Facilitation or inhibition of the oestradiol-induced gonadotrophin surge in the immature female rat by progesterone: effects on pituitary responsiveness to gonadotrophin-releasing hormone (GnRH), GnRH self-priming and pituitary mRNAs for the progesterone receptor A and B isoforms.

Progesterone can either facilitate or inhibit the oestradiol (E(2))-induced gonadotrophin surge. We have previously developed immature female rat models to characterise and investigate the mechanisms of progesterone inhibition or facilitation. The aim of the present study was to determine the role of pituitary responsiveness to gonadotrophin-releasing hormone (GnRH) and GnRH self-priming under conditions of progesterone-facilitation and progesterone-inhibition, and whether the underlying mechanisms reflect changes in mRNAs encoding the A and B isoforms of the progesterone receptor (PR) in the pituitary gland. Pituitary responsiveness to GnRH, determined by measuring the luteinising hormone (LH) response to one i.v. injection of GnRH, was decreased by 60-80% (P < 0.001) in the progesterone-inhibition model. GnRH self-priming, estimated as the increment in the LH response to the second of two injections of GnRH separated by 60 min, was also significantly reduced (P < 0.05) in this model. In the progesterone-facilitation model, the LH response to GnRH injection was increased 2.5-3-fold (P < 0.05), an effect suppressed by the progesterone receptor antagonist, mifepristone. Progesterone-facilitation of LH release and increased pituitary responsiveness to GnRH were blocked by sheep anti-GnRH serum injected i.v. immediately after insertion of progesterone implants. The PR-B mRNA isoform, measured by solution hybridisation/RNase protection assay, was the predominant form in the pituitary of the immature female rat. PR-B was increased by E(2) and decreased by progesterone in both models. Thus, in immature female rats, progesterone-inhibition of the E(2)-induced LH surge is due to significant reduction in pituitary responsiveness to GnRH as well as in the magnitude of GnRH self-priming. Progesterone-facilitation of the E(2)-induced LH surge is due to increased pituitary responsiveness to GnRH, which is mediated by PR, and depends on endogenous GnRH release. The differences between progesterone-facilitation and progesterone-inhibition are not due to differences in regulation of pituitary PR-B mRNA.

Rapid stimulatory effect of activin-A on messenger RNA encoding the follicle-stimulating hormone beta-subunit in rat pituitary cell cultures.

Activin, a dimer of the beta-subunits of inhibin, stimulates FSH secretion by cultured rat pituitary cells. Both the cell content of FSH and total FSH (secreted plus intracellular) are increased by activin, suggesting an effect on FSH biosynthesis. To test this idea directly, we examined the effect of purified human activin-A of recombinant DNA origin (rhactivin-A) on steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures prepared from adult male rats. A preliminary study of the time course of rhactivin-A action indicated that the first significant effect on FSH secretion was observed at 6 h, with maximal stimulation occurring at 24-72 h. A small (20-30%), but significant, increase in LH secretion was also observed by 24 h. For RNA analysis, pituitary cell-cultures were treated for 2-72 h with a maximally effective concentration (50 ng/ml) of rhactivin-A. FSH secretion in rhactivin-A-treated cultures was elevated by 2- to 2.5-fold. Intracellular FSH increased gradually from 24-72 h. Recombinant human activin-A stimulated FSH beta mRNA levels at all times examined; FSH beta mRNA levels in activin-treated cultures were already twice those in control cultures at 2 h, and the magnitude of this effect remained constant up to 72 h. Recombinant human activin-A brought about small increases in secretion and cell content of LH and free glycoprotein alpha-subunit and in LH beta and alpha mRNAs at various times. Thus, the increases in gonadotropin release and cell content stimulated by rhactivin-A can be accounted for by increases in the gonadotropin subunit mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Repression of glycoprotein hormone alpha-subunit gene expression and secretion by activin in alpha T3-1 cells.

