ICI 182,780 acts as a partial agonist and antagonist of estradiol effects in specific cells of the sheep uterus

We assessed the ability of ICI 182,780 (ICI) to block the estradiol (E2) responses of genes within the sheep uterus. Ovariectomized ewes in the ‘ICI+E2’ treatment group received a uterine infusion with 10⁻⁷ M ICI for 14 h, an injection of 50 μg E2 6 h after the infusion started, and were hysterectomized 18 h postinjection. Other groups received only ICI or E2, or neither treatment (‘Con’). Both E2 and ICI increased the wet weight of dissected endometrium: averaging 10.0±1.2 g for ICI+E2, ICI, and E2 groups compared to 6.8±0.6 g for Con. Slot blot analyses of endometrial RNA showed that estrogen receptor- (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin, actin and c-fos mRNAs responded to E2 treatment: the first five increased an average of 60% while the last decreased 38%. In situ hybridization identified more subtle ICI effects: agonistic up-regulation of GAPDH mRNA in superficial endometrial cells, and antagonistic down-regulation of ER and PR mRNAs in the inner layer of the myometrium. Thus, we conclude that the agonist versus antagonist effects of ICI relative to those of E2 are a function of the gene examined as well as the specific cell within the uterus.     

The antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive tract and long-term decreased fertility without testicular atrophy

Background  

Estrogen receptors (ER) have important physiological roles in both the female and male reproductive systems. Previous studies using the estrogen receptor-α knockout mouse (αERKO) or antiestrogen treatment in adult rodents have shown that ERα is essential for normal function of the male reproductive tract. In the present study, time-response effects of the antiestrogen ICI 182,780 were determined to better understand ERα function in the adult male.     

Estrogen and antiestrogen effects on neonatal ovine uterine development

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor alpha (ERalpha) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERalpha regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17 beta (E2) valerate (EV), an ERalpha agonist, 3) EM-800, an ERalpha antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERalpha, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E2 does not regulate endometrial gland differentiation or development. Although ERalpha does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERalpha does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.     

Effects of the antiestrogen fulvestrant (ICI 182,780) on gene expression of the rat efferent ductules

The efferent ductules express the highest amount of estrogen receptors ESR1 (ERalpha) and ESR2 (ERbeta) within the male reproductive tract. Treatment of rats with the antiestrogen fulvestrant (ICI 182,780) causes inhibition of fluid reabsorption in the efferent ductules, leading to seminiferous tubule atrophy and infertility. To provide a more comprehensive knowledge about the molecular targets for estrogen in the rat efferent ductules, we investigated the effects of ICI 182,780 treatment on gene expression using a microarray approach. Treatment with ICI 182,780 increased or reduced at least 2-fold the expression of 263 and 98 genes, respectively. Not surprisingly, several genes that encode ion channels and macromolecule transporters were affected. Interestingly, treatment with ICI 182,780 markedly altered the expression of genes related to extracellular matrix organization. Matrix metalloproteinase 7 (Mmp7), osteopontin (Spp1), and neuronal pentraxin 1 (Nptx1) were among the most altered genes in this category. Upregulation of Mmp7 and Spp1 and downregulation of Nptx1 were validated by Northern blot. Increase in Mmp7 expression was further confirmed by immunohistochemistry and probably accounted for the decrease in collagen content observed in the efferent ductules of ICI 182,780-treated animals. Downregulation of Nptx1 probably contributed to the extracellular matrix changes and decreased amyloid deposition in the efferent ductules of ICI 182,780-treated animals. Identification of new molecular targets for estrogen action may help elucidate the regulatory role of this hormone in the male reproductive tract.     

Alterations in mammary gland development following neonatal exposure to estradiol,transforming growth factor α, and estrogen receptor antagonist ICI 182,780

