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Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain.

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.

Animals↗

Indomethacin inhibits lordosis induced by ring A-reduced progestins: possible role of 3alpha-oxoreduction in progestin-facilitated lordosis.

Progestins with a delta-4-3-keto configuration bind to the progestin receptor (PR) and facilitate estrous behavior in estrogen-primed rats. Some ring A-reduced progestins [5alpha-dihydroprogesterone (alphaDHP), allopregnanolone, and epipregnanolone] are more potent estrus-inducing agents than progesterone when iv injected despite their lower affinity for the PR. Yet the estrus-inducing action of such progestins is reduced by the antiprogestin RU486, suggesting that binding to the PR is required for this effect. Because allo- and epi-pregnanolone are oxidized to alpha- and betaDHP, respectively, by 3alpha-hydroxysteroid oxo-reductase (3alphaHSOR), part of their estrus-inducing action may occur through the binding of such DHPs to the PR. Conversely, because 3alphaHSOR reduces alpha- and betaDHP to allo- or epi-pregnanolone, both of which exert membrane effects, the estrus-inducing effect of DHPs may involve actions independent of the PR. To test these possibilities we assessed the effect of indomethacin, a blocker of 3alphaHSOR, on the estrus-inducing action of such progestins. Because indomethacin also inhibits cyclooxygenases, we selected a dose and treatment schedule that does not interfere with prostaglandin-mediated brain processes (e.g., LHRH release). Indomethacin did not significantly modify the effect of progesterone or megestrol acetate on lordosis. Yet, it significantly reduced the action of all ring A-reduced progestins. Results suggest that: (a) oxidation is essential for lordosis facilitation by 3alpha-pregnanolones and (b) reduction of 3-keto progestins generates 3alpha-hydroxy metabolites which synergize with processes triggered by occupation of the PR by 3-keto progestins. The possible participation in this response of other events influenced by indomethacin (e.g., prostaglandin or melatonin synthesis) is discussed.

3-Hydroxysteroid Dehydrogenases↗

A prospective, randomized trial of gonadotropin-releasing hormone agonist plus estrogen-progestin or progestin "add-back" regimens for women with leiomyomata uteri.

Treatment of women with myomas with GnRH agonists (GnRH-a) for 3-6 months will result in profound hypoestrogenism, a significant but temporary reduction in uterine volume, and menstrual suppression. Long-term (i.e. > 6 months) treatment with a GnRH-a is not recommended because of accelerated bone resorption and the presence of hypoestrogenic symptoms. In this 2-yr study, women with myomas were treated with GnRH-a plus one of two steroid "add-back" regimens to minimize adverse sequelae of chronic hypoestrogenism. Fifty-one premenopausal women with large, symptomatic uterine myomas all received the GnRH-a, leuprolide acetate depot (LAD), every 4 weeks for 12 weeks at which time the women were randomized to receive LAD plus either an estrogen-progestin or progestin-only add-back regimen for an additional 92 weeks. Efficacy parameters assessed included serial uterine volumes, hemoglobin concentrations, and hematocrits; safety parameters evaluated included serial bone mineral density measurements, lipid profiles, and medication-related symptoms. This report analyzes the first 52 weeks of study data. Mean uterine volume decreased to 64% of pretreatment size at 12 weeks of LAD treatment in both groups. The estrogen-progestin add-back group had no significant regrowth of uterine volume, which was 75% of pretreatment size at treatment week 52; in contrast, the progestin add-back group had a mean uterine volume of 92% of pretreatment size by treatment week 52. Both groups demonstrated significant improvements in mean hemoglobin concentrations and hematocrits. The progestin add-back group had a significant decline in mean high density lipoprotein-cholesterol concentration, which was not seen in the estrogen-progestin add-back group. Finally, after a significant 3% bone loss during the first 12 weeks of treatment, bone mineral density stabilized in both add-back regimen groups. GnRH-a/steroid add-back regimens provide a useful long-term treatment strategy in women with large, symptomatic uterine myomas and may obviate the need for surgical intervention in selected cases. The estrogen-progestin add-back regimen was superior or equal to the progestin add-back regimen in all efficacy and safety parameters assessed.

Adult↗

The FK506-binding immunophilin FKBP51 is transcriptionally regulated by progestin and attenuates progestin responsiveness.