The alpha T3-1 cell line, a GnRH-responsive gonadotroph cell line developed by targeted oncogenesis in transgenic mice, was used to study regulation of the glycoprotein hormone alpha-subunit by activin. Transient transfection assays established that activin suppressed transcription of both the human and mouse alpha-subunit genes. Initial studies demonstrated that activin decreased transcription of -846 and -180 human alpha-subunit-luciferase constructs by about 30%, but that inhibin and follistatin were without effect. Subsequent studies to localize sequences mediating responses to activin were carried out using a series of 5'-deletions (-507 to -133) of the mouse alpha-subunit promoter fused to luciferase. The luciferase activity of the -507-base pair construct was decreased by 60-70% in the presence of activin, and follistatin prevented this decrease. There were significant stepwise losses of activin responsiveness when sequences between -507 and -424, -424, and -288, and -288 and -205 base pairs were eliminated. Clustered point mutations of the mouse alpha-subunit gene, shown previously to reduce basal expression and GnRH responsiveness, were tested to further identify sequences mediating activin repression. Constructs containing a mutated (-337 to -330) pituitary glycoprotein hormone basal element (PGBE) showed significant loss of activin responsiveness in the context of both the native promoter (-507 to +46) and a minimal promoter downstream of the -507 to -205 region of the mouse alpha-subunit gene, whereas mutation of sequences (-406 to -399) in the GnRH-response element had no effect. Multimers of the PGBE element (-344 to -300) were insufficient to mediate a full activin response when linked to a minimal promoter. When added together with GnRH to transfected cells, activin abolished the stimulatory effect of GnRH on alpha-transcription. Secretion of free alpha-subunit by alpha T3-1 cells decreased 10-50% after exposure to activin for approximately 20 h, and steady state levels of alpha-subunit messenger RNA (mRNA) decreased by about 20-25% after 24-72 h. As changes in activin sensitivity could modulate its action, activin receptor II mRNA levels were measured by Northern blot hybridization at various times after activin (or inhibin) treatment. The three species of ActRII mRNA present in alpha T3-1 cells (approximately 6, 3, and 0.5 kilobases) were unaffected up to 72 h by these treatments. These observations provide the first demonstration that activin regulates a gonadotropin subunit gene at the level of transcription. Suppression of transcription of the mouse alpha-subunit gene by activin appears to involve several segments of the alpha-promoter, one of which is in the region of the PGBE. Thus, alpha T3-1 cells may provide a favorable system to further identify the DNA sequences and nuclear factors through which activin acts to alter transcription.

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin.

In primary cultures of rat pituitary cells, inhibin and follistatin reduce steady state levels of FSH beta mRNA to less than 10% of control within 4-6 h, while activin increases this mRNA 2- to 3-fold after 2-4 h of treatment. The effects of these three gonadal polypeptide hormones on the LH beta and common alpha-subunit mRNAs are more gradual and of lesser magnitude. The present study was designed to determine whether inhibin, activin, and/or follistatin act at the posttranscriptional level by altering the stability of the gonadotropin subunit mRNAs. To determine the decay rates of FSH beta, LH beta, and alpha-subunit mRNAs, primary pituitary cell cultures were treated for 1-24 h with either of two transcriptional inhibitors, actinomycin-D or 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB), in the presence or absence of recombinant human inhibin-A, recombinant human activin-A, or purified bovine follistatin. The decay of preexisting gonadotropin subunit mRNAs was followed by Northern blot analysis. Levels of LH beta and alpha-subunit mRNAs remained constant or increased during the 24-h exposure to transcriptional inhibitors; therefore, it was not possible to calculate their half-lives. The stability of these mRNAs was not altered by inhibin, activin, or follistatin. In contrast, FSH beta mRNA turned over rapidly: the estimated half-life was 2.6 +/- 0.19 h (mean +/- SEM of eight determinations) after actinomycin-D treatment and 1.9 +/- 0.14 h (mean +/- SEM of 12 determinations) after DRB treatment. When new RNA synthesis was blocked by either actinomycin-D or DRB, there were no significant effects of inhibin, activin, or follistatin on the stability of FSH beta mRNA (n = 2-4 for each hormone). The decay of FSH beta mRNA in the presence of inhibin or follistatin alone, however, was even more rapid than that determined after the administration of transcriptional inhibitors (P < 0.005). After an initial lag of 1-2 h, the half-life of FSH beta mRNA was 0.88 +/- 0.15 h (n = 4) or 0.62 +/- 0.11 h (n = 3), in the presence of inhibin or follistatin, respectively. The most likely interpretation of these results is that inhibin/follistatin reduces steady state levels of FSH beta mRNA by inducing a labile protein that accelerates the degradation of this mRNA species, and the synthesis of this protein is blocked by actinomycin-D or DRB treatment. It is not clear at present whether inhibin, follistatin, and activin have additional effects on transcription of the gonadotropin subunit genes.