High fetal/early postnatal levels of estrogen increase breast cancer risk, but the mechanisms remain unknown. Growth factors, such as transforming growth factor α (TGFα), may participate as secondary modifiers in this process. We characterized a modulatory role of early postnatal exposure to 17β-estradiol (E₂) on the developing mammary gland morphology by treating intact female CD-1 mice with physiological doses of E₂ (2–4 μg), human recombinant TGFα (4 μg), or an estrogen receptor (ER) antagonist ICI 182,780 (20 μg) during postnatal days 1–3. Early postnatal exposure of E₂ stimulated mammary ductal growth by days 25 and 35, but by day 50 this was inhibited. The level of differentiation from terminal end buds (TEBs) to the lobulo-alveolar units (LAUs) also was reduced by day 50. The number of TEBs was increased throughout most of the development in the female mice exposed to E₂ during early life. An exposure to TGFα or ICI 182,780 between postnatal days 1 and 3 stimulated ductal growth, formation of TEBs, and the differentiation of mammary epithelial structures. ICI 182,80 treatment also caused hyperplastic lobular-like structures in 54-day-old females. Thus, neonatal exposure to TGFα and ICI 182,780 induced both similar (increase in TEBs) and different (increase/decrease in lobulo-alveolar differentiation) developmental changes in the mouse mammary gland, when compared with an exposure to E₂. A unique feature of the postnatal E₂ treatment was that it inhibited ductal migration by days 50–54. Our data suggest than an exposure to E₂ on postnatal days 1–3, possibly combined with secondary epigenetic alterations, leads to various changes within the developing mammary tree. These changes may be potential prerequisites for mammary tumorigenesis. J. Cell. Physiol. 170:279–289, 1997. © 1997 Wiley-Liss, Inc.     

Sex differences in the analgesic effects of ICI 182,780 and Flutamide on ureteral calculosis in rats

To better define the involvement of gonadal hormones in the sex differences observed in experimental visceral pain, we administered antagonists of estrogen receptors (ICI 182,780 [ICI]) or androgen receptors (Flutamide [FLU]) to adult male and female rats suffering from artificial ureteral calculosis. Subjects were divided into groups and treated with one of the substances (ICI, FLU) or sweet almond oil (OIL, vehicle) for 5 days, starting 2 days before surgery. On day 3, animals underwent surgery, with half receiving an artificial calculosis (Stone) and half only a sham procedure. The animals' behavior (number and duration of ureteral crises) and blood hormone levels (estradiol and testosterone) were determined in all groups. In OIL-treated rats the number and duration of crises were higher in females than in males. The administration of ICI or FLU resulted in hormonal effects in males and behavioral effects in females. In males ICI treatment increased estradiol plasma levels and FLU increased testosterone plasma levels; in females ICI and FLU treatments both decreased the number and duration of the ureteral crises. These results, confirming previous findings of higher sensitivity of females than males to urinary tract pain, showed the modulatory effects of estrogen and androgen antagonists on the behavioral responses induced by pain but only in females.     

Differential estrogen and antiestrogen responsiveness of the uterus during development in the fetal, neonatal and immature guinea pig

After 2-day estradiol treatments, wet weight increases in fetuses, newborns and immature guinea pigs by (means +/- SEM) 75 +/- 4%, 170 +/- 16% and 234 +/- 25%, respectively; while after 3-day tamoxifen treatments they are 83 +/- 11%, 157 +/- 35% and 127 +/- 9%, respectively. During the same periods, estradiol increases the uterine content of DNA while the effect of tamoxifen on uterine DNA decreases throughout development. Histologically, both estradiol and tamoxifen induce in the fetus an increase in the size of the stroma and myometrium. Estradiol or tamoxifen, respectively, increase the luminal epithelial cell height by (means +/- SEM) 95 +/- 2% and 67 +/- 2% in fetuses, 286 +/- 20% and 100 +/- 2% in newborns and 260 +/- 10% and 138 +/- 4% in immature animals. Luminal epithelial cell number increases in fetuses, newborns and immature animals by (means +/- SEM) 167 +/- 10%, 248 +/- 50% and 76 +/- 15%, respectively, after estradiol treatments and 160 +/- 20%, 69 +/- 15% and 17 +/- 5%, respectively, after tamoxifen treatments. Uterine epithelial growth invading the stroma was observed in both estradiol- and tamoxifen-treated fetuses. In neonatal or immature animals, estradiol increases the size and the number of endometrial glands, while tamoxifen has progressively less effects on endometrial glands and on the myometrium. It is concluded that: 1) the estradiol-induced uterotropic effect increases progressively in fetal, neonatal and immature animals; and 2) throughout development, tamoxifen has progressively weaker estrogenic properties than estradiol.     

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

Background  

The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown.     

