Interactions of Catecholestrogens with Cytoplasmic and Nuclear Estrogen Receptors in Rat Pituitary Gland and Hypothalamus

Abstract: The affinity of a series of catecholestrogens for 7S cytoplasmic receptor proteins from hypothalamus and pituitary gland of ovariectomised rats was assessed in vitro by a competitive charcoal binding assay at 4°C. The equilibrium dissociation constants ( K _i) of catecholestrogens 4-hydroxyestradiol, 4-hydroxyethynylestradiol, 2-hydroxyestradiol, 2-hydroxyethynylestradiol, and 4-hydroxyestrone were of the same order ( K _i 0.3–0.6 n m ) as those of estradiol and ethynylestradiol ( K _i: 0.1 n m ). Methylation of 2-hydroxyestradiol led to a substantial loss of binding affinity. Tritium-labelled receptor complexes were demonstrated in KCl extracts of purified nuclei from pituitary and hypothalamic tissue 1 h after intravenous injection of 0.1 mCi tritiated 2- or 4-hydroxyestradiol. These macromolecular complexes sedimented in the 5-6S region of 5–20% (w/v) sucrose gradients containing 0.4 m -KCl. Further evidence for the translocation of estrogen receptors by catecholestrogens into the nuclei of rat pituitary and hypothalamus was the increase in nuclear receptor concentrations, measured by exchange assay, 1 h after the intraperitoneal injection of 0.1 mg unlabelled catecholestrogen. Administration of 4-hydroxyestradiol and 4-hydroxyethynylestradiol increased nuclear receptor concentrations to the same maximal levels as those following application of the same dose of estradiol or ethynylestradiol, whereas the respective 2-hydroxylated compounds exhibited only 60–70% of the maximal translocating capacity. The in vivo translocating capacities of the various catecholestrogens tested at this dose correlated well with their binding affinities for cytosol receptors determined in vitro.     

Regulation of estrogen-stimulated lordosis behavior and hypothalamic progestin receptor induction by antiestrogens in female rats

Two estrogen antagonists, CI-628 (CI) and tamoxifen (TX), were used to examine the relationship between estrogen priming of lordosis behavior and progestin receptor induction in the hypothalamus-preoptic area (HPOA) of ovariectomized female rats. Lordosis behavior was assessed by measuring lordosis quotients (LQ) in response to injection of 2 micrograms of estradiol benzoate (EB) followed 48 hr later by 500 micrograms of progesterone (P). Behavior testing began 4 hr after P injection. The effects of antiestrogens were assessed by injecting CI and TX (1-2 mg) from 0 to 48 hr prior to EB. Levels of cytosol progestin receptor in the HPOA were determined by quantifying the specific binding of 0.5 nM [3H]R5020 to cytosols from animals receiving the same EB and antiestrogen treatments used in behavioral testing. TX given concurrently with or CI given 2 hr before EB abolished both lordosis behavior and induction of HPOA progestin receptors. In contrast, CI given 12 hr prior to EB abolished lordosis but permitted a 95% elevation in the concentration of progestin binding sites in the HPOA. TX or CI given 48 hr before EB resulted in moderate levels of lordosis (mean LQs from 56 to 69) and induction of HPOA progestin receptors from 85 to 130% above noninjected controls. However, CI given 24 hr prior to EB produced less than a 40% increase in brain R5020 binding even though lordosis behavior was equivalent to that seen in the 48-hr animals (mean LQ = 53). These data indicate that the effects of antiestrogens on female sexual behavior and on the synthesis of brain progestin receptors depend on which antiestrogen is used and the time interval between administration of estrogen and antiestrogen. They also demonstrate that under some conditions estrogen induction of cytosol progestin receptors in the HPOA can be dissociated from estrogen priming of lordosis behavior in rats.     