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures.

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells.

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation and inhibition of the estrogen-induced luteinizing hormone surge in the rat by progesterone: effects on cytoplasmic and nuclear estrogen receptors in the hypothalamus-preoptic area, pituitary, and uterus.

The possibility that inhibition and facilitation of estrogen-induced LH surges by progesterone could be associated with effects of this steroid on the concentration, subcellular distribution, or molecular properties of estrogen receptors in the hypothalamus-preoptic area (HPOA) and pituitary was investigated. Immature rats (28 days old) that received Silastic capsules containing estradiol-17 beta in oil (150 micrograms/ml) at 0900 h had LH surges between 1700 and 2000 h on day 29. This treatment led to depletion of cytoplasmic estrogen receptors (to 25-35% of control levels) and their accumulation in the nucleus. Insertion of crystalline progesterone implants concomitantly with estradiol implants or 24 h later resulted in blockade or enhancement of the LH surge, respectively. Progesterone administered in either mode did not significantly after the levels of estrogen receptors in the HPOA and pituitary; however, progesterone did suppress the quantity of both cytoplasmic and nuclear estrogen receptors in the uterus when administered in conjunction with estradiol for 24 h oe for 8 h after 24-h estrogen priming. The binding affinity and sedimentation properties in sucrose gradients of cytoplasmic estrogen receptors were unchanged by progesterone treatment. The specificity of the effects of progesterone on LH secretion was examined. Testosterone, dexamethasone, and the synthetic progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadien-3,20-dione) also inhibited LH surges when injected 8 h after placement of estradiol implants in 28-day-old rats. Only progesterone and R5020 brought about premature and sustained LH release when given to estradiol-primed rats at 0900 h on day 29. None of these compounds interfered directly with the binding of [3H]estradiol to cytoplasmic or nuclear receptors. In conclusion, modulation by progesterone of the estrogen-induced LH surge does not seem to result from effects on neural and hypophysial estrogen receptors. In contrast, suppression of uterine estrogen receptors by progesterone may account in part for antagonism by this steroid of estrogen-stimulated uterine growth.

Progesterone modulation of the luteinizing hormone surge: regulation of hypothalamic and pituitary progestin receptors.

