amount of tissue which is comparable to that needed for immunocytochemical procedures.

Breast Neoplasms↗

PubMed1989

Studies on the subcellular mechanisms mediating the negative estradiol effect on GnRH-induced LH-release by rat pituitary cells in culture.

Cultured pituitary cells from adult female rats were treated for 4 or 24 h in the absence or presence of E2 (10(-9) mol/l) with increasing concentrations of the transcription inhibitor actinomycin-D or the translation inhibitor puromycin. During the last 4 h of incubation, LH release was stimulated with 5 X 10(-10) mol/l GnRH. The positive E2 effect observed after 24 h treatment with the steroid was clearly abolished by actinomycin-D at concentrations greater than or equal to 10(-10) mol/l and by puromycin at concentrations greater than or equal to 10(-5) mol/l. These findings indicate that the positive E2 effect on GnRH-induced LH release is fully dependent on intact mRNA and protein synthesis. The negative E2 effect observed after 4 h treatment with the steroid was not affected by actinomycin-D and abolished by puromycin only at concentrations greater than or equal to 10(-4) mol/l. Similar results were obtained, when cells had been treated for 4 h with actinomycin-D or puromycin before the 4 h E2 treatment started. Thus, the negative E2 effect seems to be independent of mRNA synthesis and dependent on protein synthesis to a lesser extent than the positive E2 effect. In an attempt to identify positively the subcellular mechanism via which E2 exerts its negative effect, several steps in the GnRH stimulus secretion coupling mechanism were checked whether or not they are modulated by E2. The negative effects of E2 (10(-9) mol/l) on LH release induced by GnRH (10(-10), 10(-9) mol/l) and by the activators of voltage dependent Ca2+ channels K+ (64 mmol/l) or veratridine (3.3 X 10(-5) mol/l) were comparable to those of the calcium antagonist verapamil (10(-6) mol/l). These findings supported the speculation that E2 might act on the Ca2+ channels. The LH release induced by the Ca2+ ionophores A 23 187 (10(-4) mol/l) or ionomycin (6.6 X 10(-5) mol/l), however, was also significantly reduced by 10(-9) mol/l E2, indicating that the steroid modulated a mechanism secondary to the increase of intracellular Ca2+. Also GnRH (10(-9), 10(-8) mol/l) induced accumulation of [3H]inositol phosphates was not influenced by E2 (10(-9) mol/l) treatment, though the steroid exerted a significant negative effect on the LH release by these cells, indicating that phosphatidylinositol-4,5-biphosphate breakdown is not the point of attack for the estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Acute facilitory action of progesterone on gonadotropin secretion of perifused rat pituitary cells.

The purpose of this study was to investigate the kinetics and estrogen dependence of the facilitory progesterone action on LH and FSH secretion from pituitary cells under dynamic culture conditions. Anterior pituitary cells obtained from ovariectomized or intact adult Wistar rats were cultivated on Cytodex 1 microcarrier beads. The perifusion experiments were performed with four separate perfusion chambers. The cells of chambers I + II had been pretreated with medium containing vehicle (0.1% ethanol), those of chambers III + IV with medium containing 1 nmol/l estradiol for 48 h. After perfusion was started, each of the chambers was challenged with an initial 2-min GnRH (1 nmol/l) pulse. Chamber I was further perifused with medium containing vehicle, chamber II with medium containing vehicle + 100 nmol/l progesterone, chamber III with medium containing 1 nmol/l estradiol, and chamber IV with medium containing 1 nmol/l estradiol + 100 nmol/l progesterone. Three further GnRH pulses were administered at 50-min intervals to each of the chambers. In estradiol-primed cells from intact rats, progesterone induced a positive effect on LH and FSH secretion after 50 min of exposure to progesterone. After 100 min of progesterone treatment, LH and FSH release were enhanced to 420 and 500 per cent, respectively. When such cells were not primed with estradiol, a slight though insignificant positive action of progesterone on LH release was present after 50 and 100 min of treatment, whereas FSH secretion was not influenced. The facilitory effect of progesterone occurred only after 100 min when estradiol-primed cells from ovariectomized rats were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment.

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.

Animals↗

PubMed1988

An improved micromethod for the determination of biochemically active estrogen and progesterone receptors in parallel to comparative histological examination of a single piece of tissue.