An overview of the development of combined oral contraceptives containing estradiol: focus on estradiol valerate/dienogest

Natural estrogens such as estradiol (E(2)) or its valerate ester (E(2)V) offer an alternative to ethinyl estradiol (EE). E(2)-containing combined oral contraceptives (COCs) have demonstrated sufficient ovulation inhibition and acceptable contraceptive efficacy. However, earlier formulations were generally associated with unacceptable bleeding profiles. Two E(2)V-containing preparations have been approved to date for contraceptive use: E(2)V/cyproterone acetate (CPA) (Femilar(?); only approved in Finland and only in women >40 years or women aged 35-40 years in whom a COC containing EE is not appropriate) and E(2)V/dienogest (DNG; Qlaira(?)/Natazia(?)). The objective of the current review is to provide an overview of the development of COCs containing natural estrogen, highlighting past issues and challenges faced by earlier formulations, as well as the current status and future directions. The majority of information to date pertains to the development of E(2)V/DNG.     

Dose and duration effects of estradiol valerate on serum and lipoprotein lipids

Non-alkylated estrogens, like estradiol valerate (F2V), are widely used in the treatment of the postmenopausal hormonal deficiency syndrome. Their effects on serum and lipoprotein lipids are characterized by an increase in the lipid constituents of high density lipoproteins (HDL) and, usually, a decrease in low density lipoproteins (LDL). These effects are considered beneficial as regards atherogenesis and the risk for cardiovascular diseases. Unlike the effects of alkylated estrogens, no concomitant increase in triglycerides (TG) in serum and very low density lipoproteins (VLDL) - adverse effects - are seen in doses of up to 2 mg E2V. In order to compare the effects of 2 and 4 mg of E2V on serum and lipoprotein lipids, 19 bilaterally oophorectomized women participated in a cross-over study after a 4 week long wash-out period. To evaluate the influence of the time factor, 10 of the women continued taking 2 mg and 9 taking 4 mg of E2V respectively for an additional period of 12 weeks, resulting in a total treatment period of 24 weeks per group. The serum lipoproteins were separated by preparative ultracentrifugation, the serum and lipoprotein lipids being assessed using commercially available kits. In the cross-over part of the study, total (TC) and free cholesterol (FC) and phospholipids (PL) increased in HDL and decreased in LDL. Neither dose increased TG in serum or VLDL. These changes in the lipoprotein pattern persisted at the end of the entire study. Consequently, within the range of commonly used doses (2 and 4 mg) E2V seems to have a constant and, in terms of cardiovascular disease, favourable influence on lipoprotein metabolism irrespective of doses and periods studied.     

Systemic ICI 182,780 alters the display of sexual behaviors in the female rat

The present study investigates the effects of the antiestrogen ICI 182,780 (ICI) on the display of sexual behaviors in female rats. ICI 182,780 is a pure anti-estrogen and when given systemically, ICI is thought to act only in the periphery, and is not believed to cross the blood brain barrier. The present study examines the effects of ICI on sexual receptivity and on paced mating behavior following treatment with estradiol benzoate (EB) and progesterone (P) (Experiment 1) or with EB alone (Experiment 2). In Experiment 1, ICI (250.0 microg) did not affect the display of receptivity or paced mating behavior induced by EB and P. In contrast, in Experiment 2 female rats receiving EB alone displayed a decrease in the level of sexual receptivity following treatment with 500.0 and 750.0 microg ICI (but not 250.0 microg ICI). In addition, in Experiment 2 EB-treated female rats receiving 250.0 microg ICI spent more time away from the male rat following an intromission and were more likely to exit from the male compartment following a mount. Last, ICI had potent antiestrogenic effects on vaginal cytology (Experiment 2) and on the uterus (Experiments 1 and 2). The present study supports a role for peripheral estrogen receptors in sexual receptivity and paced mating behavior and suggests that estrogen receptor activation may decrease the aversive sensation associated with sexual stimulation.     

Plasma gonadotropin patterns characterizing the development of polycystic ovaries in the estradiol valerate treated rat

Polycystic ovaries (PCO) develop in female rats within 4 weeks of an injection of estradiol valerate (EV). The final morphological transition from a noncystic to a cystic state occurs in the presence of estrous (control) mean serum gonadotropin concentrations, suggesting that gonadotropin patterns rather than mean concentrations are causal to PCO. We have examined plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns in female rats at estrus and on days 5, 11, 16, and 21 following EV treatment. Estrous animals displayed large amplitude LH pulses of short duration, interspersed among frequent, low amplitude pulses of similar duration, and infrequent, broad-based LH episodes. Following EV treatment, there was a progressive decline in the frequency and magnitude of the large amplitude LH pulses, such that by day 16, they ceased altogether. Conversely, the frequency of the low amplitude pulses increased such that the total pulse frequency remained constant. The onset of this pattern coincides with the wave of atresia that precedes the emergence of cystic follicles. The pulsatile plasma pattern of FSH seen at estrus remained unaltered at all time intervals examined after EV treatment. We conclude that there is a specific LH pattern responsible for the generation and maintenance of the PCO condition.     