We have investigated the possible role of hypothalamic and pituitary progestin receptors (PR) in modulation of the estradiol-induced LH surge by progesterone in the immature rat. Rats (28 days old) that received Silastic implants containing estradiol in oil at 0900 h had LH surges approximately 32 h later. Progesterone implants were inserted concurrently with estradiol capsules or 24 h later, leading to inhibition or facilitation of the LH surge, respectively. Cytoplasmic and nuclear PR were measured by in vitro exchange assays, using near-saturating concentrations of [3H]R5020 (3H-labeled promegestone; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), 1, 8, 24, and 32 h after insertion of progesterone or blank implants. Kd values of complexes between [3H]R5020 and PR were 0.5-1 nM (cytoplasmic), and 2-3 nM (nuclear). The sedimentation rates of these complexes in sucrose gradients were 7-8S (cytoplasmic) and 3-4S (nuclear). In rats treated concurrently with estradiol and progesterone for 1 or 8 h, cytoplasmic PR were depleted to 40-60%, and this was accompanied by slight increases in nuclear PR. In control rats treated with estradiol and blank implants, there was no induction of either cytoplasmic or nuclear PR in the hypothalamus-preoptic area for up to 48 h; however, in the pituitary and uterus of these animals, PR increased significantly in both compartments (2- to 3-fold at 24 h, 3- to 5-fold at 32 h, and 4- to 7-fold at 48 h). Administration of progesterone either to inhibit or facilitate LH surges almost completely blocked the inductive effect of estradiol on cytoplasmic PR, but the absence of PR from the cytosol could not be accounted for by their presence in the nucleus. In the hypothalamus-preoptic area of estradiol-treated control rats, neither cytoplasmic nor nuclear PR increased significantly for up to 48 h. The low levels of specific [3H]R5020 binding in pituitary and uterine cytosols from progesterone-treated rats appeared to be due mainly to a decrease in the number of binding sites, rather than to an effect on binding affinities. The 7-8S peak of cytoplasmic PR was considerably reduced in rats treated for 48 h with estradiol and 24 h with progesterone. These results are consistent with the notion that hypothalamic and pituitary PR are involved in modulation of the LH surge by progesterone and point primarily to a pituitary site of action for progesterone facilitation.

Physical properties of androgen receptors in brain cytosol from normal and testicular feminized (Tfm/y hermaphrodite) mice.

Our previous results had shown that brain cytosol from androgen-insensitive, testicular feminized (Tfm/y) mice contains a reduced amount (20-25%) of androgen receptors, compared to normal female or castrated male mice, with unchanged affinity for dihydrotestosterone (DHT). We have now used various physicochemical techniques to ask whether there is a qualitative difference between these residual receptors and those in normal brain. When androgen receptors labeled with [3H]DHT in concentrated crude brain cytosol were analyzed by density gradient centrifugation and Agarose gel filtration in buffers containing 0.4-0.5 M KCl, Tfm/y receptors appeared to be smaller and more symmetrical than those from their normal siblings; however, if wild-type receptors were partially purified or prepared from slightly more dilute homogenates, their properties approached those of Tfm/y receptors in crude cytosol. This suggested that receptor molecules in concentrated cytosol from normal mice were aggregated to a greater extent than were those from the mutant. The molecular weight (54,000) and axial ratio (3:1 for prolate or oblate ellipsoid) calculated for Tfm/y receptors, therefore, may provide estimates for normal receptors as well. By DNA-cellulose chromatography, Tfm/y and female cytosol receptors were both resolved into two components, eluting at about 0.13-0.15 and 0.22-0.24 M NaCl. On DEAE-cellulose columns, both were eluted as a single major species at approximately 0.08-0.10 M KCl. Thus, excluding their state of aggregation, presumably resulting from their different concentrations in crude cytosol, Tfm/y and normal receptors were substantially identical as concerns the physical parameters examined in this study.

Effects of progesterone on the estradiol-induced follicle-stimulating hormone (FSH) surge and FSH beta messenger ribonucleic acid in the rat.