FKBP51 and FKBP52 are large molecular weight FK506-binding immunophilins that have diverse biochemical functions. Best studied is the role that they play as components of steroid hormone receptors. Differential display and gene array screens have identified FKBP51 as a progestin-inducible gene. Here we demonstrate progestin enhancement of FKBP51 mRNA and protein in T-47D cells. FKBP51 mRNA and protein levels were increased 3-fold by 20 nM R5020. Induction of FKBP51 mRNA was unaffected by 1 micro g/ml cycloheximide but was blocked by the progestin receptor (PR) antagonist RU486 (1 micro M). Reporter plasmids containing 3.4 kb and 427 bp of 5'-flanking sequences of the human FKBP51 protein gene (FKBP5) exhibited regulation by progestin in T-47D cells. A construct containing 19 bp of upstream sequence demonstrated diminished basal activity and no stimulation by R5020. To test whether elevated FKBP51 affects progestin responsiveness, HepG2 cells were transfected with human FKBP51, PR, and mouse mammary tumor virus-luciferase plasmids, and treated with R5020 (0.03-10 nM). Expression of FKBP51 increased the EC(50) for PR transactivation by 3.2-fold. Expression of FKBP51 from squirrel monkey, a New World primate with naturally occurring progestin resistance, increased the EC(50) more dramatically (11.7-fold vs. control). Expression of FKBP51 bearing a double-point mutation in the tetratricopeptide repeat domain had no effect on PR transactivation. These results suggest that increased expression of FKBP51 by progestin may attenuate progestin responsiveness in hormone-conditioned cells. Furthermore, overexpression of FKBP51 in the squirrel monkey may be a contributing cause of progesterone resistance in this species.

Animals↗

Multiple rapid progestin actions and progestin membrane receptor subtypes in fish.

Progestin hormones exert rapid, nongenomic actions on a variety of target tissues in fish. The induction of oocyte maturation and the progestin membrane receptor (mPR) that mediates this action of progestins have been well characterized in fishes. Progestins also act on Atlantic croaker spermatozoa via an mPR to rapidly increase sperm motility. Preliminary results indicate that progestins can also exert rapid actions in the preoptic anterior hypothalamus (POAH) in this species to down-regulate gonadotropin-releasing hormone (GnRH) secretion. Recently, we reported the cloning, sequencing and characterization of a novel cDNA in a closely related species, spotted seatrout, that has the characteristics of the mPR involved in the progestin induction of oocyte maturation. Three distinct mPR subtypes, named alpha, beta, and gamma, have been identified in both fishes and mammals. The tissue distribution of the mPRalpha protein in seatrout suggests the alpha-subtype mediates progestin actions on GnRH secretion, sperm motility and oocyte maturation. However, mPRbeta antisense experiments in zebrafish oocytes suggest the beta-subtype also participates in the control of oocyte maturation in zebrafish.

Animals↗

Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells.

Recently, a unique family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRalpha and mPRbeta isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). The zebrafish mPRalpha and a previously uncharacterized mPRbeta isoform were stably expressed in nuclear progesterone receptor-deficient mammalian breast cancer cells, MDA-MB-231. Expression and surface localization of the receptors were verified by flow cytometry, biotin surface labeling, and Western blotting. Plasma membrane proteins from mPRalpha- or mPRbeta-transfected cells showed high affinity (mPRalpha, K(d) 7 nM; mPRbeta, K(d) 12 nM), saturable, displaceable, single-binding sites specific for 17,20beta-DHP, whereas negligible specific 17,20beta-DHP binding was observed in nontransfected cells. Progestin treatment caused significant activation of mitogen-activated protein kinase (MAPK) within 5 min in cells transfected with either of the receptors as measured by western blotting and flow cytometry. The rank order of the potencies of several progestins in activating MAPK via mPRalpha and mPRbeta was the same (17,20beta-DHP>progesterone >4-pregnen-17,20beta,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20beta-DHP, was also the most potent inhibitor, among the progestins tested, of adenylyl cyclase activity in cells transfected with either of the receptors. This progestin significantly decreased cAMP levels in both mPRalpha- and mPRbeta-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRalpha was blocked by pertussis toxin, implying activation of a G(i) protein, while sensitivity to pertussis or cholera toxin was not shown with mPRbeta-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRalpha and mPRbeta signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

Animals↗

A progestin antagonist blocks vaginocervical stimulation-induced fos expression in neurones containing progestin receptors in the rostral medial preoptic area.

