New Concepts on the Action of Oestrogens in the Uterus and the Role of the Eosinophil Receptor System

Two receptor systems for oestrogens have been demonstrated in the uterus: the cytosol-nuclear receptor system and the eosinophil receptor system. It has been proposed that the cytosol-nuclear receptor system mediates the genomic response to oestrogens in the uterus, while the eosinophil receptor system is thought to mediate the uterine edema and other early oestrogenic responses in the uterus. Cortisol is known to decrease drastically the number of eosinophils in the blood and therefore to limit their availability for migration to the uterus. The present results show that cortisol also drastically reduces both the oestrogen-induced uterine eosinophilia and the uterine wet weight responses, but does not interfere with the oestrogen-induced uterine RNA and protein increases. Oestradiol-17 beta has a higher affinity than oestriol for the cytosol-nuclear receptors and is now found to be the more potent oestrogen in inducing the genomic activation in the uterus. Estriol has a higher affinity than oestradiol-17 beta for the eosinophil receptors, and therefore, oestriol is the stronger oestrogen in inducing those oestrogenic effects which are mediated by the eosinophil receptor system. We conclude that the eosinophil receptor system for oestrogens is a new system, independent of Jensen's cytosol-nuclear receptor system, and this eosinophil receptor system is involved in the mechanism of oestrogen action in the uterus.     

Pregnancy effects on distribution of progesterone receptors, oestrogen receptor alpha, glucocorticoid receptors, Ki-67 antigen and apoptosis in the bovine interplacentomal uterine wall and foetal membranes

Until recently, studies dealing with the uterus of the pregnant cow focus primarily on the placentome or on early and late pregnancy. Thus, there is a paucity of information about many aspects of the interplacentomal uterine wall including adherent foetal membranes. Corresponding tissue specimens were collected at the slaughterhouse and in animals undergoing premature caesarean section. Two specimens per month of pregnancy were assessed immunohistochemically for progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, Ki-67 protein and TUNEL procedure was performed. The latter two methods were employed in three animals each per months 1 and 2, 3 and 4, 7 and 8 and in six animals undergoing caesarean section at days 274 and 275 post insemination or during spontaneous labour. Results indicate that proliferation and apoptosis are of minor importance for tissue homeostasis since both can histochemically be detected only sporadically. Thus, at the sites investigated here, cellular hypertrophy plays an important role for tissue growth during pregnancy. Progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, however, exhibit cell type and pregnancy stage specific distribution patterns within the tissues assessed. Progesterone receptor immunoreactive scores remained fairly unchanged during pregnancy. Oestrogen receptor alpha scores, however, generally decreased and glucocorticoid receptors increased with ongoing gestation. Progesterone receptors and oestrogen receptor alpha were present in endometrial stroma and in myometrial smooth muscle cells during whole pregnancy. Oestrogen receptor alpha was detectable during whole pregnancy also in uterine glands. Progesterone receptors were, however, present at a very low level at the latter site only during months 1-3 and 6-9. Oestrogen receptor alpha and glucocorticoid receptors may also mediate uterine blood flow since they were present in the tunica media of uterine blood vessels. Results of the present study indicate, that progesterone and its receptor play an important role during whole gestation, mainly for uterine quiescence. Glucocorticoids and their receptors - possibly in cooperation with oestrogens and decreasing amounts of the oestrogen receptor alpha - should trigger processes initiating parturition, such as endometrial prostaglandin production. Further studies - including the periparturient period - should help to understand the exact role of the extraplacental compartment of the uterine wall for the initiation and progress of parturition.     

