Flavonoids inhibit estrogen binding to rat alpha-fetoprotein.

There is considerable interest in the role(s) of plant-derived compounds such as bioflavonoids in regulating steroid hormone action in mammals, and in particular, the possible effects of the bioflavonoids on the growth of steroid-dependent breast and prostate tumors and on possible abnormal development of steroid-sensitive tissues. Studies of the hormone-like actions of bioflavonoids often use fetal or neonatal rats, which contain high levels of serum alpha-fetoprotein (AFP), a protein that binds estradiol with a Kd approximately 5 x 10(-9) M. Interaction of bioflavonoids with AFP could affect the availability of estrogens to estrogen-responsive cells, as well as the actions of bioflavonoids. These considerations motivated us to study the effect of several flavonoids (quercetin, rutin, naringenin, chrysin, apigenin, kaempferol, myricetin, morin, fisetin) and isoflavonoids (daidzein, genistein) on estrogen binding to rat AFP. We found that naringenin, a flavanone, and quercetin and kaempferol, flavonols, inhibit estrogen binding to AFP with apparent Kds of about 5 x 10(-7) M. To our surprise, the two isoflavonoids, daidzein and genistein, have Kds of about 5 x 10(-6) M for AFP. This 10-fold [correction of 1Q-fold] difference in affinity for AFP between flavonoids and isoflavonoids suggests that AFP has a specificity for the flavonoid structure. Moreover, the affinities of bioflavonoids for rat AFP are sufficiently high to suggest that flavonoids and isoflavonoids could modulate estradiol and estrone binding to rat AFP in vivo, when present at dietary levels. Additionally, the potency of the plant estrogens may be altered by binding to AFP. The flavonoids that we tested have different hydroxyl and glucoside substituents on the A, B, and C rings, which allows us to define some of the spatial requirements for binding to AFP. We find that 5,7-hydroxyl groups in ring A and a 4'-hydroxyl group in ring B are important for binding to AFP. This information, combined with molecular modeling studies, may elucidate the molecular basis for recognition of flavonoids and estrogens by AFP. Also, these findings indicate that the flavonoid levels in the diet need to be considered in studies of the effects of various xenobiotics and endocrine manipulations on experimental animals, particularly during development when serum estrogen binding protein concentrations are often elevated. Finally, bioflavonoids should be useful tools for understanding the variety of estrogen actions initiated by different structural classes of estrogens.

Effects of toremifene on neonatal rat uterine growth and differentiation.

In the developing rodent uterus, the estrogen agonist activity of triphenylethylene antiestrogens such as tamoxifen alters uterine luminal epithelium morphology and inhibits uterine gland genesis. We examined uterine growth and differentiation in female offspring from date-mated Sprague-Dawley rats given the structurally related antiestrogen, toremifene, by s.c. injection in 10 microl of sesame oil on postnatal days (PND) 1-5, 10-14, or 20-24. Toremifene given on PND 10-14, a period of rapid uterine gland differentiation, caused a dose-related increase in uterine weight, tripled luminal epithelium cell height, and completely inhibited uterine gland development on PND 14 at doses of 10 microg or higher. Based on this dose-response analysis, a 10-microg dose of toremifene was chosen to assess uterine development after neonatal exposure (PND 1-5). Uterine weights and luminal epithelium cell heights were significantly increased by toremifene on PND 5 but returned to control levels by PND 26. Uterine gland numbers were reduced to 50% those of controls on PND 26. Dose-related uterine weight and luminal epithelium cell height increases were also observed in rats given toremifene on PND 20-24. This estrogen agonist activity of toremifene, revealed primarily in the uterine luminal epithelium, indicates that toremifene is developmentally toxic.

ICI 182,780 inhibits endogenous estrogen-dependent rat uterine growth and tamoxifen-induced developmental toxicity.

