In vitro effects of cytosolic inhibitor and opiates on the binding of [3H]oestradiol to nuclear type II binding sites of rat uterus and hypothalamus

The effect of cytosolic ultrafiltrates prepared from intact rat uteri, brain hemispheres and hypothalami and of some opiate analogues on oestradiol binding to nuclear type II sites in rat uterus and hypothalamus was studied. Opiate binding in nuclear fraction of rat uteri was also evaluated. Both uterine and hypothalamic low affinity nuclear oestradiol binding was inhibited by filtrate from uteri, while only hypothalamic nuclear binding was decreased in presence of hypothalamic filtrate. Filtrate from brain was ineffective on nuclear oestradiol binding of the studied tissues. Concentration dependent inhibition of uterine nuclear oestradiol binding could be demonstrated by some opiate analogues in vitro. Specific low affinity nuclear binding of opiate antagonist naloxone and agonist dihydromorphine was observed in rat uteri which could be inhibited by uterine filtrate and oestradiol but not by hypothalamic filtrate or other steroids. Present findings support the probable intracellular interplay of opiates and oestradiol action and suggest that cytosolic inhibitor factor might be involved.     

Characterization of uterine sex steroid receptors in the pig and their variation during the oestrous cycle

The present study establishes and validates an in vitro binding and exchange assay for tissue receptors for oestradiol (E) and progesterone (P) in pig uterus. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The relative concentrations of the receptors were measured in dissected samples from endometrium and myometrium obtained at late prooestrus, oestrus, and luteal phases of the oestrous cycle. The Scatchard analysis of the oestradiol and R 5020-receptor complex displayed linearity and indicated a single class of high affinity, low capacity binding sites. Significant variations were seen in the binding of E and P to their cytosolic and nuclear receptors, following the changes in the circulating levels of the hormones in blood plasma during the oestrous cycle. Both tissue components, i.e. endometrium and myometrium followed a similar pattern when related to the stage of the oestrous cycle considered. The ERc increased from prooestrus, reaching a maximum at standing oestrus, thereafter decreasing. The concentration of ERn increased from prooestrus towards the early luteal phase, with a significant reduction by day 8 of the cycle. The amounts of PRc were maximal at standing oestrus, remaining high during the early luteal phase, while the PRn showed a linear increase from oestrus onwards throughout the luteal phase.     

On the mechanism of opioid-oestradiol interactions

Characteristics of opioid binding and possible relationships between oestradiol and opioid binding sites were studied in rat oestrogen sensitive tissues(uterus, preoptic area-anterior hypothalamus, median eminence-basal hypothalamus). Naloxone (Nal) and oestradiol (Oe) bindings were assessed by in vitro saturation analyses. In 800 g supernatants of both uterine and hypothalamic tissues homogenates high affinity (Kd: 2-4 X 10(-9) M) and low capacity [3H]Nal binding sites were found. These binding sites were sedimented from 800 g supernatant by further centrifugation at 10(5) g for 1 h. In competition studies [3H]Nal binding was completely prevented by morphine, while met-enkephalin and leu-enkephalin caused only a partial inhibition. [3H]Nal binding was increased by ovariectomy and decreased by Oe treatment (10 micrograms/100 g b.wt) in both tissues. The cytoplasmic [3H]Oe binding in the studied tissues seems to be affected by the naloxone binding system. After in vitro saturation of naloxone binding sites by naloxone the [3H]Oe binding to low affinity sites (type II) in hypothalamus as well as in uterus has been increased by 8- and 2-fold, respectively. These results indicate the presence of specific [3H]Nal binding in rat uterus with similar properties to those found in the hypothalamus. Furthermore an interaction between opioid and oestradiol receptor systems could be also suggested.     

Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

The binding of [6,7-(3)H]oestradiol-17beta to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.     

Oestradiol and progesterone: soluble receptor levels and metabolism in the uterus of the ovariectomized ewe

Ovariectomized ewes received injections designed to mimic to some extent oestradiol and progesterone secretion during early pregnancy (maintenance progesterone), during oestrus (oestrous oestradiol) and during the luteal phase of the previous cycle (priming progesterone). The animals were killed at times equivalent to 1, 4 or 7 days after oestrus in those animals which had received oestrous oestradiol. The level of soluble oestradiol and progesterone receptors in whole uterus, and [3H]oestradiol and [3H]progesterone metabolism by uterus minces were measured. Oestradiol receptor level was highest on day 1 in those animals receiving oestrous oestradiol with no significant effect at any stage of the inclusion or omission of priming or maintenance progesterone. Progesterone receptor level was also high on day 1 in those animals receiving oestrous oestradiol with high levels maintained to day 4. Again, inclusion of priming or maintenance progesterone was without effect. In animals not receiving oestrous oestradiol the level of both receptors was uniformly low. Metabolism of [3H]oestradiol was low and not affected by treatment. [3H]Progesterone metabolism, although more variable, was also low and not affected by treatment.     