Inhibition of non-genomic responses to oestrogen in the rat uterus by testosterone propionate

Testosterone propionate (50 mg/kg), administered together with oestradiol, inhibited the oestrogen-induced uterine eosinophilia, deep endometrial oedema and the increase in uterine wet weight, 6 h after treatment. The same dose of the androgen decreased the number of eosinophils in the blood and increased their degranulation, explaining the effect of testosterone in the uterus. The high doses of the androgen used were in the range of the doses reported by others to block selectively the oestrogen-induced increase in uterine peroxidase content but not other responses to oestrogen or the cytosolic oestrogen receptor translocation to the nucleus. The dissociation by high doses of testosterone of the oestrogen-induced uterine eosinophilia, wet weight increase and oedema from other responses to oestrogen in the absence of any measurable effect of testosterone upon cytosolic-nuclear oestrogen receptors supports the idea that uterine eosinophilia and oedema are oestrogenic responses regulated by mechanisms different from those of the genomic responses, and is in agreement with the hypothesis of the mediation of uterine oedema by eosinophils.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia.

The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.     

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 μM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 μM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 μM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.     

Counter current transfer of PGF2 alpha in the mesometrial vessels as a mechanism for prevention of luteal regression in early pregnancy

A new concept has been presented on the mechanism protecting the corpus luteum during oestrous cycle, early pregnancy and pseudopregnancy induced by oestrogens. The concept is based on the recently discovered mechanism of back transfer of prostaglandin F2 alpha from the broad ligament vasculature into the uterus and on the participation of oestrogen in this process. The morphological facilitates for counter-current transfer of PGF2 alpha in the area of mesometrial vasculature and ability the uterus to bind PGF2 alpha were presented. It has been concluded that the process of PGF2 alpha back-transfer from mesometrial vasculature into the uterus may reduce in uterine venous blood the amplitude of PGF2 alpha pulses and by this way may reduce the penetration of prostaglandin into subovarian area and from there to the corpus luteum.     

Intracellular relationships of the oestrogen receptor in the rat uterus and hypothalamus during the oestrous cycle.

Simultaneous measurements were made of the specific oestrogen receptor in the nuclear and cytosol fractions prepared from the uterus and hypothalamus of 50--81-day-old female rats undergoing a 4-day oestrous cycle. In the uterus, the content of nuclear receptor fluctuated in concert with known cyclic changes in the secretion of oestrogen, being maximal at pro-oestrus. Over the period of 50--81 days, the nuclear content at all phases increased with age, again corresponding to known age-related increases in ovarian secretion of oestrogen. This age-related increase in nuclear content, averaged from the values of the different phases in each age group, was related to equivalent increases in uterine wet weight, an increase of 1 pmol of receptor being accompanied by an increase of 80--90 mg. The concentration of cytosol receptor was maintained constant, with respect to wet weight, throughout the cycle and with age, irrespective of changes in nuclear content. In the uterus of normal mature females, translocation of receptor into the nucleus did not lead to depletion of cytosol receptor, suggesting a process of continuous replenishment/synthesis. In the hypothalamus, the nuclear content of oestrogen receptor was also maximal at pro-oestrus. In contrast with the uterus, the content of hypothalamic cytosol receptor was minimal at this phase and reflects depletion of the cytosol receptor, possibly as a result of translocation. The extent of translocation was low compared with that in the uterus and did not alter with age during the age-period studied. This low nuclear binding of the receptor in vivo is discussed in relation to the presence of a cytosol factor, present in limiting amounts, which in vitro mediates the binding of cytosol receptor to oligo(dT)-cellulose. The difference in the physiological response of the uterus and of the hypothalamus to oestrogens may be related to the extent of nuclear binding of receptor.     

