Estradiol-17β Receptor Research Articles

Variations in hepatic estradiol-17β receptor concentrations during the annual reproductive cycle of diploid and triploid female catfish, Heteropneustes fossilis (Bloch)

Concentrations of hepatic estradiol-17β (E2) receptors (ER) in cytosolic and nuclear fractions were evaluated in diploid and triploid female catfish Heteropneustes fossilis (Bloch) during four different reproductive periods of a complete reproductive cycle. Basal level of ER concentration was noted in the resting period of both diploids and triploids. Receptor level gradually elevated through the preparatory period and reached a peak in the pre-spawning period in both diploids and triploids. However, ER concentrations were overall reduced in triploid to that of diploid females. In a single point assay, in diploids, ER concentration showed about a 3-fold rise ( p < 0.001) in the cytosolic and a 4-fold rise ( p < 0.001) in the nuclear extracts from resting to the pre-spawning period. In triploids, only a 2-fold rise was observed both in cytosolic ( p < 0.01) and nuclear ( p < 0.05) ER concentration during the same span. Finally, a sudden fall of receptor level was observed in the spawning period in both the ploidy groups with a lower concentration in the triploids. The K d value did not differ between the females of diploids (cytosolic 1.12 ± 0.21 nM and nuclear 6.9 ± 0.9 nM) and triploids (cytosolic—1.13 ± 0.17 nM, nuclear—6.8 ± 2 nM). However, B max of the diploid showed about double the value than triploid females both in the cytosolic (diploid—367.4 ± 33.24 pmol/mg protein, triploid—187.3 ± 13.20 pmol/mg protein, p < 0.001) and nuclear extracts (diploid—946 ± 66 pmol/mg DNA, triploid—558 ± 98 pmol/mg DNA, p < 0.01) of liver. Lower E2 binding capacity and lower amount of E2 receptors of triploid catfish liver with a stunted vitellogenic status could be one of the major causes for reduced gonadal development and sterility in female triploids.

Estradiol-17β receptors in cytosolic fraction of chondrosarcoma

Estradiol-17β receptors were detected in the cytosolic fraction of the majority of bone chondrosarcomas by protamine sulfate sedimentation of the hormone-receptor complex. The absolute number of receptors in tumors was higher in men than in women, but chondrosarcomas with estradiol-17β receptors more often occurred in women. The highest level of receptors was revealed in highly differentiated chondrosarcomas. No relationships between tumor location, size, type of the process, and the number of receptors was detected. We can state with assurance that the absence of estradiol-17β receptors in chondrosarcomas in women is a favorable prognostic sign with regard to metastases or local tumor relapses.

Detection of Estrogen Receptor mRNA in Trout Pineal and Retina: Estradiol-17β Modulates Melatonin Production by Cultured Pineal Photoreceptor Cells

Pineal photoreceptor cells produce the neurohormone melatonin, a major "Zeitgeber" of the organism. This compound is involved in the control of development, growth, sexual maturation, and seasonal reproductive cycles. The present study reports that the photosensitive pineal organ and the retina of the trout express a 3.5-kb mRNA corresponding to the estradiol-17β receptor. The effects of estradiol-17β on melatonin production by cultured pineal photoreceptor cells were also studied. Under a light/dark (LD:12/12) cycle, these cells maintained a rhythmic secretion of melatonin, with higher amounts being released during the dark phase. The amplitude of the rhythm tended to increase with time in culture. Application of estradiol-17β during the dark phase of an LD cycle (i.e., for 12 hr) affected melatonin release in a dose-dependent manner; low concentrations (10 -10 to 10 -8 mol/liter) were inhibitory, whereas high concentrations (over 10 -7 mol/liter) were stimulatory (when compared to the values obtained at 10 -9 mol/liter). When estradiol-17β was given continuously for several 24-hr LD cycles, the inhibitory effect observed during the first dark phase disappeared in subsequent dark periods. In the presence of estradiol-17β, at concentrations ranging from 10 -10 to 10 -5 mol/liter, a high amplitude rhythm in melatonin secretion returned more rapidly than in controls. The present results suggest that estradiol-17β receptors are expressed in the fish pineal and retina and that estradiol-17β modulates melatonin secretion by cultured pineal photoreceptors. Sex hormones seem therefore to exert a control on the pineal. Pineal photoreceptors appear as multieffector cells which transduce information from both physical (photoperiod) and internal (chemical) factors.