Vaginocervical stimulation (VCS) has a variety of effects on the brain, physiology and behaviour. Previous work demonstrated that a progestin antagonist blocked neuronal response to VCS (i.e. Fos expression) in the absence of progesterone in some neurones, and suggested that some of the effects of VCS on the brain are mediated by ligand-independent activation of progestin receptors (PRs). Although it had been reported previously that some of the cells in which VCS induces Fos expression also contain PRs, it had not been determined if a progestin antagonist blocked Fos expression in these particular neurones. The purpose of this experiment was to determine if a progestin antagonist decreases Fos expression specifically in cells that also express PRs in the preoptic area and ventromedial hypothalamus. As has been shown previously, VCS increased Fos-immunoreactive (ir) expression in the particular areas studied. In the rostral medial preoptic area, VCS increased Fos expression in cells that coexpressed PRs, as well as in cells that do not. However, in the caudal medial preoptic area, VCS only increased Fos expression in cells that did not coexpress PRs. Injection of the progestin antagonist, RU 486, decreased Fos expression in the rostral, but not caudal medial preoptic area, and it decreased Fos expression only in cells that coexpressed PR-ir. In contrast to a previous report, in the present study, the progestin antagonist did not inhibit VCS-induced Fos expression in the ventromedial hypothalamic area. The results of this experiment suggest that the progestin antagonist inhibits VCS-induced Fos expression in some neurones by blocking PRs, and they provide further support for the idea that VCS influences neuronal response in some cells by ligand-independent activation of PRs in those cells.

Animals↗

RU486, a progestin antagonist, binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins.

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.

Binding, Competitive↗

The concentration of faecal progestins during the oestrous cycle in Nkone cows and the effect of duration of storage of faecal samples at room temperature on faecal progestin levels.

The objectives of this study were to determine whether ovarian function in Nkone cows could be monitored by measuring progestin concentrations in faeces and to assess the effect of duration of storage at room temperature on faecal progestin concentrations. Faecal and blood samples were obtained once a day for 26 days from 21 Nkone cows whose oestrous cycles had been synchronized. Faecal samples from each cow were divided into five portions that were kept at room temperature for 0, 6, 12, 24 and 48 h, respectively, and then frozen. After centrifuging the blood to recover plasma and extracting steroids from the faeces, analysis of progesterone (P4) was carried out using solid-phase radioimmunoassay. The faecal progestin and plasma progesterone profiles corresponded well and were positively correlated (r = 0.70, p < 0.01). Faecal progestin concentrations decreased with increasing duration of storage at room temperature during both the follicular and luteal phases (p < 0.01). In both cases, the decline in faecal progestin concentrations followed an exponential pattern. The progestin concentrations in faeces after 48 h of storage at room temperature were higher (p < 0.05) during the peak luteal phase than in the follicular phase.

Animals↗

Distribution of progestin-concentrating cells in rat brain: colocalization of [3H]ORG.2058, a synthetic progestin, and antibodies to tyrosine hydroxylase in hypothalamus by combined autoradiography and immunocytochemistry.

The anatomical distribution of progestin-concentrating cells in rat brain was investigated by thaw-mount autoradiography. Fifteen 23-day-old ovariectomized and adrenalectomized female Holtzman rats were injected sc with 5 micrograms 17 beta-estradiol in oil/100 g BW daily for 4 days. On the fifth day, all animals were injected iv with 1 microgram/100 g BW [3H]ORG.2058, a synthetic progestin. The animals were killed after 30 or 60 min, and the brains were frozen and processed for autoradiography. Nuclear concentration of radioactivity was found in certain cells of the forebrain and midbrain and was prevented by prior injection (15 min) of excess unlabeled ORG.2058. In the preoptic-septal region, progestin-labeled neurons were observed in the nucleus (n.) septi lateralis, n. interstitialis striae terminalis, n. preopticus medialis, n. preopticus lateralis, n. preopticus periventricularis, and n. preopticus suprachiasmaticus. In the hypothalamus, labeled neurons were seen in n. periventricularis hypothalami, n. arcuatus hypothalami, n. ventromedialis hypothalami, n. dorsomedialis hypothalami, n. premammillaris ventralis, n. premammillaris dorsalis, and lateral hypothalamus. In the extrahypothalamic region, a few labeled cells were found in the central, medial, and cortical nuclei of the amygdala, the organum subforniculare, the lateral geniculate nucleus, the parietal and entorhinal cortex, and the central gray of the midbrain. Some of the progestin-concentrating cells were characterized with combined autoradiography and immunocytochemistry. Tyrosine hydroxylase-containing cells of the arcuate nucleus and the hypothalamic periventricular nucleus (group A12) showed nuclear concentration of radioactivity. Tyrosine hydroxylase cells in groups A11, A13, and A14; the substantia nigra (group A9); and the ventral tegmental area (group A10) did not show nuclear concentration of [3H]ORG.2058. The autoradiographic study demonstrates specific progestin-binding sites in nuclei of neurons in select areas of the rat brain. The results of combined autoradiography and immunocytochemistry suggest, for the first time, a direct action of progestin on tuberoinfundibular dopaminergic neurons.