Uterine type II estrogen-binding sites are not of eosinophil origin

A recent report by Lyttle et al. (Lyttle, C. R., Medlock, R. L., and Sheehan, D. M. (1984) J. Biol. Chem. 259, 2697-2700) suggested that nuclear type II sites in the rat uterus are of eosinophil origin and may represent [3H]estradiol binding to eosinophil peroxidase. To further evaluate this hypothesis we examined the response of nuclear type II sites to estrogen under conditions where eosinophils are not present. Results of our experiments show that physiological levels of estradiol-17 beta (10 nM for 72 h) will stimulate nuclear type II sites in highly purified cultures (21-25 days; 4 passages) of rat uterine stromal and myometrial cells. The magnitude of the response of type II sites to estradiol in these stromal (4-fold) and myometrial (80-fold) cell cultures was essentially identical to that observed in the uterine cell types following in vivo estrogen treatment. Since these highly purified cultures of uterine cells were prepared from the uterus of a 21-day ovariectomized rat which is devoid of eosinophils, we conclude that estradiol stimulation of nuclear type II sites is a direct intracellular response to estrogen which occurs independent of eosinophil accumulation. Furthermore, we have found that type II sites in the rat uterus are not peroxidase. This was demonstrated by experiments which show type II sites are present in the 39,000 X g supernatant fraction of uterine cytosol, whereas peroxidase activity is quantitatively recovered in the crude mitochondrial (39,000 X g) pellet. Likewise, the small amount of peroxidase activity (approximately 10%) in the total homogenate which contaminates our nuclear pellet preparations was extracted (98-100%) with 0.5 M CaCl2. Type II estrogen-binding sites (95-100%) remained associated with the nuclear pellet fraction after peroxidase extraction. Therefore, stimulation of cytosol and nuclear type II sites by estrogen in the rat uterus is a direct intracellular response to the hormone unrelated to eosinophil accumulation and/or peroxidase activity.     

Seminal oestrogens in the boar: origin and functions in the sow

The boar produces considerable amounts of oestrogens in the Leydig-cells also occurring in semen. Very high oestrogen concentrations are measurable in the fluid of the tubuli, which contribute the main part of seminal oestrogens. Additionally, the accessory sex glands add 22% of the unconjugated oestrogens and 12% of conjugated oestrogens to the ejaculate. Concentrations vary considerably according to season and individuals. So far a maximum of 15.3 micrograms was measured in one ejaculate. Infusion of oestrogens (simulation of the oestrogen content of an ejaculate) at oestrus through a catheter into the uterus lumen leads to an increase of the myometrial contraction-frequency for 3 h. Additionally "inseminations" with oestradiol-17 beta (Oe 2), oestrone (Oe 1), and oestrone-sulfate (Oe 1-S) (naturally occurring in the ejaculate) in 10-micrograms amounts at oestrus may lead to an increase of PGF2 alpha concentrations in the uterine veins within a few minutes. This increase may be found after "insemination" with each of the three steroids but not after saline. A parallel rise of the "inseminated" oestrogen is measurable in the uterine vein plasma and may reach concentrations up to several thousand pg/ml. This rise also leads to significantly increased concentrations in peripheral plasma for about 30 min after the "insemination" of Oe 2 (increase of Oe 2 and Oe 1-S) and Oe 1 (Oe 1 and Oe 1-S). Consequences of seminal oestrogens for sperm transport and the timing of ovulation are discussed.     

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.     

Role of early and late oestrogenic effects on implantation in the mouse

Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17 beta) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized-progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.     

Uterine trauma and limb defects

The temporary clamping of the uterine blood vessels on one side of the uterus during late pregnancy in the rat (days 14-16) results in hemorrhage and tissue necrosis in the extremities of the fetuses from the experimental side and occasionally from the control side. A further series of experiments showed that similar fetal hemorrhage followed the temporary clamping (45 minutes) of the uterine wall or uterine fat, excluding major uterine vessels; handling the uterus for 5 minutes; and stretching of the uterine blood vessels. A low incidence of fetal hemorrhage was also associated with laparotomy alone, but the fetuses were unaffected by extensive handling of the uterus through the abdominal wall or by intraperitoneal anesthesia. Fetal hemorrhage was also induced by a short episode of severe maternal hyperthermia but not by a high dose of ethanol given by gavage. These results suggest that a range of uterine trauma may result in fetal hemorrhage, perhaps through a common mechanism.     

