Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism.

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses.

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.

Estrogen action in the mouse uterus: the influence of the neuroendocrine-adrenal axis.

Estrogen receptor levels were measured in uteri from ovariectomized-hypophysectomized mice. Cytosolic estrogen receptor levels had a range of 1.65-3.83 X 10(-13) mol/100 micrograms DNA and varied significantly with time after surgery. Nuclear receptor levels also varied with time and had a range of 0.26-1.19 X 10(-13) mol/100 micrograms DNA. Microsomal receptor levels varied from 0.24-0.72 X 10(-13) mol/100 micrograms DNA. Concurrent temporal changes in uterine or vaginal epithelial histology were not observed. Binding properties of the estrogen receptor from the ovariectomized-hypophysectomized mouse uterus were determined; Scatchard analysis of saturation binding data showed one class of binding sites (Kd = 1.32 nM; n = 276 fmol/mg protein) with values similar to those obtained from ovariectomized-adrenalectomized mouse uterus. The ability of the ovariectomized-hypophysectomized mouse uterus to respond to estrogen stimulation was determined in uterine wet weight and DNA synthesis assays; uteri from ovariectomized-hypophysectomized mice responded to estrogen in the same way as uteri from ovariectomized mice. Uteri from ovariectomized-adrenalectomized mice showed a similar growth response to estrogen stimulation. The temporal pattern of estrogen receptor distribution was determined in uteri from ovariectomized-adrenalectomized mice; there were two peaks of nuclear receptor translocation at 1 and 8 h, but the rate of cytosol receptor replenishment was slower than that previously seen in ovariectomized mouse uteri. Circulating estrogen and progesterone levels were measured in ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice at different times after surgery. There was no significant difference in systemic estradiol levels among the groups. However, there were significant differences in progesterone levels; mean values from ovariectomized, ovariectomized-hypophysectomized, and ovariectomized-adrenalectomized mice were 6.5, 2.1, and 0.029 ng/ml, respectively. The differences in progesterone levels correlate with previously reported differences in uterine estrogen receptor levels. Thus, data from this system suggest that adrenal-derived progesterone may play a greater role in regulating estrogen receptor levels in target tissues than was previously thought.

Estrogen action in the mouse uterus: adrenal modulation of receptor levels.

Adrenal regulation of estradiol receptor levels was demonstrated in the ovariectomized mouse uterus. After ovariectomy, cytosol receptor levels initially followed a cyclic pattern, with a periodicity of approximately 5 days and a range of range of 2.25-4.80 x 10(-13) mol estrogen receptor/100 micrograms DNA. Nuclear receptor levels showed marked fluctuations initially, but stabilized after day 15 at approximately 0.5 x 10(-13) mol/100 micrograms DNA. Microsomal receptor levels also changed with time, but at a lower level than the cytosol and nuclear fluctuations. Concurrent cyclic changes in uterine or vaginal histology were not observed. After simultaneous ovariectomy and adrenalectomy, cytosol estradiol receptor levels remained low (less than or equal to 1.5 x 10(-13) mol/100 micrograms DNA) from 5-24 days, with no fluctuations. Nuclear and microsomal receptor levels changed, but to a smaller extent than those in ovariectomized mice. Concurrent histological changes were not observed. The residual cytosolic estrogen receptor remaining in the ovariectomized-adrenalectomized mouse uterus was characterized. Scatchard analysis of saturation binding data showed one class of binding sites with a Ka of 6.56 nm-1 (n = 350 fmol/mg protein). This cytosol receptor sedimented at 8S on low salt sucrose density gradients. Receptor specificity was assessed by competitive equilibrium binding. The rank order of ligands was 17 beta-estradiol = diethylstilbestrol greater than nafoxidine. Progesterone and 5 alpha-dihydrotestosterone showed no appreciable binding activity. After the injection of estradiol (10 micrograms/kg), 80-90% of the total available estrogen receptor translocated to the nuclear compartment. This translocated receptor could stimulate uterine hypertrophy and hyperplasia, as assessed in 1- and 3-day bioassays, and was able to initiate progesterone receptor synthesis. From these results, we conclude that the adrenal can modulate uterine estrogen receptor levels.