ESTROGEN RECEPTOR CONTENT OF REGIONS OF THE HYPOTHALAMUS AND PITUITARY OF THE GILT FROM FOUR TO TWENTY-EIGHT WEEKS OF AGE

Estradiol-17β receptors (E2R) in 100 000 × g supernatant fractions from the hypothalamus and pituitary were determined for groups of five gilts, slaughtered at 4-wk intervals from 4 to 28 wk of age. Tissue samples were separated into the anterior hypothalamus (AH), the central hypothalamus (CH), posterior hypothalamus (PH), anterior pituitary (AP) and posterior pituitary (PP). After saturation binding assays were conducted, E2R concentrations were determined by Scatchard analysis. When expressed on a fmol mg−1 protein basis there was no increase in receptor concentration with age (P &gt; 0.05); however, AP had higher concentrations of receptors than the AH, CH or PH (10.6 ± 1.4 vs. 6.5 ± 0.9, 6.7 ± 0.7 and 6.2 ± 0.6, P &lt; 0.01). Posterior pituitary receptor levels remained low during maturation of the gilt with affinity constant (Ka) values similar to that obtained for the AP. Affinity constant values were found not to change with age; however, AP values were greater than that of the hypothalamus samples (55.2 ± 2.7 vs. 4.8 ± 0.2, × 108M−1). On a total tissue basis AH and CH had significant increases in receptor content from 4 to 28 wk (41.4 to 138.2 and 31.6 to 163.4 fmol, P &lt; 0.05). It was also determined that the soluble protein content per gram wet tissue decreased with age in all regions of the hypothalamus and therefore may mask increases in receptor numbers. Keywords: Estradiol, receptor, gilt, hypothalamus, pituitary, swine

Serum LH, progesterone and estradiol-17β levels throughout the ovarian cycle, during the early stage of pregnancy and after the parturition and the abortion in the common marmoset,Callithrix jacchus

In the ovarian cycle of common marmosets, serum progesterone began to increase at two to three days after estradiol-17β or LH surge, attained a peak of 25–70 ng/ml and then declined to a level of under 2 ng/ml before the ensuing rise in estradiol-17β and LH. Serum estradiol-17β increased to 700–5,500 pg/ml during the luteal phase, synchronizing with progesterone. It is suggested that the corpus luteum secreted estradiol-17β as well as progesterone. The cycle length as determined from the interval between successive LH surges was approximately 28 days. During the luteal phase, the levels of progesterone and estradiol-17β were higher than in Old World monkeys and women, but marmosets were not accompanied by any clinical symptoms due to excessive progesterone and estradiol-17β. This suggests that such unresponsiveness to progesterone and estradiol-17β in marmosets reflects the small amount of estradiol-17β receptor and presumably also the lower function of the post receptor system. Recovery of the post-partum ovarian cycle in two marmosets differed from that observed in Old World monkeys and women. The first LH surge was found on the ninth and tenth day after parturition and the first ovulation led to the next pregnancy. This suggests that the suckling stimulus of newborns in the common marmoset does not cause any delay in recovery of the ovarian cycle. In three cases of abortion, the recovery of the ovarian cycle was almost the same as that in the case of normal parturition: the first LH surge appeared on the 10th, 14th, and 34th day after abortion.

Direct evidence of in vitro phosphorylation-dephosphorylation of the estradiol-17β receptor. role of Ca 2+-Calmodulin in the activation of hormone binding sites

The calf uterus estradiol-17β receptor is a phosphoprotein and its hormone binding activity is regulated by two endogenous enzymes both of which have been purified and characterized in detail: a nuclear phosphatase that inactivates the hormone binding sites of the receptor, and a cytosol kinase that activates these sites. Here we report that the kinase is stimulated by Ca 2+ and calmodulin. Direct evidence is presented that the receptor is phosphorylated by the kinase and dephosphorylated by the phosphatase.

Computer-aided assessment of receptor status in human breast cancer

Computer-aided analysis of simulated cytosolic estradiol-17β receptor binding data using Scatchard and nonlinear Mass Action models showed the latter to be superior. Simulation studies of binding data, incorporating the magnitude and uncertainties associated with inefficiencies of separating free from bound hormone, nonspecific and specific binding, allowed the prediction of uncertainties associated with estimates of receptor content. Realistic values for the minimum detectable receptor concentration that can be distinguished from zero with a given probability were also obtained and may be used as a cutoff value for selection of patients for hormone therapy. This improved reliability in quantification should more clearly define the prognostic value of these receptor assays with respect to adjuvant therapy for primary cancer following surgery or anti-estrogen treatment for recurrent disease, thereby improving the management of breast cancer. Computer simulations and regression analyses were performed on a PDP11 34 using programs written in FORTRAN IV.