Metabolism of [4-14C]oestradiol by oestrogen-induced uterine peroxidase

1. An enzyme that catalyses the metabolism and binding of [4-(14)C]oestradiol to protein and to other high-molecular-weight substances in the presence of H(2)O(2) was shown to be absent from the uteri of immature rats and to be induced by physiological doses of oestrogen or pregnant-mare-serum gonadotrophin. 2. The pH optimum, stability to heat and other characteristics of the uterine enzyme system as well as its subcellular distribution were determined. 3. The increase in the ability of uterine preparations to convert [4-(14)C]oestradiol into water-soluble products as a result of oestrogen treatment was accompanied by an increase in peroxidase and NADH oxidase activities and was inhibited by actinomycin D and cycloheximide. 4. The results support the proposal that the increase in peroxidase activity after oestrogen treatment might be part of an adaptive response of the uterus permitting it to bind and inactivate oestrogens and thus limit the duration of their effect upon this target tissue.     

MECHANISM OF EOSINOPHILIA

Eosinophil leucocyte production was studied in the bone marrow of normal rats and rats given single injections of Trichinella spiralis larvae which stimulated eosinopoiesis. the development sequence of eosinophils in the bone marrow was based on morphological criteria combined with studies of the extent of eosinophil labelling after injections of tritiated thymidine. the proliferative compartment contained at least three recognizable steps in eosinophil development which were defined.
There was a delay of 23 hr after injection of larvae before the proportion of eosinophils in the bone marrow increased and it had doubled by 49 hr. the mitotic index increased by a factor of 3 after stimulation. Estimates of the cell cycle parameters were made for marrow eosinophils 1-3 days after stimulation, using the technique of analysing labelled mitoses. the results were compared with a similar group of normal rats, and were processed by using a computer program. Marrow eosinophil cell cycle time was 30 hr in normal rats and 9 hr in stimulated rats, and this acceleration was associated with a reduced spread of cell cycle times. the number of eosinophil cell divisions and the transit times for each compartment in normal and stimulated rats were estimated. This showed that the stimulus may have resulted in five or six additional divisions among the youngest eosinophils in the dividing compartment. From these figures an outline of eosinopoiesis in the marrow of normal and stimulated rats is proposed.     

Progesterone therapy results in partial reversibility of uterine abnormalities of the adult anovulatory rat.

The effects of progesterone therapy (5 mg, administered subcutaneously daily for 6 days) on the abnormal uterus of adult anovulatory Wistar rats have been studied. These rats, rendered anovulatory by neonatal treatment with testosterone propionate or clomiphene citrate, displayed severe hyperplasia and metaplasia of the uterine luminal epithelium and a disproportionately high content of nuclear oestrogen receptor, as a result of constant oestrogen stimulation unrelieved by progesterone [White, Moore, Elder & Lim (1981) Biochem. J. 196, 557-565]. Progesterone therapy resulted in the virtual elimination of the hyperplasia and metaplasia and a corresponding decrease in the content of nuclear oestrogen receptor with the proportion of the unoccupied nuclear receptor being increased to values exhibited by normal cyclic females. There was also a decrease in the content of progestin receptors, a putative index of oestrogenic stimulation. Further, in the testosterone-treated group, progesterone therapy resulted in the restoration of oestrogen receptor translocational responses to oestradiol stimulation. Progesterone treatment of these anovulatory rats thus provides a model system for investigating the biochemical mechanisms underlying progestin antagonism and regulation of oestrogen-stimulated cell proliferation.     

2-Fluoro-oestradiol does not cause uterine refractoriness but inhibits oestradiol-induced implantation in rats

An intravenous injection of 2-fluoro-oestradiol simultaneously with an implantation-inducing dose of oestradiol reduced the number of implantation sites in delayed implanting hypophysectomized rats maintained with progesterone. Administration of 2-fluoro-oestradiol 1 h before or after oestradiol had no effect. Furthermore, injection of as much as 500 ng 2-fluoro-oestradiol 48 h before administration of oestradiol failed to have any effect upon implantation, i.e. failure to block implantation was correlated with failure to induce the uterine refractory state. These results suggest that conversion of primary oestrogens to catechol oestrogens could be important for implantation as well as for the induction of the oestrogen refractory state in the uterus.     

Development and characteristics of an oestrogen sulphotransferase in placenta and uterus of the pregnant mouse. Comparison between mouse and rat.