To assess the effects of the steroidal antiestrogen ICI 182,780 on postnatal uterine development, female Sprague-Dawley rats were given s.c. injections of ICI 182,780 (0.1-100 micrograms/rat) on each of postnatal days (PND) 10-14. ICI 182,780 inhibited uterine growth, as measured by uterine weight, in a dose-dependent manner but had no effect on either uterine luminal epithelium hypertrophy or gland genesis. Immunohistochemical analysis revealed that ICI 182,780 (10 micrograms) markedly reduced uterine estrogen receptor (ER) immunoreactivity in all uterine cell types while tamoxifen (10 micrograms) increased ER immunoreactivity, most notably in the luminal epithelium. In addition, tamoxifen increased uterine weight and induced luminal epithelium hypertrophy but inhibited uterine gland genesis--outcomes also seen with synthetic estrogens such as diethylstilbestrol. To test the hypothesis that these effects are a consequence of the estrogen agonist activity of tamoxifen, rats were cotreated with ICI 182,780 (10 micrograms, PND 8-14) and tamoxifen (10 micrograms, PND 10-14). ICI 182,780 greatly reduced or completely blocked tamoxifen-induced uterine weight gain, luminal epithelium hypertrophy, tamoxifen-induced ER immunoreactivity, and the inhibition of uterine gland genesis. ICI 182,780 given daily on PND 1-5 did not alter PND 5 uterine weight or uterine differentiation on PND 26. We conclude that postnatal exposure to ICI 182,780 does not affect uterine growth or differentiation at an age when the uterus is not dependent on estrogen for growth, i.e., PND 1-5, but does inhibit later endogenous estrogen-dependent uterine growth. The blockade of tamoxifen-induced uterine developmental alterations by ICI 182,780 demonstrates that these tamoxifen effects result from its estrogen agonist activity.

The effects of phytoestrogens on neonatal rat uterine growth and development.

Phytoestrogens found in clover, alfalfa, and soybeans have caused reproductive toxicity in several mammalian species. Other estrogens, such as diethylstilbestrol (DES), are developmental toxicants, reducing uterine estrogen receptor (ER) concentration, altering uterine growth, and eliciting reproductive tract abnormalities in the rat. The present study examines the effects of the phytoestrogens coumestrol and equol on the developing rat uterus. Various doses of these compounds were injected sc on postnatal days (PND) 1-5 or 1-10 to ascertain their effects on uterine weight and ER levels, and on PND 10-14 to determine their effects on uterine weight and gland genesis. Coumestrol (PND 1-5) was about 10(-3) as potent as DES in increasing uterine weight (wet or dry) while equol increased dry weight only, with a potency of 10(-5) that of DES. Although the 10 and 100 micrograms doses of coumestrol (PND 1-5 or 1-10) initially increased uterine wet weight, by PND 20 uterine weights either equaled or fell significantly below controls. The 100-micrograms dose of coumestrol (PND 1-5 or 1-10) reduced ER levels at all ages, while the 10-micrograms dose was not as effective. Equol (PND 1-5 or 1-10) did not affect ER levels. Premature uterine gland genesis occurred by PND 9 for the PND 1-5 100-micrograms coumestrol dose. When given on PND 10-14 (the critical period of gland genesis), 10 micrograms and 100 micrograms of coumestrol and 10 micrograms DES greatly increased uterine weight, while no effect was elicited by equol. Although coumestrol and equol inhibited uterine gland genesis in a dose-dependent manner, neither abolished gland genesis as did 10 micrograms of DES or tamoxifen. These data demonstrate that coumestrol elicits uterine biochemical and morphological toxicity much like DES. Equol decreased uterine gland number without increasing uterine wet weight or luminal epithelial hypertrophy, which is inconsistent with either an estrogenic or antiestrogenic action in the uterus.

Effects of coumestrol and equol on the developing reproductive tract of the rat.

The phytoestrogens, coumestrol and equol, are weakly estrogenic. Here, we have examined their ability to induce responses in the neonatal rat uterus. Potent estrogens such as diethylstilbestrol (DES) and 17 beta-estradiol which initially double uterine weight on postnatal Day (PND) 5 when given on PND 1-5 subsequently reduce both uterine growth and gland development at later ages. In this study, Sprague-Dawley pups were treated neonatally (PND 1-5) with various doses of coumestrol and equol, and sacrificed at different ages to determine alterations in biochemical and morphological endpoints. Other rats were injected with the same compounds during the critical period of gland genesis (PND 10-14) to examine their effects on gland development. At the 100 micrograms coumestrol dose, on PND 1-5, premature gland development and increased uterine weight were observed. However, at later ages, uterine weight was significantly lowered and there was a severe suppression in the estrogen receptor (ER) levels. Equol lowered uterine weight at the later ages but did not affect ER levels. When given on PND 10-14, both coumestrol and equol caused a dose-dependent inhibition of gland genesis though not as severe as either DES or tamoxifen. Coumestrol was about 10(3) more potent than equol as an estrogen and behaved much like DES with respect to its effects on uterine weight, glands, and ER levels. At the doses used in this study, equol failed to demonstrate either estrogenic or antiestrogenic activity.

Progesterone and estradiol interaction in the regulation of rat uterine weight and estrogen receptor concentration.

