Wendell W. Leavitt

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.

RU486 is not an antiprogestin in the hamster

The biological activity and progestin receptor binding activity of the synthetic steroid RU486 (RU38486; 17-beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17-alpha-(1-propynl++ +)- estra-4,9-diene-3-one) were investigated in the hamster. RU486 demonstrated no antiprogestational activity in the female hamster in that it was ineffective in blocking decidualization or interrupting early pregnancy. Competitive binding assays showed RU486 did not compete from hamster uterine progestin receptor. It is concluded that hamster uterine progestin receptor has unique steroid binding specificity.

Progestin-induced down regulation of nuclear estrogen receptor in uterine decidual cells: Analysis of receptor synthesis and turnover by the density-shift method

The density-shift method was used to study the effect of the synthetic progestin, R5020, (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) on the turnover and synthesis of nuclear estrogen receptor in hamster decidual cells. Newly-synthesized receptor was labeled with dense [2H, 13C, 15N] amino acids and separated from pre-existing receptor by density-gradient centrifugation. Progestin increased receptor turnover within 3 h of treatment and blocked estradiol-induced receptor synthesis at 6 h and 9 h. Thus, progestin down regulates estrogen receptor by increasing receptor turnover and inhibiting estrogen-induced receptor replenishment.

Progesterone-modulation of estrogen action: rapid down regulation of nuclear acceptor sites for the estrogen receptor.

Our previous studies demonstrated that progesterone down regulates the occupied form of nuclear estrogen receptor (Re). Using the density shift method, we discovered that progestins stimulate the turnover of nuclear Re within 3 h of treatment, and Re synthesis is suppressed subsequently. Thus, the primary site of progestin action in down-regulating Re is the stimulation of nuclear Re turnover followed by the inhibition of Re replenishment. A major breakthrough in our understanding of how progestin controls Re turnover was made by studying nuclear acceptor sites for Re that were found to decrease markedly within 2 h of progestin treatment. These and other results indicate that progestin induces a factor called the Re regulatory factor (ReRF) which acts to block nuclear Re acceptor sites, and this in turn decreases nuclear Re retention on chromatin acceptor sites, leading to an enhanced turnover (or processing) of nuclear Re.

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.

Cell Biology of the Endometrium

The uterus is an extremely dynamic organ the normal function of which is orchestrated by a regular procession of cellular and molecular events that occur in response to changing levels of ovarian hormones secreted during the female reproductive cycle. This is well exemplified by the human menstrual cycle as depicted in Figs. 1 and 2. The menstrual cycle is named for the one overt indication of the cyclic nature of female reproductive function, that is, the periodic discharge of blood from the vagina, which results from sloughing of the endometrium. The average menstrual cycle is 28 days in length. The first part of the cycle, the proliferative phase, is the phase when follicles grow. Ovulation occurs about midway through the cycle (day 14), and the remainder of the cycle, the secretory phase, reflects corpus luteum function.

Temporal effects of progesterone domination on estrogen and oxytocin receptors in hamster uterus

The purpose of this study was to determine whether progesterone (P)-induced down regulation of estrogen receptors (Re) and oxytocin receptors (ROT) changes with the time of P exposure. Ovariectomized hamsters were given s.c. Silastic implants of estradiol (E2) and P for 4, 8 and 16 days. Cytosol and nuclear Re were measured at low temperature with the pyridoxal phosphate exchange assay, and ROT was assayed in the membrane fraction by [3H] oxytocin binding. Nuclear Re and ROT were down regulated throughout the 16-day P exposure period, but cytosol Re (and total Re) increased progressively from 4 to 16 days indicating that the down regulation of cytosol Re escapes P control with time. This conclusion was supported by P withdrawal studies in which P implants were removed for 6 or 12 h. P withdrawal resulted in equivalent recovery responses of nuclear Re and ROT after 4, 8 and 16 days of P exposure. Although cytosol Re recovery to P withdrawal occurred at 4 and 8 days, no response was obtained after 16 days of P exposure. Uterine weight increased during steroid treatment, and morphometric analysis of the P-dominated uterus revealed significant increases in the cross sectional area of the endometrium and myometrium with time of P exposure. Cytological examination of the uterus showed prominent secretory changes in the epithelial compartment on day 16 with accumulation of secretion in the uterine lumen. These results demonstrate that P can chronically down regulate nuclear Re and ROT. However, the control of cytosol Re varies with the time of P exposure, and cytosol Re levels become refractory to P domination by 16 days. The present observations indicate that the escape of cytosol Re from P control may be associated with the proliferation of one of more uterine cell populations such as glandular and luminal epithelial cells.