An improved radioreceptor assay of unfixed cryostat sections of human target tissues has been developed. Sections collected on glass coverslips were immediately incubated with 5 nM concentrations of either tritiated estradiol-17 beta for estrogen receptor (ER) or ORG 2058 for progesterone receptor (PR) determination. For quantitation, receptor-bound and free hormone were separated by isoelectric focusing (IEF). The assay allows the determination of steroid hormone receptors and comparative histological examinations in immediately neighbouring serial sections of a single piece of tissue. Biochemically, the validity of the assay procedure was evidenced by Scatchard analysis, by ligand and tissue specificities, by the linear relations of receptor and protein concentrations and the number of sections per test tube. Diagnostically, we compared the routine (6 point DCC-Scatchard) procedure for breast cancer analysis with the section method. A good correlation for ER and a less pronounced correlation for PR was found. Statistically, the precision of the method was verified by low deviations of duplicate determinations, low day-to-day variations and low inter-assay variations.

Breast Neoplasms↗

PubMed1988

Inhibitory actions of keoxifene on luteinizing hormone secretion in pituitary gonadotrophs.

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.

Animals↗

PubMed1988

Quantitative determination of oestradiol-17 beta hydroxysteroid dehydrogenase: increased sensitivity by HPLC separation of the hormones permits the measurement of enzyme activity in cryostat sections.

The activity of the oestradiol-17 beta hydroxysteroid dehydrogenase in human endometrial and breast cancer specimens was determined by the NAD-dependent conversion of oestradiol-17 beta to oestrone. The sensitivity of the determination was improved by the separation of the hormones by HPLC. We are now able to determine oestradiol-17 beta hydroxysteroid dehydrogenase quantitatively in cryostat sections. A clear correlation of serum progesterone levels and oestradiol-17 beta hydroxysteroid dehydrogenase activity in the endometrium was demonstrated, and we found a more than 30-fold increase in enzyme activity after the progesterone surge. In contrast, in breast cancer samples, we found no correlation between oestradiol-17 beta hydroxysteroid dehydrogenase and the measured serum parameters.

17-Hydroxysteroid Dehydrogenases↗

PubMed1987

Evidence of a gene-dosage effect for the glucocorticoid receptor in trisomy 18 mice.

The amount of cytosolic glucocorticoid receptor in liver of Ts18, Ts16, and Ts19 vs euploid mouse fetuses was studied after incubation of [3H]dexamethasone with cytosol followed by isoelectric focusing on polyacrylamide gels. In addition, corticosterone concentrations and enzyme activities of alanine aminotransferase and tyrosine aminotransferase were measured in the cytosol of the livers. The amount of glucocorticoid receptor in the cytosol fractions of the livers was always higher in the Ts18 than in the euploid fetuses of the same litter. It was also significantly (P less than 0.0005) higher if pooled data from different litters were analyzed. The ratio of the glucocorticoid receptor in Ts18 vs euploid mice varied between 1.3 and 4.7, with a mean of 2.1. In contrast, the glucocorticoid receptor levels in Ts16 and Ts19 fetuses were not different from the corresponding euploid controls. Comparing the corticosterone levels of the three trisomies tested with the corresponding euploid fetuses, no significant differences were found, indicating that the markedly elevated cytosolic glucocorticoid receptor concentrations in Ts18 were not due to different corticosterone levels. This finding is consistent with the assignment of the glucocorticoid receptor gene to chromosome 18 in the mouse. There was no correlation between glucocorticoid receptor levels and the activity of the two glucocorticoid inducible enzymes tested in the liver of mouse fetuses.

Alanine Transaminase↗

PubMed1987

Chemiluminescent labels for steroid immunoassays.

During the last few years non-isotopic techniques have been developed to measure steroid hormones. One of these methods is the luminescence immunoassay. Three classes of substances have been found to yield reasonable results, i.e. aminophthalhydrazides, aminonaphthohydrazides and acridiniumesters. Examples of application are shown.

Estriol↗

PubMed1987

Metabolism of exogenous 4- and 2-hydroxyestradiol in the human male.

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.

Adult↗

PubMed1986

Effects of estradiol and some antiestrogens (clomiphene, tamoxifen, and hydroxytamoxifen) on luteinizing hormone secretion by rat pituitary cells in culture.

