Thomas G. Muldoon

Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretion

Estradiol secreted by the maturing follicle is the primary trigger for the surge of gonadotropins leading to ovulation. Progesterone has stimulatory or inhibitory actions on this estrogen-induced gonadotropin surge depending upon the time and dose of administration. The administration of progesterone to immature ovariectomized rats primed with a low dose of estradiol induced a well-defined LH surge and prolonged FSH release, a pattern similar to the proestrus surge of gonadotropins. A physiological role of progesterone is indicated in the normal ovulatory process because a single injection of the progesterone antagonist RU 486 on the day of proestrus in the adult cycling rat and on the day of the gonadotropin surge in the pregnant mares serum gonadotropin stimulated immature rat resulted in an attenuated gonadotropin surge and reduced the number of ova per ovulating rat. Progesterone administration brought about a rapid LHRH release and an decrease in nuclear accumulation of estrogen receptors in the anterior pituitary but not the hypothalamus. The progesterone effect was demonstrated in vitro in the uterus and anterior pituitary and appears to be confined to occupied estradiol nuclear receptors. In in vivo experiments the progesterone effect on estradiol nuclear receptors appeared to be of approximately 2-h duration, which coincided with the time period of progesterone nuclear receptor accumulation after a single injection of progesterone. During the period of progesterone effects on nuclear estrogen receptors, the ability of estrogens to induce progesterone receptors was impaired. Based on the above results, a model is proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion.

Use of the direct linear plot to estimate binding constants for protein-ligand interactions.

Abstract The direct linear plot (Eisenthal and Cornish-Bowden[1974] Biochem. J. 139 , 715–720) for the determination of enzyme kinetic constants has been assessed as a means of describing specific steroid-protein interactions. In the rat uterine cytoplasmic estrogen receptor system, determination of the equilibrium dissociation constant (KD) and of the total number of ligand-binding sites (Bmax) has been made, and the results are in good agreement with those obtained by Scatchard and Lineweaver-Burk plot analyses. The usefulness of the direct linear plot lies in the speed and simplicity with which it can be constructed and interpreted.

Structure — activity relationships of some unique estrogens related to estradiol are predicted by fit into dna

The estrogenic activity of 11 beta-acetoxy estradiol, 11 beta-hydroxy estradiol, 11 alpha-hydroxy estradiol and 9 beta-estradiol was compared to estradiol using the restoration of uterine weight and prevention of LH rise in immature ovariectomized rats as endpoints of the assay. There was a good correlation between results using the two methods and estrogenic activity was found to be in the following order: 11 beta-acetoxy estradiol greater than estradiol greater than 9 beta-estradiol greater than 11 beta-hydroxy estradiol greater than 11 alpha-hydroxy estradiol. The biological activities of these compounds could be explained on the basis of stereochemical complementarity to the structure of DNA.

The role of steroid hormones in the regulation of gonadotropin secretion.

SUMMARY An acute rise in serum gonadotropins occurs in the Immature rat on castration indicating the role of gonadal steroids in gonadotropin regulation. In the castrated male rat testosterone brings about restoration of gonadotropins to intact levels at doses that are “physiological” as judged by seminal vesicle and ventral prostate weights. The suppression of LH takes place at lower doses than FSH. Estradiol prevents the post-ca

Microsomal receptor for steroid hormones: functional implications for nuclear activity.