Progesterone Regulation of Protein Synthesis and Steroid Receptor Levels in Decidual Cells

Steroid hormones influence gene transcription in target cells through a process involving hormone binding to specific intracellular receptor proteins. Much has been learned about the macromolecular events regulated by steroid hormones in target cells that synthesize and secrete hormone-specific protein products, and progesterone action can be attributed to the regulation of gene expression and the formation of specific messenger RNA molecules for these export protein^.^ The nature of the interaction between the receptor-hormone complex and the acceptor (effector) sites in the target cell nucleus that control gene expression, however, remains largely unknown. It is generally believed that a relationship must exist between the number of nuclear receptor sites and hormone-dependent gene tran~cription.~ Evidence is needed to verify, however, that such a relationship actually exists, and it is not certain whether hormone action is the result of receptor binding and retention by nuclear acceptor ~ i t e s , ~ ’ ~ receptor “processing” in the n~c leus , ”~ or other events.’ Thus, we need to learn more about receptor regulation and processing in order to learn how hormone action is mediated at the level of gene expression. In this chapter, we will review new approaches to the study of progesterone action in the decidualized rodent uterus. Implantation of the blastocyst in the wall of the sensitized uterus leads to proliferation of the underlying endometrial stromal cells to form the decidua. The decidual reaction can be induced experimentally to form “deciduoma” in the absence of a fertilized ovum by traumatization of the endometrium during the sensitive period. A unique feature of the deciduomal reaction in the rodent uterus is that pro-

Decidual Cell Function: Evidence for A Role in the Regulation of Serum CBG and A 60Kda Protein During Early Pregnancy in the Hamster

Several serum proteins increase in titer during pregnancy. We tested the hypothesis that decidual cells may signal the production of certain serum proteins in the hamster. Measurement of serum CBG by equilibrium binding using either [3H]-progesterone or [3H]-cortisol in conjection with ion exchange chromatography showed that decidualization increased CBG levels. Two-dimensional gel electrophoresis revealed that a 60 kDa++ protein increases markedly in the serum of the hormonally pseudopregnant (PSP) animal soon after artificial induction of decidualization on PSP day 4. The 60 kDa serum protein remains low in the nondecidualized PSP animal, but it increases in the pregnant animal. A photoaffinity labeling procedure was used to covalently bind [3H]-androstadienolone to CBG. Fluorography of 2D gels run under denaturing conditions established that the 60 kDa protein did not bind steroid as did CBG (69 kDa). To determine whether decidual cells could induce the 60 kDa and CBG proteins, different numbers of decidual cells were injected IP into PSP recipients. A single injection of 50 x 10(6) decidual cells induced both serum proteins within 48h, whereas the same number of hamster fetal cells was ineffective. Thus, these results demonstrate that hamster decidual cells induce a 60 kDa protein of unknown function and serum CBG. Since the decidual cell itself does not appear to be the source of either protein, it follows that the decidual cell signals the synthesis and secretion of these proteins elsewhere in the body, most likely in the liver. To our knowledge, this is the first demonstration that the decidual cell regulates serum CBG and other proteins in this manner.

Novel Mechanisms for Regulation of Mammalian Estrogen and Progesterone Receptors

Numerous studies have demonstrated that steroid hormone receptors serve as mediators of hormone-induced gene expression (1–9). In general, an increase in occupied receptor levels and the retention of these receptors by chromatin binding sites (acceptor sites) (10–18) is required for specific induction of gene products. Although estrogen-dependent stimulation of estrogen receptor (Re) (19–21) and progesterone receptor (Rp) (22–27) synthesis appears to follow accepted models for hormone-dependent protein induction, the mechanism for progesterone-dependent decreases is Re and Rp levels is not understood. In this chapter, we will present and discuss recent findings from our laboratory concerning estrogen and progesterone modulation of intracellular Re and Rp levels, and from our results formulate a hypothesis for the mechanism of progesterone-induced Re down-regulation.