Primary pituitary cell cultures from adult female rats were incubated for 4 or 24 h with various concentrations of estradiol (E2) or the antiestrogens (AE) tamoxifen (TMX), 4-hydroxytamoxifen (OH-TMX), and clomiphene citrate (CC). The luteinizing hormone (LH)-response of these cultures to gonadotrophin releasing hormone (GnRH) was monitored. Treatment for 4 or 24 h with high concentrations (10(-5) M) of the AE significantly decreased the GnRH-induced LH-release by the gonadotrophs. The negative E2-effect, which is observed in this model after 4 h was enhanced and the positive E2-effect, which occurs after 24 h, was completely reversed into a negative effect by these high AE-concentrations. Treatment of pituitary cells with increasing concentrations of E2 (10(-13)-10(-6) M) or AE (10(-12)-10(-5) M) for 24 h led first to a dose dependent increase of the LH-response to 5 X 10(-10) M GnRH. At higher E2- or AE-concentrations this positive effect was lost, resulting in bell shaped dose-response curves. The following maximal effective concentrations (EDmax) were found: E2 = 10(-10) M, OH-TMX = 10(-9) M, CC = 10(-7) M, TMX = 10(-6) M. Incubation of pituitary cells for 24 h with concentrations of AE near their EDmax and stimulation with increasing concentrations (10(-11)-10(-7) M) of GnRH resulted in significant increases of LH-secretion over a wide range of GnRH-concentrations. It is concluded that AE possess marked intrinsic activities on pituitary LH-secretion: at extremely high concentrations they suppress the GnRH-induced release of the gonadotrophin. At lower concentrations they increase the pituitary LH-response to GnRH in a manner which is qualitatively indistinguishable from that of E2.

Animals↗

PubMed1986

Importance of A-ring substitution of estrogens for the physiology and pharmacology of reproduction.

Estrogens substituted in the ortho-position of the phenolic hydroxy-group with an additional hydroxy- or methoxy-group are quantitatively important estrogen metabolites; first isolated and identified from the urine of man and rodents have been demonstrated in blood and different organs, e.g. the pituitary and hypothalamus. The physiological importance of the preeminent representatives of this group, the 2- and 4-hydroxyestrogens, the so-called catecholestrogens, is still equivocal. For example, numerous in vivo investigations in rodents have demonstrated that gonadotrophin secretion is influenced by these catecholestrogens. However, depending on the position of the A-ring substituent, major potency differences have been observed. The significant discrepancies between the quantitative and qualitative effects of catecholestrogens in in vitro and in vivo experiments have been presented and explained on the basis of different receptor affinities and the pharmacokinetics of catecholestrogens. An array of A-ring-substituted steroid model substances has been tested with respect to the effects of 2- and/or 4-substitution on stimulation or blockade of the estrogenic potency.

Animals↗

PubMed1986

Short-term effects of oestradiol and 4-hydroxyoestradiol on gonadotrophin-releasing hormone induced luteinizing hormone secretion by rat pituitary cells in culture.

Dispersed pituitary cells from adult female rats were preincubated for different time periods (0-12 h) in the absence or presence of 10(-9)M oestradiol (E2) or 4-hydroxyoestradiol (4-OHE2). Then the media were changed and the cells incubated for 4 h with either vehicle, or E2, or 4-OHE2 and additionally with different concentrations (10(-11)-10(-7) M) of gonadotrophin-releasing hormone (GnRH). Treatment of pituitary cells with E2 for 4 h (i.e. no preincubation with E2) significantly decreased the LH-response to GnRH at concentrations greater than or equal to 10(-10) M of the decapeptide. During a transition time of approximately 10 h (i.e. in cultures preincubated with E2 or vehicle for 2, 4, 6 or 8 h and then coincubated with E2 or vehicle and GnRH for 4 h) no differences between E2- and vehicle-treated cultures were observed. After 14 and 16 h of E2-treatment (i.e. 10 or 12 h preincubation and 4 h coincubation with GnRH) the LH-responses to GnRH in these cultures were significantly higher than in the respective controls. A nearly identical reaction pattern was observed when 4-OHE2 was used instead of E2. In a second series of experiments dispersed rat pituitary cells were suspended in a carrier gel and continuously perifused with medium, using small chromatography columns. When these cells were exposed for 4 min to 10(-9) M GnRH at 60 or 48 min intervals, they reacted with reproducible pulsatile LH-discharges during at least 6 subsequent stimuli with the decapeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Interactions of methylestrogens with cytoplasmic and nuclear estrogen receptors in rat pituitary gland, hypothalamus and uterus.