Neonatal exposure to estradiol valerate programs ovarian sympathetic innervation and follicular development in the adult rat

A single injection of estradiol valerate (EV) to 14-day-old rats (when the ovarian follicle population has been already established) disrupts cyclicity, increases the activity of key enzymes of androgen biosynthesis, and develops polycystic ovary by a causally related increase in ovarian noradrenaline (NA). The current study examined an early window of ovarian development to look for a specific stage of development at which estradiol can induce such changes in sympathetic activity and follicular development. A single dose of EV applied to rats before the first 12 h of life rapidly increases (after 24 h) the ovarian expression of nerve growth factor (Ngfb) and p75 low-affinity neurotrophic receptor (Ngfr) mRNAs. When adults, rats presented early vaginal opening, disrupted cyclicity, appearance of follicular cyst, absence of corpus luteum, and infertility. Total follicles decreased, mainly due to a reduced number of primordial follicles, suggesting that estradiol acts in the first stages of folliculogenesis, when primordial follicles are organizing. These changes paralleled a 6-fold increase in NA concentration. No changes in NA content were found in the celiac ganglia, suggesting a local, non-centrally mediated effect of estradiol. Surgical section of the superior ovarian nerve (the main source of sympathetic nerves to the ovary) to rats neonatally treated with EV decreased intraovarian NA, delayed vaginal opening, and blocked the development of follicular cyst and that of preovulatory follicles. Therefore, we can conclude that early exposure to estradiol permanently modifies ovarian sympathetic activity and causes profound changes in follicular development, leading to the polycystic ovary condition.     

Pharmacokinetics and biotransformation of estradiol valerate in ovariectomized women

Estradiol valerate is well suited for treatment of the characteristic symptoms accompanying menopause in women. The pharmacokinetics and biotransformation of estradiol valerate were studied in women following intravenous, intramuscular and oral administration. Intravenously given, estradiol valerate is split by enzymatic hydrolysis into 17 beta-estradiol and the fatty acid. The estrogen arising in vivo from the steroid ester is subject to intermediate metabolism. The metabolites are eliminated at a nearly constant rate mainly in urine. The biotransformation of estradiol valerate was not different following intravenous or intramuscular injection. Orally given estradiol valerate is subject to an extensive first pass metabolism. Daily repeated oral administration does not result in accumulation of 17 beta-estradiol and its metabolites.     

ICI 182,780 has agonistic effects and synergizes with estradiol-17 beta in fish liver, but not in testis

Background  

ICI 182,780 (ICI) belongs to a new class of antiestrogens developed to be pure estrogen antagonists and, in addition to its therapeutic use, it has been used to knock-out estrogen and estrogen receptor (ER) actions in several mammalian species. In the present study, the effects and mechanism of action of ICI were investigated in the teleost fish, sea bream (Sparus auratus).     

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.     

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.     

Embryotoxicity of sex steroidal hormones in nonhuman primates: II. Hydroxyprogesterone caproate, estradiol valerate

Two sex steroid compounds which have been used clinically for parenteral supportive therapy of pregnancy were examined for embryotoxic effects in rhesus and cynomolgus macaques. Hydroxyprogesterone caproate (HPC) alone or in combination with estradiol valerate (EV) were administered intramuscularly (i.m.) to pregnant monkeys at 7-day intervals between 20 and 146 days of gestation and fetuses were examined following cesarean section at 150 +/- 2 days. HPC alone was tested in both species at doses ranging from 0.01 X to 10 X the human dose equivalent (HDE); only rhesus monkeys were exposed to the HPC + EV combination at 0.1 X to 10 X HDE. Total embryolethality resulted following the administration of HPC alone and combined with EV at 1 X and 10 X HDE in rhesus monkeys; the level of abortions in cynomolgus monkeys exposed to HPC (0.1 X to 1 X HDE) was comparable to controls. A small number of nonspecific malformations and developmental variations observed in cynomolgus fetuses after HPC exposure were considered to be incidental findings. No anomalies were found in surviving rhesus monkey fetuses treated with HPC + EV. The results indicate that long-term in utero exposure to the progestin, HPC, alone or in combination with EV in rhesus and cynomolgus monkeys, is embryolethal but not teratogenic at doses up to ten times the human therapeutic dose.