Hormone binding of estradiol-17β receptor: Evidence for its regulation by cytoplasmic phosphorylation and nuclear dephosphorylation. Prevention of dephosphorylation by antiestrogens

1. Hormone binding activity of estradiol-17β receptor appears to be regulated in vitro by a phosphorylation-dephosphorylation process: receptor inactivated by a nuclear phosphatase is reactivated by cytoplasmic activity requiring ATP. 2. The enzyme activating hormone binding has been purified and characterized from calf uterus cytosol. It is absolutely dependent on ATP, it is strongly stimulated by MgCl 2 as well as by CaCl 2, it shows high affinity for inactive receptor ( K m ~ 0.3 × 10 −9 mol of estradiol-17β binding sites/I) and it sediments through sucrose density gradient at 6 S. 3. Estrogen receptor translocated into nuclei by estradiol-17β in vivo is inactivated during incubation of nuclei at 25°C whereas receptor translocated by tamoxifen and nafoxidine is stable in the same conditions. The difference in stability, previously observed in intact cells [1], is due to inability of the nuclear phosphatase to inactivate receptor complexed with antiestrogens. 4. On the basis of these and previous results [2–5] it is suggested that phosphorylation of inactive receptor in cytosol and dephosphorylation of active receptor in nuclei regulate hormone binding of receptor in vivo. Non steroidal antiestrogens seem to interfere with this regulation by preventing nuclear dephosphorylation of receptor.

Hydroxylapatite “batch” assay for estrogen receptors: Increased sensitivity over present receptor assays

A “batch” hydroxylapatite procedure for the adsorption of the uterine estradiol 17β-receptor complex is described. Characterization with respect to washing efficiency, binding specificity, competition, adsorption time, sensitivity and stability against increasing KC1 ionic strength were included. Equilibrium parameters obtained by Scatchard analysis were compared to the range of values found in the literature. K ta and receptor site concentration per uterus obtained by this “batch” technique were found to be well within the range described by these reported values. This technique is particularly advantageous due to its wide range of operational sensitivity (capable of detecting specific estradiol-17β binding to a cytosol fraction containing from 5 to 500 μg protein per 225 μl). The assay is run entirely at low temperature (0–2°C). In addition this technique depends on a homogeneous insoluble chemical, hydroxylapatite, which can be obtained in analytical grade quantities of uniform particle size, shows little affinity for free steroid, can be readily packed or resuspended, and appears independent of changes in concentrations of KC1 up to 2500 mM. Additional considerations include the effect of temperature during assay, the importance of empirical correction for non-specific binding, the contributions of binding information on the calculation of equilibrium parameters and statistical evaluation of random error and assay repeatability.

Differential effects of the anti-estrogen MER-25 and of three 5α-reduced androgens on mounting and lordosis behavior in the rat

Four experiments were performed in order to evaluate further the hypothesis that androgen must be aromatized to estrogen for the activation of masculine sexual behavior in the male rat. In Experiment 1 it was found that the anti-estrogen MER-25 failed to disrupt mounting behavior in castrated males which simultaneously received testosterone propionate (TP). However, in Experiment 2 it was found that MER-25 as weil as 3β-androstanediol effectively activated masculine behavior in castrated males treated simultaneously with dihydrotestosterone propionate. Both MER-25 and 3β-androstanediol had previously been shown to display an affinity for cytoplasmic estradiol-17β receptors present in male rat anterior hypothalamus. In Experiments 3 and 4, performed with ovariectomized females, it was found that whereas MER-25 antagonized the stimulatory effect of estradiol benzoate (EB) on lordosis behavior, 3β-androstanediol did not. In addition, 5α-dihydrotestosterone and 3α-androstanediol, two compounds which had previously been shown to have almost no affinity for estradiol-17β receptors in the hypothalamus, both inhibited the stimulatory effect of EB on lordosis. It is concluded that the fact that anti-estrogens suppress lordosis induced in females with either EB or TP, but fail to disrupt TP-induced mounting behavior in male rats does not argue against the aromatization hypothesis for masculine sexual behavior.

389. Pituitary sensitivity to LRF and estradiol-17β receptor population during the rat estrus cycle

This study was designed to confirm preliminary work indicating a direct effect of estradiol on the pituitary by examining the pituitary sensitivity to LRF at a series of critical time periods during the estrous cycle. Rats were bled each morning (11.00) during the 4-day estrous cycle and additionally at 14.00 18.00 and 22.00 on diestrus-2 and noon 14.00 16.00 18.00 and 20.00 on proestrus. After each bleeding LRF (50 ng) was injected and blood collected 10 60 and 120 minutes postinjection. Blood FSH response to LRF was minimal. Serum LH showed both maximal and prolonged response on proestrus; the response on the other days was acute and of short duration and the order of magnitude was estrus greater than diestrus-1 greater than diestrus-2. Pituitary and hypothalamic cytosol receptor populations measured by protamine precipitation and Scatchard plot analysis showed a significant decrease starting on proestrus followed by an increase on estrus. This pattern correlates well with plasma estradiol levels found at this time in accordance with the current concept of receptor depletion by nuclear translocation followed by subsequent cytoplasmic receptor resynthesis. The changes in pituitary receptor population correlating with its sensitivity to LRF indicates further a direct effect of estradiol on the pituitary. This finding has been confirmed by us using an isolated pituitary preparation. (FULL TEXT)