The in vitro affinities to cytoplasmic estrogen receptors of the methylestrogens 2-methylestradiol-17 beta, 4-methylestradiol-17 beta and 4-hydroxy-2-methylestradiol-17 beta, which are useful probes to test the biological importance of 2- or 4-hydroxylation of estradiol-17 beta (catecholestrogen formation), have been determined in hypothalamic, pituitary and uterine tissue of the ovariectomized rat. Moreover, the in vivo capacity of these compounds to translocate estrogen receptors into the cell nucleus of pituitary and uterine tissue has been studied. Methylestrogens exhibited estrogen receptor affinities which were not significantly different from the binding affinity of estradiol-17 beta. When given at a high dose (100 micrograms/animal) their nuclear translocation capacity was equal (4-methylestradiol-17 beta) or even higher (2-methylestradiol-17 beta) than that of estradiol-17 beta. However, at a low dose (5 micrograms/animal) 4-methylestradiol was completely ineffective in both the pituitary gland and the uterus, and 2-methylestradiol-17 beta was less potent than estradiol-17 beta in the pituitary gland.

Animals↗

PubMed1984

[Postmenopausal bleeding following the application of an estrogen-containing hair lotion--concomitant endocrine changes].

Application of a commercially available hair lotion containing high concentrations of oestradiol led to uterine bleeding in a 76-year old patient. Histological examination of the endometrium showed typical proliferation. Oestradiol blood levels were in the normal range of the follicular or luteal phase, LH- and FSH-serum concentrations were suppressed. After discontinuing the application of the assay used (20 pg/ml) and gonadotrophins rose to normal postmenopausal levels. These findings prove that after local application of oestradiol on the scalp, percutaneous resorption of the steroid is significant and systemic oestrogen effects have to be taken into account.

Age Factors↗

PubMed1984

Biphasic modulation of pituitary sensitivity to GnRH by oestrogens: the effects of A- and D-ring substitution on LH release in cultured pituitary cells.

The sensitizing effect of oestrogens on GnRH-stimulated LH release was evaluated in pituitary cells from adult female rats, cultured for 2 days in the presence of 10(-13) to 10(-6) M concentrations of oestradiol and selected A- and D-ring modified oestrogens. With almost all steroids tested, bell-shaped dose-response curves with comparable LH-maxima but different ED max values were obtained for the LH response to a submaximal GnRH stimulus (5 X 10(-10) M). Maximal LH response to 5 X 10(-10) M GnRH were found at the following oestrogen concentrations: oestradiol and 4-hydroxyoestradiol = 10(-11) M; 2-methyloestradiol = 10(-9) M; 2-hydroxyoestradiol = 10(-8) M; with 4-methyloestradiol no significant maximum was observed. When cells were pretreated with 10(-13), 10(-11) and 10(-9) M oestradiol, or 4-hydroxyoestradiol, or 2-hydroxyoestradiol, and exposed to increasing concentrations of GnRH (10(-11) to 10(-7) M), an almost 10-fold decrease in the ED50 for GnRH was observed after pretreatment with 10(-11) M oestradiol and 4-hydroxyoestradiol. With 2-hydroxyoestradiol at this concentration, the sensitizing effect was much less pronounced. Increasing the steroid concentration to 10(-9) M slightly decreased the effect of oestradiol and 4-hydroxyoestradiol, whereas it increased the effect of 2-hydroxyoestradiol. Thus, at the target cell 4-hydroxyoestradiol has the same potency as oestradiol, while 2-hydroxyoestradiol is significantly less active. The sensitizing effect of oestradiol is only slightly decreased by the presence of a methyl group in position 2, but is markedly reduced by a methyl group in position 4. Our results also demonstrate the high sensitivity of the pituitary to oestrogen-induced enhancement of GnRH-stimulated gonadotrophin release, as well as the decrease of the positive effect at high oestrogen concentrations. The bell-shaped dose-response curves for oestrogen action should be taken into account when evaluating the effects of oestrogens and their derivatives upon gonadotrophin release from the pituitary gland.

Animals↗