Animals↗

Progestin radiopharmaceuticals labeled with technetium and rhenium: synthesis, binding affinity, and in vivo distribution of a new progestin N2S2-metal conjugate.

We have prepared and evaluated three metal conjugates of a progestin-monoamine-monoamide (MAMA') bisthiol chelate system. These conjugates of rhenium and technetium-99 and -99m, are structural analogs of the bisamino-bisthiol (BAT) conjugates we have described recently, but the MAMA' chelate, being more polar than the BAT system, gives a conjugate that is much less lipophilic, having an octanol-water partition coefficient that is nearly 80-fold lower. In competitive binding assays, the Re- and 99Tc-MAMA'-progestin conjugates bind to the progesterone receptor with affinities greater than that of progesterone itself, and in a direct binding assay, the equilibrium dissociation constant (Kd) of the 99mTc-MAMA' conjugate was 0.97 nM. As is typical for 11 beta-substituted progestins, these conjugates also have substantial binding affinity for glucocorticoid receptors. In tissue distribution studies in immature female rats, the progestin-99mTc-MAMA' conjugates show selective uptake for principal target tissue (such as uterus) over that of blood and nontarget tissue (such as muscle); these uptake ratios reach maximum levels of 5 and 4, respectively. Uptake by fat, liver, and kidney is quite high; however, only the uptake in uterus is displaceable upon coinjection of the selective progestin ORG2058. Metabolism studies show that the radioactivity in the uterus is essentially unmetabolized out to 4 h, while liver activity is completely due to metabolites. Other tissues show an intermediate fraction of unmetabolized conjugates that decreases with time. The in vivo behavior of the progestin-99mTc-MAMA' conjugate is similar to that of the labeled BAT conjugate: its uptake selectivity is somewhat greater than that of the BAT conjugate, but its target tissue uptake is lower. Factors that may be responsible for limiting the target tissue uptake properties of these conjugates are their moderate affinity for progesterone receptor, their substantial binding to glucorticoid receptors, and their large overall molecular size.

Animals↗

Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns.

BACKGROUND: In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. METHODS: In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. RESULTS: Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. CONCLUSION: Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

Animals↗

Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites.

Putative progestin receptors have been characterized in brain and pituitary tissue from untreated and estrogen-primed ovariectomized-adrenalectomized rats. The properties of these sites appear indistinguishable from those of cytoplasmic progestin receptors from the uterus: 1) sedimentation coefficient of 7S, which is reduced by half in the presence of 0.3 M KCl; 2) specificity of binding which strongly favors synthetic and natural progestins as opposed to glucocorticoids, androgens, and estrogens; 3) a dissociation constant for binding the synthetic progestin [3H]R5020 (17 alpha, 21-dimethyl-19-norpregna-4, 9-diene-3,20-dione) of 0.3 nM; and 4) similar rates of formation and dissociation of the [3H]R5020-receptor complexes. In these respects, the estrogen-inducible and noninducible receptors of the brain also appear to be indistinguishable from each other. Estrogen induction of progestin receptors is apparent in uterus (6-fold), pituitary (8-fold), mediobasal hypothalamus (4-fold), and preoptic area (4-fold), all estrogen receptor-containing areas. The corticomedial amygdala does not show an estrogen effect on progestin receptor levels even though it contains estrogen receptor sites. The midbrain of the rat doeogen-insensitive receptors in the brain is relatively low and of the same order of magnitude as in nonstimulated hypothalamus, preoptic area, and pituitary, yet variations are seen among the estrogen-insensitive structures, with lowest levels occurring in cerebellum (6-7 fmol/mg protein) and highest levels occuring in cerebral cortex (approximately 25 fmol/mg protein). These findings are discussed in relation to the actions of progesterone which do and do not require estrogen priming and in relation to intracranial progesterone implantation studies.