Inefficient transmission of H5N1 influenza viruses in a ferret contact model

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two na?ve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other na?ve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.     

Oestradiol and progesterone receptors in the pig oviduct during the oestrous cycle

The concentrations of the tissue receptors for oestradiol (E) and progesterone (P) in the porcine oviduct at different stages on the oestrous cycle have been investigated by in vitro binding and exchange methods. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The concentrations of ERc and ERn were two-fold higher in the ampulla as compared to the isthmus. The amount of ERc in the isthmic portion of the oviduct did not vary throughout the oestrous cycle. However, the ampullar ERc concentrations increased during prooestrus, showed a maximum at standing oestrus, thereafter decreasing. Significant variations in the amount of oviductal ERn were observed. Despite the differences in ERn amounts between segments, the concentration of ERn increased significantly during late prooestrus, attaining a three-fold elevation and remaining elevated during the period of standing oestrous and early luteal phase (days 3-4), thereafter returning to basal levels. No significant variations in the amount of isthmic PRc were found throughout the period studied. The ampulla, however, showed a significant increase in PRc concentrations during standing oestrus, thereafter decreasing. The concentrations of PRn in isthmus and ampulla were of about the same magnitude and varied significantly during the oestrous cycle, increasing in concentration from standing oestrous onwards. The temporal relationships between the variations in levels of oestradiol and progesterone receptors in oviductal tissues and those of the circulating plasma levels were established. The data obtained in this study suggest a relationship between the changes in the levels of oestradiol and progesterone oviductal binding during the first days of the oestrous cycle, and the gamete and embryo transport throughout the oviduct in the porcine species.     

Oestradiol receptors in non-neoplastic gynaecomastic tissue of phenotypic males.

Cytoplasmic oestradiol receptors have been reported in breast tumor tissue but not in the uninvolved tissue from the same patients. Since gynaecomastic tissue is highly predisposed to neoplastic transformation, such receptors were looked for in this tissue from 13 phenotypic males of whom 6 had Klinefelter's syndrome. High affinity saturable binding (Kd approximately 10(-10) M) of 3H-oestradiol-17 beta was found in the breast tissue from 12 of these patients by gel elution and dextran-coated charcoal techniques. The concentration of binding sites were found to range from 16 to 359 fmol/mg cytosol protein. Previous studies by the present authors showing steroid aromatising capacity and the present finding of specific oestradiol receptors in a 'high-risk tissue' in the absence of any histologically detectable neoplasms might be relevant in elucidating the natural history of breast cancer in such individuals.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

Nuclear oestrogen receptors in rat liver. Development of assay conditions, characterization and the translocation of oestrogens in vivo.

A method for the determination of specific oestrogen-receptor binding sites in rat liver nuclei is described. Nuclear receptors showed a high affinity for oestradiol (Kd approximately 3 x 10(-9)M), a low capacity, and a distinct specificity for substances with known oestrogenic and anti-oestrogenic activity. No sex differences were seen in the concentrations of nuclear receptors from either vehicle- or ethynyloestradiol-pretreated rats. Only a limited number of binding sites could be extracted with 0.4 M-KCl. The remaining sites, which were solubilized by sonication and treatment with deoxyribonuclease I, sedimented at 3-4 S. Of four oestrogens tested (oestradiol, ethynyloestradiol, diethylstilboestrol, tri-p-anisylchloroethylene), ethynyloestradiol was the most effective translocation agent in vivo, nuclear uptake occurring at doses below 1 microgram/rat; changes in salt extractability of nuclear receptors occurred at doses lower than those required to achieve absolute increases in nuclear receptor concentrations.     