tration rise of gonadotropins in the male rat at a dose level of @3% of the testosterone dose. Using a variety of techniques, it has been demonstrated that receptors for estradiol are found in the hypothalamus and pituitary of both male and female rats in similar concentrations. No such high affinity receptors are found for androgens in either sex although androgens appear to interfere with estrogen binding. The regulation of gonadotropin secretion in the male thus may be mediated by conversion of androgens to estrogens and the interference. manifested by them in estrogen binding. The conversion to estrogens appears to be of importance in the control of FSH because androgens not convertible to estrogens such as Sa-dihydrotestosterone and 5a-androstane-3cc. 17b-dial are even less effective than testosterone in suppressing FSH. However, for the suppression of LH, such a conversion does not appear necessary. Conclusive evidence for a positive feedback effect of estradiol on the pituitary leading to greater sensitivity to LHRH in the release of LH has been provided by studies in the cyclic rat and a pituitary stalk-sectioned rat model. Progesterone in the absence of estrogens does not have any effect on gonadotropins. In the presence of estrogens the effect may be stimulatory or inhibitory depending on dose. Although the relationship between the gonads and pituitary secretion of gonadotropins has been studied by several investigators, the precise interactions involved and the role played by individual gonadal steroids in the regulation of gonadotropin secretion are still poorly understood. This paper further explores the intricate relationship between gonadal steroids and gonadotropin secretion in the immature and mature rat. The immature rat turned out to be an excellent animal preparation for some of the initial studies because of the sensitive pituitary-gonadal axis in both the male and female rat. Furthermore, the absence of daily cyclic changes in the female was an additional advantage. A possible drawback to the use of the immature animals is the change of sensitivity of the hypothalamic-pituitary axis to the feedback effect of steroids at puberty suggested by Byrnes and Meyer in 1951 Cl], and confirmed by McCann and Ramirez in 1964[2], using bioassay techniques for LH. This concept was challenged by Swerdloff et al. in 1972 [3]. Recent work in our laboratory [S] has confirmed a dramatic change in the amount of estradiol required for preventing the post-castration

Comparison of the accuracy of the Scatchard, lineweaver-burk and direct linear plots for the analysis of steroid-protein interactions

Target tissues for steroid hormones are responsive by virtue of and to the extent of their content of functional intracellular receptors. Recent years have seen a shift in considerations of the cellular dynamics and distribution of these receptors, with current views favoring predominant intranuclear localization in the intact cell. This paper summarizes our analyses of the microsomal estrogen and androgen binding capability of rat uterine and ventral prostate tissue, respectively; these studies have revealed a set of high affinity sites that may act as a conduit for estrogen traversing the cell en route to the nucleus. These sites have many properties in common with cytosolic receptors, with the salient difference of a failure to activate to a more avid DNA-binding form under conditions which permit such activation of cytosolic receptors. The microsomal estrogen-binding proteins also have appreciable affinity for progesterone, another distinction from other known cellular estrogen receptor species. Various experimental approaches were employed to demonstrate that the microsomal receptors were not simply cytosol contaminants; the most convincing evidence is the recent successful separation of the cytosolic and microsomal forms by differential ammonium sulfate precipitation. Discrete subfractionation of subcellular components on successive sucrose gradients, with simultaneous assessments of binding capability and marker enzyme concentrations, indicates that the major portion of the binding is localized within the vesicles of the endoplasmic reticulum free of significant plasma membrane contamination. The microsomal receptors are readily solubilized by extraction with high- or low-salt-containing buffers or with steroid. The residual microsomes following such extraction have the characteristics of saturable acceptor sites for cytosolic estrogen-receptor complexes. The extent to which these sites will accept the cytosolic complexes is equal to the concentration of microsomal binding sites extracted. These observations suggest three possible roles for the microsomal receptor-like proteins: (a) modulation of estrogen access to nuclear binding sites; (b) formation of functional complexes which diffuse to other extranuclear sites to alter non-genomic cellular processes; (c) regulation of nuclear concentration of estrogen-receptor complexes by virtue of producing microsomal acceptor sites for uptake of free or loosely associated nuclear complexes, previously thought to exist in the cytoplasm.

Androgen receptor dynamics in the rat ventral prostate

Abstract Determination of equilibrium dissociation constants and concentration of specific high-affinity binding sites for the interaction between a steroid and its protein receptor has been examined as a function of the graphical method of analysis. Using data from 17β-estradiol association with rat uterine cytosol as a basis for the experimental range of standard deviations encountered, a series of pseudorandom data sets were generated by computer. These data were used to examine the efficacy with which graphical presentations by the method of Scatchard, Lineweaver and Burk, or the direct linear plot would allow accurate determinations of the binding parameters. Of the three methods, the direct linear plot was consistently superior.

Factors controlling estrogen receptor levels in normal mouse mammary tissue.