Pharmacokinetics of free catecholestrogens and catecholestrogen benzoates

To provide a definite basis for studies on the biological effects of exogenously administered catecholestrogens, the time courses of the concentrations of these estrogens in serum, pituitary and CNS-tissues were studied in male rats after s.c. injection of either 150 μg of 4-hydroxyestradiol or 2-hydroxyestradiol (dissolved in 200 μl sesame oil/ethanol/ascorbic acid; 97.5/2.5/0.1; vol/vol/wt) or equimolar amounts of 4-hydroxyestradiol 3,4-dibenzoate or 2-hydroxyestradiol 2,3-dibenzoate (dissolved in 200 μl sesame oil). The injection of free catecholestrogens resulted in bolus-like elevations of the serum and tissue concentrations of the respective compound (max. values up to 9 ng/ml, half-life below 1 h) whereas the injection of catecholestrogen benzoates gave lower (max. values about 1 ng/ml) but prolonged elevations (half-life approx. 24 h and 32 h for 4-OHE₂ and 2-OHE₂) of the respective free catecholestrogen.     

Effect of estrogen on lipoprotein lipase activity and cytoplasmic progestin binding sites in lean and obese Zucker rats

Injections of 5 micrograms estradiol benzoate (EB) for 5 days resulted in decreases in the rate of body weight gain in both lean (Fafa) and obese (fafa) Zucker rats. EB administration also resulted in significant induction of cytoplasmic progestin binding sites in both hypothalamic-preoptic area (H-POA) and adipose tissues from rats of both genotypes. However, EB treatment significantly decreased lipoprotein lipase (LPL) activity in adipose tissue from lean, but not obese, Zucker rats and the same treatment increased LPL activity in the uteri from lean, but not obese, Zucker rats. The data are discussed in terms of the metabolic and reproductive dysfunctions observed in the genetically obese rat.     

Thyroidal modulation of androgenic expression in mice submandibular gland

We investigated the influence of testes and thyroid gland on the maintenance of biochemical parameters and of [3H]R1881 binding sites of adult mice submandibular gland (SMG). Castration (Cx) performed at beginning of puberty prevented sex-dependent SMG development without interfering with maximal androgen binding capacity. Thyroidectomy (Tx) had strong effects on SMG, mainly by lowering the number of androgen binding sites. All alterations could be fully reverted after treatment with testosterone (5 mg/animal, single dose) or with thyroxine (T4, 250 micrograms/animal per day during 5 days). The effects of Cx on SMG could be reverted by therapy with testosterone, T4, or with both hormones (testosterone + T4) in a non-synergistic fashion. It is shown the importance of thyroidal activity on the physiological maintenance of androgen receptors in the murine SMG; the role played by thyroid gland seems to be essential for the full expression of the androgen-dependent SMG activity in adult mice.     

Gonadal steroids and anterior lobe dynorphin in the male rat

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.     

Effect of the catecholestrogen 2-hydroxyestradiol on the renin-angiotensin system in the rat

The effects of the catecholestrogen 2-hydroxyestradiol (250 and 500 micrograms/day, each for 7 days) on plasma renin substrate (PRS), activity (PRA) and concentration (PRC) were studied in male rats as compared with those of estradiol (250 micrograms/day, for 7 days) and vehicle alone (for 7 days). Pre-treatment levels of PRS, PRA, PRC and the PRA/PRC ratio were similar in four groups. After vehicle treatment, PRS, PRA, PRC and the PRA/PRC ratio remained unchanged. Estradiol treatment, however, produced an increase in PRS, an increase in PRA but no change in PRC. The PRA/PRC ratio after estradiol treatment was high. On the other hand, 2-hydroxyestradiol treatment caused no increase in PRS at a daily dose of 250 micrograms and a slight but significant increase in PRS at a daily dose of 500 micrograms. This treatment also produced increases in PRA as well as PRC at the two daily doses. These increases in PRA and PRC tended to be higher at a daily dose of 500 micrograms than at a daily dose of 250 micrograms. The PRA/PRC ratios after 2-hydroxyestradiol treatment were unaltered at the two daily doses. It is concluded that, while 2-hydroxyestradiol is less active in increasing PRS than estradiol, the compound is capable of increasing PRC.     