Nuclear and cytosol steroid hormone receptor levels in the myometrium during pregnancy in the sheep

Methods for the measurement of nuclear receptors for oestradiol and progesterone in sheep myometrium have been established. Scatchard analysis of nuclear receptors gave dissociation constants (nM) on days 0 and 112 of pregnancy of 1.95 and 1.76 for oestradiol and 4.20 and 4.12 for progesterone, respectively. The concentration of nuclear and cytosol high-affinity receptors for oestradiol and progesterone has been determined during the first 112 days of gestation; and possible roles of oestradiol and progesterone in the regulation of myometrial hypertrophy and function are discussed.The rate of hypertrophy, as measured by changes in protein : DNA and RNA : DNA ratios, was maximal during days 56–84 and declined thereafter. The level of cytosol oestradiol receptor decreased rapidly between day 0 (oestrus) and day 28, and then more slowly between days 28 and 112, when expressed per unit of cytosol protein. However, when expressed per unit of DNA the level increased after day 28 to a peak at day 84, then decreased markedly to day 112. The level of nuclear oestradiol receptor declined from a peak at oestrus to very low levels on days 56–84, then increased markedly to day 112. The concentration of cytosol progesterone receptor declined from a peak at oestrus to low levels on days 28–112. The changes in the level of nuclear progesterone receptor were more complex; the level increased between oestrus and day 28, declined markedly to day 56, then increased again to high levels on days 84–112.The data suggest that oestradiol does not have any important role in stimulating myometrial growth, since the level of nuclear receptor for oestradiol was low when the rate of hypertrophy was maximal. The changes in nuclear progesterone receptor level were less clearly separated, temporally, from changes in rate of hypertrophy, and the possible influence of progesterone on myometrial growth remains unclear.     

Changes in the density of peripheral benzodiazepine binding sites in genital organs of the female rat during the oestrous cycle

The affinity and the density of peripheral-type benzodiazepine binding sites (PBzS) in tissues from the genital organs of female rats were studied during the oestrous cycle. When comparing PBzS density on the day of oestrus to PBzS density on the day of pro-oestrus, a significant increase was observed in the ovary (1.9-fold), oviduct (2.4-fold) and uterus (1.7-fold), but not in the kidney. Serum oestradiol also increased to a maximum on the day of pro-oestrus. The ovarian and uterine PBzS density and serum concentrations of oestradiol and progesterone were measured every 8 h between the days of dioestrus and pro-oestrus. Ovarian and uterine PBzS density increased to a maximal value at 01:00 and 09:00 h, respectively, on the day of pro-oestrus. However, a significant increase in PBzS density occurred in the ovary (P less than 0.02) and uterus (P less than 0.001) at 09:00 h on the day of pro-oestrus as compared to 09:00 h on the day of dioestrus. These changes were associated with an increase in serum oestradiol and progesterone concentrations. The affinity of PBzS in all tissues examined remained unaltered during the oestrous cycle. This study demonstrates that changes associated with the oestrous cycle occur in the density of PBzS in various genital organs.     

A novel binding site for a synthetic progestagen in breast cancer cells

A novel, high-affinity saturable binding site for the synthetic 19-nor testosterone progestagen, 17 alpha-ethinyl-13 beta-ethyl 17 beta-hydroxy-4,15-oestradiene-3-one (gestodene) has been detected using a sensitive affinity chromatography technique. This binding site is present in a range of malignant breast-derived cells lines, regardless of the presence of oestrogen and progesterone receptors, but is absent from endometrial carcinoma cells that contain both oestrogen and progesterone receptors. Competition studies show that this binding is not attributable to the receptors for the progestagens, androgens, glucocorticoids or mineralocorticoids. Cytosolic gestodene binding is refractory to competition with oestradiol but nuclear gestodene binding is completely abolished by oestradiol. The binding of oestradiol to the oestrogen receptor is reduced 40-50% by competition with gestodene. Non-dissociating polyacrylamide gel electrophoresis and size-exclusion high performance liquid chromatography reveal that this binding activity is associated with a protein of mean molecular mass 47 +/- 9 kDa. Ligand binding studies with a range of other cell lines indicates that this binding site appears to be specific to breast cancer cells. These data show the presence of a partly oestrogen competable novel binding protein in breast cancer cells which does not appear to be due to any of the conventional steroid receptors.     