Autologous down-regulation of hormone receptors has been shown for several steroid hormones. We have previously shown estradiol (E2) regulates estrogen receptor (ER) in ovariectomized (OVX) rats. These studies have been extended to investigate the interaction between progesterone (P) and E2 in the regulation of ER and uterine weight. We implanted Silastic capsules containing varying concentrations of E2 (0.0005 mg E2/ml to 5.0 mg E2/ml of sesame oil) into adult female Sprague-Dawley rats one week after OVX. Simultaneously with implantation, P injections were started (sc in sesame oil) at doses of 1-40 mg/day for three days. In the absence of P, the 0.05 mg E2/ml implant significantly increased total ER levels (measured by cytosol and nuclear exchange assays) by 25%, while the two highest concentrations of E2 (0.5 and 5.0 mg E2/ml) significantly decreased cytosol and total ER levels by at least 40%. No P dose altered ER levels in OVX rats or in rats given E2 implants of 0.01 mg E2/ml or lower. At E2 implant concentrations of 0.05 mg E2/ml and higher, P decreased total ER levels 30%-50% compared to the appropriate E2-only controls. P increased uterine weight by 25% in OVX controls and in rats treated with E2 implants of 0.01 mg E2/ml and below. In contrast, P inhibited uterine weight gain induced by 0.05-5.0 mg/E2 ml implants by 20%-30%; maximal inhibition occurred at 5 mg/day of P and above. These data demonstrate that P increases uterine weight but does not alter ER concentration in rats with low E2 levels (OVX or low E2 concentration implants) but decreases uterine weight and down-regulates ER at higher E2 levels, regardless of whether ER is up-regulated or down-regulated by E2.

Long-term effects of postnatal exposure to diethylstilbestrol on uterine estrogen receptor and growth.

Diethylstilbestrol (DES) treatment of female rats on postnatal days (PND) 1-5 reduces uterine growth, estrogen receptor (ER) level and gland number by PND 25, while daily DES treatment on PND 1-25 increases uterine growth 4-fold, further reduces ER level and completely suppresses gland formation. We now report the persistence of these effects in adults. By PND 60, uterine weight was 70% of controls in rats injected with DES on PND 1-5 but only 10% of controls in rats injected PND 1-10 or longer. In fact, uterine weights were the same on PND 10 and 60. Uterine gland numbers were reduced to 30% of controls in all DES-treated rats regardless of exposure length; however, luminal and glandular epithelial cell heights were reduced to less than 50 and 70%, respectively, of controls when DES was given on PND 1-25 but not when given on PND 1-5. Ovariectomy 7 days prior to sacrifice on PND 60 reduced uterine weight in controls by 67% and in rats injected with DES on PND 1-5 by 53%, but had no effect in rats injected with DES on PND 1-10. DES exposure at either PND 1-5 or 1-10 lowered ER levels by 35-50% at both 60 and 90 days. Treatment with a high dose of estradiol (E2) 1 week before sacrifice significantly down-regulated ER to the same concentration in all treatment groups at PND 60 and 90. Following E2 treatment, all groups also showed increased uterine weight at PND 60 and 90. These data show there is a short period of development (PND 5-10) in which further DES exposure indirectly inhibits uterine growth.

Estradiol down-regulation of the rat uterine estrogen receptor.

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.

Short-term effects of physiological and pharmacological doses of estradiol on estrogen receptor and uterine growth.

Estradiol (E2) regulation of estrogen receptor (ER) concentrations has been shown to be both time- and dose-dependent. E2 concentrations of 0.5 mg/ml or greater contained in Silastic capsules suppressed uterine ER concentrations after one day's exposure. In this study, we looked at the effects of physiological (1.0 and 10.0 micrograms subcutaneous injections) and pharmacological (5.0 mg/ml implants) doses of E2 on ER concentrations at times less than 24 hours. The implanted rats had maximum E2 plasma levels of approximately 2000 pg/ml for at least six hours which fell to around 800 pg/ml by 12 hours where they remained up to 24 hours. The physiological doses resulted in plasma levels at one hour of 2000 pg/ml (10 micrograms dose) and 250 pg/ml (1 microgram dose) both of which fell to less than 60 pg/ml by six hours. All treatments caused maximal ER suppression by six hours; however, the implants caused a greater reduction in ER levels than either of the physiological doses. The reduction of ER levels was due primarily to a decrease in the "cytosolic" receptor. Despite the decrease in ER, all doses caused a significant and equivalent increase in uterine weight at six hours, however, only the implanted animals maintained the maximal uterine weight gain through 24 hours. This maintenance of uterine weight appears to be correlated with a small but significant increase in the nuclear ER level over this same time period. Thus, while E2 can cause a short-term suppression of its receptor concentration with no effect on short-term uterine weight gain, uterine growth is positively correlated with the level of nuclear ER.