Down-regulation of progestin receptors in guinea pig brain: new findings using an immunocytochemical technique

Progesterone injection in estradiol-primed, ovariectomized guinea pigs results in down-regulation of hypothalamic progestin receptors determined by in vitro binding assays. In order to determine if progesterone also decreases immunostaining of progestin receptors and if progestin receptors are down-regulated preferentially in particular neuroanatomical areas, ovariectomized guinea pigs were injected with doses of estradiol benzoate (10 micrograms at 42 h before progesterone injection) and progesterone (500 micrograms at 4, 12, or 24 h before perfusion) that reliably induce the expression of lordosis and subsequent behavioral refractoriness to progesterone. Progestin receptor-immunoreactive cells were counted in sections from discrete parts of the preoptic area and hypothalamus. As expected, estradiol dramatically increased cell nuclear, and, to a lesser extent, cytoplasmic, immunostaining in defined regions of the preoptic area and hypothalamus. By 12 h after progesterone injection, the number of progestin receptor-immunoreactive cells had decreased in some areas, but not others. The rostral and caudal aspects of the ventrolateral hypothalamus were particularly responsive showing a substantial decrease in progestin receptor-immunoreactivity by 12 h after injection. No decreases in the progestin receptor-immunoreactive cell number were observed in any of the preoptic regions examined, although obvious decreases in immunostaining intensity were seen. The results of these immunocytochemical experiments extend earlier findings from in vitro progestin binding experiments and demonstrate that as with progestin binding, progestin receptor-immunoreactivity decreases when progesterone is injected in a behavioral desensitization procedure. Furthermore, they point to the ventrolateral hypothalamus as one site in which the down-regulation of progestin receptors may be particularly responsive to progesterone.     

Estrogenic effects of zearalenone on the expression of progestin receptors and sexual behavior in female rats

Zearalenone is a resorcylic acid lactone compound that is produced by fungal infection of edible grains and is believed to influence reproduction by binding to estrogen receptors. In order to study the potential estrogenic effects of this compound in the brain, we examined the effects of zearalenone on the expression of neuronal progestin receptors and feminine sexual behavior in female rats. Ovariectomized rats were treated with zearalenone (0.2, 1.0, or 2.0 mg), estradiol benzoate, or vehicle daily for 3 days. They were then either perfused, and progestin receptors visualized by immunocytochemistry, or injected with progesterone and tested for sexual receptivity with male rats. Progestin receptor-containing cells were counted in the medial preoptic area and ventromedial hypothalamus. The two highest doses of zearalenone increased the concentration of neuronal progestin receptors, as did 10 microg of estradiol. The highest dose of zearalenone (2 mg) also induced progestin receptor staining density comparable to that of 10 microg of estradiol benzoate. In behavioral tests, ovariectomized animals treated with 2 mg of zearalenone followed by progesterone showed levels of sexual receptivity comparable to females treated daily with estradiol benzoate (2 microg) followed by progesterone. These studies suggest that, although structurally distinct and less potent than estradiol, zearalenone can act as an estrogen agonist in the rat brain.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during the acute feedback effects of estradiol

Ovariectomized (OVX), hypothalamo/pituitary-disconnected (HPD) ewes were used to ascertain the short-term effects of estradiol on the number of gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland. The time course of the study was such that measurements were made during the period of short-term negative feedback and positive feedback. Groups of 4 OVX-HPD ewes were given 250-ng pulses of GnRH each hour and an i.m. injection of oil (Group 1) or 50 micrograms estradiol benzoate in oil (Groups 2-4). Blood samples were collected from each ewe prior to treatment with estradiol or oil and again immediately before slaughter. Groups 2, 3, and 4 were killed 6, 16, and 20 h, respectively, after administration of estradiol. Amplitudes of luteinizing hormone (LH) pulses and average plasma concentrations of LH were reduced 6 h after estradiol treatment. Sixteen and 20 h after injection, the average plasma LH levels were elevated, but pulse amplitudes were similar to preinjection values. The number of GnRH receptors was significantly (p less than 0.01) increased within 6 h of estrogen treatment and further increased 16 and 20 h after treatment. Pituitary content of LH was similar in all groups. These data indicate that the number of GnRH receptors in the pituitary gland of ewes can be acutely influenced by a direct effect of estradiol. However, the magnitude and direction of the change in receptors number does not account for the changes in pituitary responsiveness to GnRH, suggesting estradiol also modifies post-receptor mechanisms that influence secretion of LH.     