Adrenalectomy↗

Progestin receptors in brain and pituitary of 20-day-old fetal mice: an autoradiographic study using [125I]progestin.

The distribution of progestin target sites in the brain and pituitary of estrogen-primed 20-day-old fetal mice was investigated by thaw-mount autoradiography. Three pregnant mice were each implanted sc with a Silastic tube containing estrogen on day 17 and ovariectomized on day 19 of gestation. Twenty-four hours after ovariectomy 10 fetuses (5 males and 5 females) were collected and each injected sc with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). For competition, two additional fetuses were injected with 20 micrograms R5020 1 h before (Z)-17 beta-hydroxy-17 alpha-(2[125I]iodovinyl)4-estren-3-one [( 125I]Progestin) to demonstrate that nuclear uptake and retention of radioactivity were specific for progestin. Two hours after injection of [125I]Progestin all fetuses were mounted, frozen, and sectioned in a cryostat. After 1-37 days of exposure, sections were developed and scanned for labeled cells. Cells with nuclear concentration were found in the male and female preoptic area, within certain nuclear groups in the basal hypothalamus, in the central gray of the midbrain, and in the pituitary. No labeling was detected in the cortex or amygdala. The results indicate that cells in certain regions of the brain and pituitary express progestin receptors at the end of gestation and suggest that progesterone is important for the normal development of these cells.

Animals↗

Progestin receptor cells in mouse cerebral cortex during early postnatal development: a comparison with preoptic area and central hypothalamus using autoradiography with [125I]progestin.

The distribution of progestin target cells in the cerebral cortex and the effect of estrogen treatment was assessed during the critical period of brain development and compared with the preoptic/central hypothalamic regions. [125I]progestin was injected into 0, 2, 8, and 12 day postnatal mice pretreated for 3 days with oil, 5 micrograms/100 g b, wt., or 100 micrograms/100 g b. wt. of estradiol dissolved in oil. Two hours after injection of radiolabeled ligand, brains were frozen and processed for thaw-mount autoradiography. At birth, labeled cells were detected in the deep (lamina VI) and intermediate (lamina V) layers of the lateral cortical regions, increased in laminae V-VI of the lateral cortex and laminae II-VI of the cingulate/paracingulate cortex at days 2 and 8, and decreased throughout the cortex by day 12. Pretreatment of animals with estradiol had no noticeable effect on the nuclear concentration of [125I]progestin in cortical cells, while estrogen weakly enhanced labeling in preoptic/central hypothalamic regions at day 2 and markedly augmented labeling in the 8 and 12 day brain. The results demonstrated that progestin receptor cells are present in the postnatal dorsal cortex, preoptic area, and hypothalamus and that the topography of cortical progestin target cells differs in part from that of estrogen target cells reported earlier.

Animals↗

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas.

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.

Adenocarcinoma↗

Intrahypothalamic effects of progestin agonists on estrous behavior and progestin receptor binding.

To investigate whether metabolism of progesterone (P) to other progestins is necessary for the facilitation of estrous behavior in estrogen-primed rats, we evaluated the behavioral effectiveness of intrahypothalamic implants of two P metabolites, 5 alpha-dihydroprogesterone (DHP) and 20 alpha-hydroxyprogesterone (20HP), and of desoxycorticosterone (DOC). We also determined whether the progestin receptor binding capacity of the steroids correlated with their behavioral efficacy. Implants of DHP and 20HP into the ventromedial hypothalamus were considerably less effective than P in activating estrous behavior; in contrast, the mineralocorticoid DOC was nearly as effective as P. Binding studies showed that P had the highest affinity for brain progestin receptors followed by DHP, DOC and 20HP. Thus there was a poor correlation between the behavioral efficacy and the progestin receptor binding properties of the steroids tested. These data suggest (1) that neither 5 alpha-reduction nor 20 alpha-hydroxylation are necessary for P activation of estrous responsiveness and (2) that the structural features required for the behavior promoting effects of P may not be identical to those required for binding to brain progestin receptors.

20-alpha-Dihydroprogesterone↗

Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells.

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.

Animals↗