Upon testosterone administration, a dose-dependent cytosolic depletion and nuclear accumulation of androgen receptors in the ventral prostate of 1-day-castrated male rats is observed. Replenishment in the cytosol is rapid with a return to control levels 3 h after testosterone stimulation. The process of nuclear retention (as measured 4-6 h post-injection) is both dose-dependent and time-dependent (there is no retention of the androgen receptor 15 h after testosterone). When assayed 1 h after testosterone, the increase in nuclear binding sites was not sufficient to conclude that the disappearance of cytosolic binding sites could be accounted for by translocation of cytosolic receptors to the nucleus. The cytosolic compartment contained more than 70% of the total cellular receptors whether testosterone was present (in the range 50-400 micrograms/100 g body wt.) or not. Nuclear processing of androgen receptors is extensive and it is dose-dependent. Turnover of prostatic androgen receptors was studied simultaneously in the cytosolic, microsomal and nuclear compartments 1, 2, 3, 4, 5 and 6 h after testosterone administration. Cytosolic and microsomal depletion-replenishment patterns are similar, displaying a nadir after 1 h and a full replenishment 3-4 h post-testosterone. Cycloheximide, but not actinomycin D, inhibits cytosolic and microsomal replenishment. Nuclear accumulation and retention of androgen receptors is insensitive to both drugs. The very rapid and RNA synthesis-independent turnover of androgen receptors in the ventral prostate suggests that testosterone regulates the receptor levels acutely by both a rapid post-transcriptional positive action and a similarly rapid negative effect on nuclear receptor half-life.

A direct effect of LHRH on anterior pituitary estrogen receptors in the female rat

Several aspects of the control of estrogen receptor populations in normal mouse mammary tissue were investigated. In C3H female mice mammary gland tissues were excised and homogenized. All subsequent manipulations were done at 0-4 degrees C. Further details of methods are given. Rising levels of mammary tissue receptor were observed at an age when estrogen levels were also rising. At an earlier age 17beta-estradiol did not cause an increase in receptor content. The level remained high and was not depressed by ovariectomy. In the adult mouse mammary tissue responded to estrogen stimulation with depletion and replenishment of cytosol receptors. Receptor levels increased during pregnancy rose still more in late pregnancy and decreased after parturition. Glucose sedimentation properties of estrogen receptor changed from 4-5S before pregnancy to an 8S form during pregnancy. In lactating animals the 8S form persisted but the 4-5S form became predominant in nonlactating mice. Cycloheximide was equally effective in inhibiting early receptor replenishment when given 3 hours after estrogen or when given at the same time or earlier. This drug did not alter 5-h receptor levels. While the roles of estrogen progesteroneand prolactin are not clear the involvement of estrogen receptor in tumorigenesis may be a control point. Some features of hormonal regulation of mammary receptors shown may find application in the loss of estrogen responsiveness during neoplastic therapy.

Inhibitory Effect of Progesterone on Occupied Estrogen Receptors of Anterior Pituitary and Uterus in Adult Rats

Abstract An analysis has been made of the effects of LHRH on anterior pituitary estrogen receptors. Dispersed pituitary cells from ovariectomized, estrogen-primed rats were incubated in the presence or absence of 1–10 ng of LHRH per pituitary equivalent for 30 min at 37°C. Following lysis, nuclear estrogen receptor concentration was observed to be significantly elevated in a dose-related fashion, while extranuclear binding was depressed by a constant factor. At 5 or 10 ng LHRH levels, the increased nuclear binding could not be explained by translocation of cytoplasmic receptor, and total cellular receptor activity was significantly enhanced. Isolated cytosol and nuclei were not subject to significant fluctuations in the presence of 1–10 ng of LHRH, although 100 or 1000 ng of LHRH did significantly stimulate nuclear binding. In separate experiments, 15 min incubations with 1 or 10 ng of LHRH were not sufficient to cause altered receptor binding, under conditions where 30 min incubation effected the above results. At 120 min of incubation, binding in the extranuclear compartment was increased dramatically over the 30-min interval, and this was entirely at the expense of the nuclear binding capacity. In vivo administration of LHRH caused an increase in nuclear anterior pituitary receptor activity at 1 h, without concomitant alteration in cytosol content. At 10 h, the intracellular distribution of receptors had shifted in a manner consistent with nuclear→cytoplasmic recycling of receptor, and the distribution mirrored that of the untreated control animals. The results indicate that LHRH has a direct stimulatory effect on nuclear estrogen receptor activity which cannot be explained by stimulation of cytoplasmic receptor depletion. Such an action could account for the self-priming effect of LHRH on its own activity, since estrogens are known to modulate (presumably through a receptor-mediated mechanism of action) the responsiveness of pituitary cells to LHRH.