Modulation of Progestin Binding Activity in Cultured Human Breast Carcinoma Cells: The Effect of Serum Type and Concentration

Abstract

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity.

MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PR_c) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PR_c to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E₂) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T₄₇D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binidng levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level the highlight the fact that serum components can alter dramatically the cellular progestin binding activity.     

Inhibition by bromoestrogens of the effects of estradiol on apomorphine-induced climbing behavior

The chronic administration of estrogens to mice or rats will result in antidopaminergic effects. Apomorphine-induced climbing behavior in mice, the result of direct stimulation of dopamine receptors in the striatal and mesolimbic regions, is a simple animal model for examining these antidopaminergic effects of estrogens. Bromoestrogens, inhibitors of catechol estrogen formation, have been utilized in order to examine the role of estrogen metabolism in dopaminergic antagonism. Mice were pretreated for 3 days with 2-bromoestradiol, 4-bromoestradiol, or 2,4-dibromoestradiol dibenzoates alone or in combination with estradiol benzoate prior to apomorphine administration. The haloestrogens did not alter the climbing-induced responses elicited by apomorphine, whereas estradiol benzoate clearly attentuated the actions of apomorphine. Furthermore, the bromoestradiol dibenzoates were effective in reversing the effects of estradiol benzoate when the two steroids (estradiol benzoate and a bromoestrogen dibenzoate) were administered simultaneously during pretreatment. Thus, the bromoestrogens are able to inhibit the antidopaminergic effects of estradiol exhibited in the apomorphine-induced mouse climbing model.     

Neurokinin A in the anterior pituitary of female rats: effects of ovariectomy and estradiol.

The effect of acute and chronic ovariectomy and the substitutive treatment with 17-beta estradiol and/or progesterone on anterior pituitary levels of neurokinin A (NKA) was studied in female rats. Acute ovariectomy did not result in significant changes of NKA in the anterior pituitary gland as compared with the levels in diestrous intact rats, but a single injection of 5 micrograms of estradiol in ovariectomized rats significantly decreased NKA levels in the anterior pituitary gland. Progesterone was without effect and did not modify the decrease of NKA in the anterior pituitary gland induced by estradiol. In rats examined 11 to 17 days after ovariectomy, NKA in the anterior pituitary gland was significantly higher than in diestrous intact rats. In the hypothalamus, ovariectomy resulted in decreased levels of NKA in the median eminence-arcuate nucleus. Estradiol significantly reduced NKA stores in the anterior pituitary gland but increased them in the whole hypothalamus and in the median eminence-arcuate nucleus. Thus, estradiol seems to be a powerful regulator of NKA stores in the adenohypophysis and also in the hypothalamus.     

Cytoplasmic progesterone receptors in the hypothalamus-preoptic area of the mouse: effect of estrogen priming

A synthetic progestin, R5020, was used to identify cytoplasmic progestin receptors in the hypothalamuspreoptic area (HPOA) of ovariectomized mice. These high-affinity receptors exhibited an apparent dissociation constant of approx. 1 nM. The receptors were specific for progestins. [3H]R5020 binding was inhibited by more than 50% with a 50-fold excess of either radioinert R5020 or progesterone. 5 alpha-Dihydroprogesterone inhibited binding to a lesser extent. 3 alpha-Hydroxy-5 alpha-pregnane-20-one and cortisol did not compete for [3H]R5020 binding. Administration of estradiol benzoate (10 micrograms), 48 h prior to death, resulted in a 54% increase in the HPOA progestin receptor concentration when compared to oil-injected controls. These data demonstrate that there are specific and saturable cytoplasmic progestin receptors in the mouse HPOA and that the concentration of these receptors is increased after estrogen treatment.     