The mouse placenta possesses a soluble oestrogen sulphotransferase activity which increases markedly from at least 12 days of gestation until term. At about 16 days of gestation, a similar activity is found in the uterus. This activity also increases until term and disappears rapidly post partum. The uterine enzyme activity appears to require the presence of the foetal unit for its onset, since unoccupied horns, whether their endometrial stromal cells are differentiated to decidual cells or not, are essentially devoid of it. Uterine cytosols from non-pregnant mice are also inactive in this respect. In late gestation, the uterine sulphotransferase is confined to the decidua basalis, the areas to which the placentas are attached. The sulphotransferase(s) of placenta and uterus has an absolute requirement for 3'-phosphoadenosine 5'-phosphosulphate, and possesses little activity in the absence of exogenous thiol groups. Stimulation is also seen in the presence of Mn2+, Mg2+ or Ca2+. Oestrone and oestradiol, and to a lesser degree oestriol, are substrates for the enzyme(s), whereas testosterone, cortisol and dehydroepiandrosterone are not. Oestrone and oestradiol at higher concentrations (1.0-1.5 microM) completely inhibit the enzyme(s). These enzymes could play a role in altering tissue concentrations of active oestrogens during gestation in the mouse. Oestrogen sulphotransferase activity is low or absent in reproductive tissues of the pregnant rat.     

Proceedings: Action of chronically administered oestradiol on the uterus of the rat

Oestradiol induces increased synthesis of RNA and DNA in the uterus of ovariectomized rats. The effects of continuously administered oestradiol on nucleotide synthesis in the uterus of the rats are reported. Ovariectomized rats were given 2 Mug oestradiol-17 beta, subcutaneously, every 8 hr until autopsy at various times 1 to 7 days after the first injection of oestradiol. (3H) uridine or (3H) thymidine was administered intraluminally 15 min before beath. Uteri were processed for autoradiography. The number of labelled cells and the average number of grains/cell were counted. (3H) uridine labelling reached a peak at 6 to 54 hr and then decreased steadily thereafter. DNA synthesis was maximal at 48 hr in all regions and minimal at 144 hr. These results indicate that oestrogen caused maximum stimulation of RNA synthesis in the rat uterus at 30 and 48 hr respectively but activity was reduced thereafter. The uterine epithelium and stroma were hypertrophied and hyperplastic when RNA and DNA synthesis were minimal. This could be due to refractoriness of the specific target tissues to continued hormonal stimulation.     

Replenishment and nuclear retention of oestradiol-17 beta receptors in rat uteri during postnatal development.

1. Uteri of 6--10-day-old rats do not show a late growth response to oestrogen (increase in rate of DNA synthesis and cell division) exhibited by fully competent (20 days or older) uteri. We posed the question whether the lack of the late growth response is due to an inability to replenish the cytoplasmic pool of oestrogen receptors or to curtailed retention of oestrogen binding in the nucleus. Uterine nuclear and cytoplasmic receptors were measured by a [3H]oestradiol-17 beta exchange assay, at 1, 3, 6, 14 and 24 h after oestrogen injection. 2. The replenishment of cytoplasmic oestrogen receptors showed a similar pattern in the uteri of 6 and 10-day-old (partially responsive) and in 20-day-old (fully responsive) rats. 3. Oestrogen was retained longer in uterine nuclei obtained from 5 and 10-day-old rats than in uterine nuclei of 20 and 25-day-old rats. 4. Oestrogen receptors resistant to 0.4 M KCl extraction (residual receptors) were found in uterine nuclei of 6 and 25-day-old rats after oestrogen injection at all the times tested. The concentration of these residual receptors during the late period (6--24 h after injection) was not significantly different in uterine nuclei of 6-day-old and 25-day-old rats. 5. We conclude that neither lack of oestrogen receptor replenishment nor curtailed retention of oestrogen binding in the nucleus is the factor which limits the complete responsiveness to oestrogen in uteri of rats during postnatal development.