Search

The effects of the antiprogestins (APs) ZK 98.299, ZK 98.734 and RU 486 on GnRH-stimulated LH secretion and their antagonistic activity on progesterone (P) actions were investigated in cultured pituitary cells from adult female Wistar rats. P (100 nM) was able to exert a facilitatory effect on GnRH (1 nM)-induced LH secretion after short-term (4 h) treatment of estradiol-primed (1 nM, 48 h) rat pituitary cells. When the APs (10 pM-10 microM) were introduced during the 4 h incubation period with P the facilitatory effect of P was totally abolished at concentrations greater than 10 nM (ZK 98.299, ZK 98.734) and greater than 1 nM (RU 486). Also the APs were shown to block the inhibitory action of P which occurs after long-term incubation of pituitary cells with this steroid. However at concentrations greater than 10 nM (ZK 98.734, RU 486) and greater than 100 nM (ZK 98.299) this antagonistic action of the APs was lost. To evaluate whether the APs have direct effects on GnRH-induced LH secretion in the absence of exogenous P pituitary cells cultivated for 48 h with or without 1 nM estradiol were incubated for 4 or 24 h with increasing concentrations of the APs (10 pM-10 microM). Four hour treatment of non-estradiol-primed cells with ZK 98.299 or ZK 98.734 was without any effect on the LH response to a 1 nM GnRH-stimulus. Only the highest concentration of RU 486 (10 microM) reduced the LH response. Twenty-four hour treatment of the cultures with the APs led to enhancement of GnRH-stimulated LH secretion by up to 113, 37 and 33% for ZK 98.734, ZK 98.299 and RU 486, respectively. When estradiol-primed cells were used for the same experiments we observed exclusively inhibitory effects on GnRH-induced LH secretion after 4 and 24 h treatment periods. It is concluded that these new APs are potent inhibitors of P-actions, but also per se they induce diverse effects on GnRH-stimulated LH secretion in cultured rat pituitary cells which have to be taken into account.

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.

Phenol Red (Phr) which is widely used as a pH indicator in cell culture media has recently been described to possess estrogenic activity in different cell types. In the present study we investigated if the dye shows such activity on LH secretion of cultivated rat pituitary cells and controlled the established effects of estradiol (E2) and keoxifene (K) in this model in the absence of Phenol Red. 24 h treatment of pituitary cell cultures with Phr led to enhancement of GnRH-stimulated LH secretion whereas 4 h treatment reduced LH secretion. When the cells received E2 instead of Phr for the indicated incubation periods we observed nearly identical results i.e. a short-term inhibitory and a long-term stimulatory effect on LH secretion. 24 h treatment of pituitary cell cultures with increasing concentrations of Phr led to a stimulatory effect on GnRH-stimulated LH secretion an effect that occurred at 10 microM got maximal at 100 microM and was lost at higher concentrations resulting in a bell-shaped dose-response curve. The inhibitory action of Phr was present at concentrations greater than or equal to 10 microM. Both effects could be blocked by the antiestrogen K indicating their specificity. K has recently been described to induce an antigonadotrophic effect in this model. Although high concentrations of the antiestrogen were still able to inhibit LH secretion this effect was not present at lower concentrations when Phr-free culture medium was used in the experiments. Thus Phr showed weak estrogenic activity in the gonadotroph. The established actions of E2 and K on LH secretion were qualitatively reproducible when Phr was excluded from the culture medium.

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.

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.

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.

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.

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.

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.

Using a competitive binding assay the effects of 2-hydroxyestradiol-17 beta, 4-hydroxyestradiol-17 beta, estradiol-17 beta and progesterone on the binding of tritiated catecholaminergic ligands to membrane preparations from rat brain and pituitary gland were studied. Up to a concentration of 10(-5) M none of the steroids tested was able to displace [3H]spiroperidol, [3H]dihydroergocryptine or [3H]dihydroalprenolol. The data suggest that the catecholestrogens do not interfere directly with the binding of catecholaminergic ligands to dopaminergic, alpha-adrenergic or beta-adrenergic receptors in the central nervous system. The view that a catechol structure is not essential for the interaction with dopaminergic receptors was further supported by the results obtained from additional studies on the competition of O-methylated and deaminated dopamine metabolites with [3H]spiroperidol binding.

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.

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.