The effect of estrogen treatment on scopolamine inhibition of lordosis.

Previous evidence indicates that the cholinergic muscarinic antagonist, scopolamine, inhibits lordosis in female rats. In the experiments reported here, the effects of various doses and repeated administrations of estrogen on the scopolamine inhibition of lordosis were examined. In the first experiment, intraperitoneal injections of scopolamine (1 mg/rat) completely inhibited lordosis in ovariectomized rats primed with low doses of estradiol benzoate (0.25 or 0.5 micrograms for 3 days) and progesterone (500 micrograms). However, scopolamine was significantly less effective in inhibiting lordosis in females primed with a higher dose of estradiol benzoate (25 micrograms for 3 days) and progesterone (500 micrograms). When hormone priming was repeated on subsequent weeks, scopolamine continued to inhibit lordosis in females that received 0.25 micrograms estradiol benzoate but was less effective in females primed with 0.5 micrograms. Scopolamine failed to inhibit lordosis in females treated with 25 micrograms estradiol benzoate on these later tests. In the second experiment, various doses of scopolamine (1, 2, or 4 mg/rat) were administered intraperitoneally to females primed with the highest dose of estradiol benzoate (25 micrograms) and progesterone (500 micrograms). Lordosis was inhibited equally by all scopolamine doses during the first week. As in the first experiment, scopolamine failed to inhibit lordosis at all doses on subsequent weeks of testing. These results indicate that the ability of scopolamine to inhibit lordosis is reduced by increasing the dose or the number of estrogen exposures. Because higher doses of scopolamine failed to restore its inhibitory effect on lordosis an upregulation of muscarinic receptors by estrogen cannot account for the reduced effectiveness of scopolamine.     

Effects of Repeated Administration of Estradiol Benzoate on Tubero-Infundibular GABAergic Activity in Male Rats

Repeated (once a day for 8 days) but not single administration of estradiol benzoate (10 micrograms/kg, s.c.) induced a sevenfold increase in anterior pituitary gamma-aminobutyric acid (GABA) concentration in male rats. GABA concentration also increased in the median eminence whereas no changes or decreases were observed in other brain regions including hypothalamic arcuate nucleus, lateral septum, hippocampus, caudate nucleus, and substantia nigra. Eight-day estradiol benzoate injection also enhanced the Vmax of median eminence glutamate decarboxylase activity without affecting the Km of the enzyme for glutamic acid. Taken together, these results suggest that repeated administration of estradiol benzoate increases the activity of the tubero-infundibular GABAergic system in male rats.     

Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.     

Androgens, progestins, and glucocorticoids induce follicle-stimulating hormone beta-subunit gene expression at the level of the gonadotrope

FSH is produced by the pituitary gonadotrope to regulate gametogenesis. Steroid hormones, including androgens, progestins, and glucocorticoids, have all been shown to stimulate expression of the FSHbeta subunit in primary pituitary cells and rodent models. Understanding the molecular mechanisms of steroid induction of FSHbeta has been difficult due to the heterogeneity of the anterior pituitary. Immortalized LbetaT2 cells are a model of a mature gonadotrope cell and express the endogenous steroid receptor for each of the three hormones. Transient transfection of each receptor, along with ligand treatment, stimulates the mouse FSHbeta promoter, but induction is severely diminished using receptors that lack the ability to bind DNA, indicating that induction is likely through direct DNA binding. All three steroid hormones act within the first 500 bp of the FSHbeta promoter where six putative hormone response elements exist. The -381 site is critical for FSHbeta induction by all three steroid hormones, whereas the -197 and -139 sites contribute to maximal induction. Interestingly, the -273 and -230 sites are also necessary for androgen and progestin induction of FSHbeta, but not for glucocorticoid induction. Additionally, we find that all three receptors bind the endogenous FSHbeta promoter, in vivo, and specifically bind the -381 site in vitro, suggesting that the binding of the receptors to this element is critical for the induction of FSHbeta by these 3-keto steroid hormones. Our data indicate that androgens, glucocorticoids, and progestins act via their receptors to directly activate FSHbeta gene expression in the pituitary gonadotrope.     