Rapid and sensitive detection of oestrogen receptors in cells and tissue sections by autoradiography with 125I-oestradiol

Summary The presence of receptors for steroid hormones in individual cells and tissue sections was assessed within 4–24 h using dry mount autoradiography with radio-iodinated oestradiol. Low affinity and nonspecific binding of steroids were significantly reduced by washing the cells or sections with diluted antiserum to oestradiol.For cells of the MCF-7 cell line variations in grain density were observed, indicating that cells of the MCF-7 cell line are heterogenous with respect to their cellular receptor concentrations of oestrogen receptors. Receptor-negative cells, such as peritoneal macrophages, did not retain oestradiol label.In tissue sections of rat and calf uterus, predominant labelling was observed on the endometrial gland cells and stroma.Oestradiol receptor binding in the uterus cytosol for both radio-iodinated and tritiated oestradiol showed the same qualitative characteristics as determined by sucrose gradient sedimentation profiles and a comparable amount of binding sites was found for both labels. The relative binding affinity of¹²⁵I-oestradiol compared to [³H]oestradiol is about 70–80%.The dry mount autoradiographic technique as presented can be used for rapid screening of heterogeneiety in oestrogen receptor distribution in cells and tissue sections, since this technique reveals differences in receptor concentrations on the single cell level.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

Pharmacological characterization of two specific binding sites for neurohypophyseal hormones in hippocampal synaptic plasma membranes of the rat

Synaptic plasma membranes containing binding sites for tritiated oxytocin and arginine vasopressin were isolated from rat hippocampus. The binding parameters for oxytocin and vasopressin sites were determined and statistically analysed. The fitted curve for oxytocin binding was compatible with a model where the ligand interacts with two classes of receptors with different capacities and affinities. The sites with low binding capacity had an apparent dissociation constant at equilibrium of 1.8 nM and a maximal binding capacity of 17 fmol/mg protein. By contrast, the Scatchard plot failed to reveal a marked heterogeneity in the population of sites labelled with [3H]vasopressin with an affinity of 1.5 nM and a maximal binding capacity of 39 fmol/mg protein. The specificity of these binding sites, tested in competition experiments, revealed that these neurohypophyseal hormones labelled two distinct populations of sites. One population with a high affinity for vasopressin, oxytocin and vasotocin, the other population with a high affinity for vasopressin and vasotocin and a low affinity for oxytocin. Adenylate cyclase activity was not affected by arginine-vasopressin or oxytocin. These receptors are compared with previously characterized peripheral receptors.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

The role of sub-optimal preovulatory oestradiol secretion in the aetiology of premature luteolysis during the short oestrous cycle in the cow

Premature regression of the corpus luteum, following the first post partum ovulation, is often preceded by sub-optimal preovulatory oestradiol secretion and accompanied by elevated levels of oxytocin receptors early in the luteal phase. We have investigated the role of preovulatory oestradiol in the control of subsequent oxytocin receptor concentration and activity by treating ovariectomised cows, over a simulated 48 h follicular phase, with high (600 microg per day) medium (300 microg per day) or low (150 microg per day) levels of oestradiol. These doses of oestradiol generated mean+/-S.E.M. plasma oestradiol concentrations of 12.1+/-1.0, 4.9+/-0.5 and 2.9+/-0.4 pg ml(-1), respectively. In Study 1 (n=4 per group), we found that by day 4 following oestrus there was a significant (P< 0.05) effect of the level of oestradiol on the inhibition of oxytocin binding activity measured in endometrial biopsy samples. This had fallen to mean+/-S.E.M. concentrations of 25+/-2 fmol per mg protein in the high group, 47+/-8 fmol per mg protein in the medium group and 65+/-12 fmol per mg protein in the low group. In Study 2, cows (n=3 per group) were treated with the same three levels of oestradiol followed by treatment with increasing levels of progesterone from days 3 to 6 following oestrus, generating mean+/-S.E.M. plasma concentrations of 2.17+/-0.18 ng ml(-1) by day 6. On day 6, there was a significant (P< 0.01) effect of the level of oestradiol on PGF(2alpha) release in response to oxytocin challenge. High, medium and low oestradiol groups exhibiting mean+/-S.E.M., increase plasma PGF(2alpha) metabolite concentrations of 10.0+/-2.2, 21.3+/-4.3 and 41.3+/-1.2 pg ml(-1), respectively, during the hour after oxytocin administration. From these results, we postulate that at the first post partum ovulation a low level of preovulatory oestradiol can result in the early generation of a luteolytic mechanism during the subsequent luteal phase due to impaired inhibition of oxytocin receptors allowing increased PGF(2alpha) release.     

Characterization of estrogen receptors in the hamster brain

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.