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method.

Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.

Estradiol-stimulated increases in uterine eosinophils and nuclear type II estrogen-binding sites are prevented by pertussis toxin.

Previous studies from several laboratories have demonstrated that estradiol treatment resulted in an increase in nuclear type II binding sites. Our previous data suggest that this increase was due to the estradiol-stimulated influx of circulating eosinophils. Therefore, we suggested that the uterine nuclear type II estrogen-binding sites were not of uterine origin. In this report we present further evidence to support this hypothesis. Treatment of immature rats with estradiol resulted in the stimulation of several uterine parameters, namely wet weight, protein synthesis, eosinophil number, peroxidase activity, nuclear type II binding sites, and the synthesis and secretion of a 180-kDa protein. The coadministration of pertussigen had no effect on the estradiol-stimulated increase in wet weight, protein synthesis, or the synthesis and secretion of the 180-kDa protein. However, pertussigen did prevent the estradiol-stimulated increase in eosinophils, peroxidase activity, and nuclear type II binding sites, demonstrating a coordinated response. Since peroxidase activity is known to be contained int he eosinophil, these data are consistent with our earlier demonstration that the type II sites are of eosinophil origin. These data also support and extend our previous findings in neonatal animals that estradiol can stimulate a growth response without a corresponding increase in the nuclear type II binding sites. These results further indicate that the estradiol-stimulated increase in eosinophils does not appear to play a key role in the control of uterine growth.

Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels.

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.

Identification of uterine nuclear type II estrogen binding sites in estrogen treated rats.

Uterine nuclear fractions from estrogen-treated rats contain both the estrogen receptor and a lower affinity estrogen binding site (type II site). In Scatchard plots of estrogen binding, two types of curves are seen. The hook-shaped form is composed of a linear component (the estrogen receptor) and a convex component (the type II site) while the curvilinear form is resolvable into two linear binding species (the estrogen receptor and a secondary site). To clarify the relationship between the two forms, we examined the curvilinear form from immature rats injected for 4 days with estradiol (E2) for type II site properties. Like the hook-shaped type II, this form could be detected in a nuclear exchange assay at both 37 and 4 degrees C, but at neither temperature in the presence of reducing agent. Additionally, the steroid specificity of the curvilinear form was identical to the hook-shaped form. The hook-shaped form was found in both immature and ovariectomized adult rats implanted for 6 days with an E2-releasing Silastic capsule to provide pharmacological E2 levels. When uteri from implanted animals displaying the hook-shaped form were mixed in various ratios with uteri lacking type II sites, the curvilinear form was produced. Animals given an E2 implant for 3 days, followed by a 3 day hormone-free period showed a curvilinear form. In vivo E2 dose-response experiments showed the curvilinear form at low E2 doses and the hook-shaped form at the high dose and in implanted animals. We conclude that curvilinear Scatchard plots result from the presence of authentic type II at lower concentrations than those giving rise to the hook-shaped form.

Inhibition of rat uterine gland genesis by tamoxifen.

We have previously shown that rat uterine gland genesis occurs rapidly and synchronously between postnatal days 9-15. Exogenous estrogens either stimulate or inhibit gland genesis depending on dose and age at administration. We therefore examined the developmental effects of the triphenylethylene antiestrogen tamoxifen, which exhibits both estrogen agonist and antagonist properties, in the postnatal rat uterus. Tamoxifen administered sc in oil on postnatal days 1-5 or days 10-14 caused dose-related inhibition of uterine gland genesis which persisted to day 26 or day 60, respectively. Tamoxifen administered on postnatal days 20-24, which is after the age of normal gland genesis, did not alter the number of preexisting glands. A 24-h exposure to tamoxifen inhibited 17 beta-estradiol (E2)-induced ornithine decarboxylase (ODC) activity measured 6 h after E2 administration in 14-day-old rats. Treatment with tamoxifen before or during the period of gland genesis also reduced uterine responsiveness to a single dose of E2 as measured by both uterine weight gain (after a 24-h exposure on days 14, 19, 22, and 26) and the pattern of E2-induced ODC activity in 26-day-old rats. Control rats respond to E2 with peaks of ODC activity at 6 and 18 h after administration. Treatment with tamoxifen on either postnatal days 1-5 or 10-14 reduced the 18-h peak to approximately half of controls but did not affect the 6-h E2-induced ODC peak. Analysis of both nuclear and translocatable cytosol estrogen receptor in uteri from 26-day-old rats indicate that neither the dissociation constant (KD) nor the number of binding sites was affected by tamoxifen treatment on postnatal days 1-5 or 10-14.