Effect of estrogen on postovulatory LH surge in baboons.

In normally cycling female baboons, an LH surge appeared prior to ovulation, in addition, another LH surge (postovulatory LH surge) was observed within two days after ovulation. An attempt was then made to determine the effect of postovulatory LH on the luteinization of corpus luteum in baboons. Injections of 300 micrograms estradiol benzoate were given at 09.00 and 16.00 hr daily for 5 days following ovulation; the plasma level of LH was increased, but plasma progestin was suppressed. These results infer that the injected estrogen (estradiol benzoate) may inhibit the luteotrophic effect of postovulatory LH on the corpus luteum, therefore, plasma progestin remains lower even though postovulatory LH is elevated.     

Anisomycin inhibition of estradiol-induced and progesterone-facilitated luteinizing hormone surges in the immature rat

These studies were designed to examine the effect of anisomycin, a potent and reversible inhibitor of protein synthesis with low systemic toxicity in rodents, on induction of luteinizing hormone (LH) surges by estradiol and their facilitation by progesterone. Immature female rats that received estradiol implants at 0900 h on Day 28 had LH surges approximately 32 h later (1700 h on Day 29). Insertion of progesterone capsules 24 h after estradiol led to premature (by 1400 h) and enhanced LH secretion. Protein synthesis was inhibited by 97%, 95%, 47%, and 16% in the hypothalamus-preoptic area (HPOA) and by 98%, 87%, 35%, and 0% in the pituitary at 30 min, 2 h, 4 h, and 6 h after s.c. injection of anisomycin (10 mg/kg BW), respectively. A single injection of anisomycin at 0, 3, 6, 9, 12, 24, 27, or 30 h after estradiol treatment significantly lowered serum LH levels at 32 h. The effect of injecting anisomycin at 0, 24, or 27 h was overridden by progesterone treatment at 24 h, but LH secretion was delayed serum LH levels were basal (10-30 ng/ml) at 1400 h but elevated (500-800 ng/ml) at 1700 h. Complete suppression of LH surges in estradiol-plus-progesterone-treated rats was achieved with 2 injections of anisomycin on Day 29 at 0900 h and again at 1200 h or 1400 h. Further experiments were designed to examine proteins that might be involved in anisomycin blockade of progesterone-facilitated LH surges. Intrapituitary LH concentrations at 1700 h on Day 29 were 70-80% higher (102 +/- 12.5 micrograms/pituitary) in rats that received 2 injections of anisomycin than in vehicle-treated controls (58.5 +/- 7.7 micrograms/pituitary). There were no significant effects of anisomycin on cytosol progestin receptors in the HPOA (7.1 +/- 1.5 fmol/tissue, anisomycin; 7.2 +/- 0.3, vehicle) or pituitary (8.3 +/- 1.3 fmol/tissue, anisomycin; 11.7 +/- 2.9, vehicle) at this time. The concentration of pituitary gonadotropin-releasing hormone receptors (GnRH-R), however, was significantly lower after anisomycin (265 +/- 30 vs. 365 +/- 37 fmol/mg protein) treatment. These results suggest that both estradiol-induced and progesterone-facilitated LH surges involve protein synthetic steps extending over many hours. Blockade of progesterone-facilitated LH surges by anisomycin appears to be due primarily to an effect on release of LH to which lowering of GnRH-R levels may contribute.