Estrogenic activity of zearalenone and zearalanol in the neonatal rat uterus.

Fusarium sp. contaminated feedstuffs elicit adverse estrogenic effects in several commercially important animal species via the mycotoxin zearalenone. An estrogenically active synthetic derivative, zearalanol, is used as an anabolic agent in cattle. Since estrogens can irreversibly alter target tissue development, we investigated the estrogenic activity of these compounds in the neonatal rat uterus. Both induced dose-dependent premature uterine growth when injected daily on postnatal days 1-5 (ED50 = 1.3 mg/kg BW). Nuclear estrogen receptor levels dramatically increased 1 hour after either a single injection on day 5 or after five daily injections. In 5-day-old animals, the translocated nuclear receptor was characterized as a single class of binding sites with a dissociation constant (KD) for estradiol (E2) of 1 nM. At 15 days, zearalanol-treated animals showed greater uterine nuclear receptor retention than zearalenone-treated animals. In 5-day-old animals, single mycotoxin doses induced five fold elevations of ornithine decarboxylase (ODC) at 6 hours. Unlike the growth response, ODC dose-response studies showed zearalanol to be about 20-fold more effective than zearalenone. Time course studies revealed that a low dose of zearalenone, but not of zearalanol, resulted in a shift in peak activity from 6 to 8 hours. These data suggest that metabolism of zearalenone may be important in short-term pharmacodynamics. In a competitive binding assay, neither compound competed [3H]E2 from the E2 binding site on alpha-fetoprotein. We conclude that the uterine growth response and ODC induction demonstrate the neonatal estrogenic action of these mycotoxins, apparently mediated via the estrogen receptor. The greater effectiveness of zearalanol in inducing ODC may be related to nuclear retention and/or zearalenone metabolism.

Eosinophils as the source of uterine nuclear type II estrogen binding sites.

Rat uterine nuclei have been reported to contain two types of estrogen binding sites (I and II). Type I is the classical high affinity, low capacity translocatable estrogen receptor, while type II is a lower affinity non-translocatable binding site. Correlation of data on hormonal specificity of induction and inhibition, tissue and cellular localization, and ontogenic appearance for type II binding sites and eosinophils suggested the hypothesis that these binding sites are associated with eosinophils. Although type II sites are reported to be highly correlated with uterine growth, premature growth can be elicited in newborn rats by five daily estradiol injections without the appearance of type II sites. Under these conditions, no eosinophils, as measured by peroxidase activity, are found. However, multiple estradiol injections on postnatal days 6-10 or 10-14 increased the levels of both type II sites and eosinophils. Eosinophils, purified from a peritoneal lavage, were found to contain a low affinity binding site with characteristics similar to type II binding sites. Short term estrogen-treated rat uteri contain only type I nuclear receptor and low levels of eosinophils while long term estrogen-treated rat uteri contain both type I and type II nuclear binding sites as well as approximately 6 X 10(5) eosinophils/uterus. The addition of this number of purified eosinophils to short term treated uteri resulted in saturation curves and Scatchard plots identical to those seen in long term treated uteri. These data indicate that the type II nuclear binding site is transported into the uterus with eosinophils following estrogen treatment.

Uterine responses to estradiol in the neonatal rat.

Although single doses of estrogens are known to be ineffective in stimulating complete uterine responses in newborn rats, repeated doses elicit toxic responses that become evident in adulthood. In this study, neonates injected daily from birth with 10 micrograms 17 beta-estradiol (E2) demonstrated significant uterine wet weight gain by day 3 and near-maximum growth (230% of control) by day 5. Elevated uterine weight can be maintained by repeated daily injections at least through day 13. Other uterine growth responses after 5 days of E2 (as percent of control) are: dry weight, 163%; DNA content, 193%; protein content, 211%; and nuclear estrogen receptor, 890%. Ornithine decarboxylase activity increased to 520% of control when measured 6 h after a single E2 injection on day 5. Histological examination of uteri from animals treated for 5 days reveals an altered stroma with evidence of circular muscle differentiation, while the lumenal epithelium, which is cuboidal in controls, becomes columnar after E2 injections. These data suggest that estrogens act in fundamentally the same manner in the neonatal uterus as in the adult uterus, although the appearance of the complete response appears to e slower in the neonate. The estrogen-induced precocious development we describe suggests that an estrogen may be involved in the normal postnatal development of uterine estrogen responsiveness. Adult toxicity, resulting from repeated neonatal estrogen dosing, may partly be a consequence of continual hormone action inducing developmentally inappropriate responses.