This study was designed to investigate the effects of progesterone on the estradiol (E2)-induced FSH surge and FSH beta messenger RNA (mRNA) using immature rat models developed previously to demonstrate inhibition or facilitation of the LH surge by progesterone. Twenty-eight day-old rats that received E2 implants at 0900 h had FSH surges about 1700 h on day 29 (32 h). In rats treated with E2 alone, serum FSH was 15.1 +/- 1.6 ng/ml at this time, while in those animals treated concurrently with E2 and progesterone, serum FSH was significantly suppressed (8.3 +/- 0.7 ng/ml, P less than 0.001). For demonstration of progesterone facilitation, rats were primed for 24 h with E2 before progesterone treatment. This led to premature and enhanced FSH secretion: at 1400 h on day 29 serum FSH was 45.5 +/- 2.7 ng/ml compared to 6.4 +/- 0.5 ng/ml in rats treated with E2 alone. To examine the effects of these dual actions of progesterone on FSH synthesis, steady state concentrations of FSH beta mRNA were measured by Northern analysis. FSH beta mRNA generally increased in parallel with FSH release. Levels of this mRNA were about 1.5-fold higher in rats undergoing E2-induced FSH surges than in rats in which the surge was blocked by progesterone. Also, at the onset of the progesterone-facilitated FSH surge, FSH beta mRNA was about 5-fold higher in animals treated with E2 and progesterone than in those treated with E2 only. On the morning after the FSH surge (48 h after E2 treatment) FSH beta mRNA was low to undetectable. In contrast, levels of FSH beta mRNA were 7- to 8-fold higher at this time in rats in which the surge was blocked by progesterone. Serum inhibin concentrations were significantly elevated (P less than 0.05) in animals treated with E2 alone for 32 h (3077 +/- 260 fmol/ml) or 48 h (2344 +/- 148 fmol/ml) compared to those treated with E2 and progesterone in the inhibition paradigm (2469 +/- 106, 1896 +/- 114 fmol/ml, respectively). After 32 h of E2 treatment in the facilitation paradigm, serum inhibin was comparable (P greater than 0.2) in rats treated for 8 h with blank implants (2592 +/- 168 fmol/ml) and those treated for 8 h with progesterone (2720 +/- 188 fmol/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen and estrogen receptors in the developing mouse brain.

Specific binding of the androgens, 5alpha-dihydrotestosterone (DHT) and testosterone, and of 17beta-estradiol by brain cytosol from mice at 3-5,9-11, and 18-23 days of age was measured by charcoal assay and glycerol gradient centrifugation and analyzed by Scatchard plots. The immature mouse brain contains putative receptors for these steroids which migrate at 8 S in gradients at low ionic strength and at 5 S in 0.5 M KCl. Investigation of estradiol binding was complicated by the presence in cytosol from 3-5 day-old mice, and to a lesser extent from 9-11 day-old mice, of the high capacity, fetoneonatal estradiol binding protein (FEBP) which is no longer detectable at 3 weeks. The rapid dissociation of the FEBP-estradiol complex under non-equilibrium conditions probably led to over-estimation of free steroid concentration and thus to an apparent increase in the affinity of 8 S receptor for estradiol with age (for female brain cytosol KD=9.5 X 10(-10)M at 3-5 days and 2.7 X 10(-10)M at 18-23 days). The number of estradiol binding sites remains relatively constant during the first 3 weeks at 7-9 fmol/mg protein, while the number of DHT binding sites in female brain increases from 3.2+/-0.3 to 6.6+/-0.9 to 9.6+/-0.3 (mean+/-SE) fmol/mg protein in the 3 age groups. Dissociation constants and numbers of sites for both DHT and estradiol binding are similar in brain cytosol from male and female mice. Testosterone and DHT compete for the same binding site, but its affinity for DHT is about twice that for testosterone. The high affinity of the brain receptor for DHT (KD=4-5 X 10(-10)M) may reflect the slow metabolism of DHT to 5alpha-androstanediols, amounting to less than 10% after 2 h at 0 C. Binding of DHT and estradiol to cytosol from brain regions was also investigated. DHT receptors increase in parallel in various regions with age; the concentration of sites in the hypothalamus-preoptic area (HPOA) is 1.2-3.4 times that in the cerebral cortex (C). The concentration of estradiol binding sites in HPOA to that in C increases about 12-fold from neonatal to adult stages, reflecting both an increase in HPOA sites and a decrease in C sites, while the concentration in the remainder of the brain shows little change. Androgen and estrogen receptors in brain cytosol from immature mice can be distinguished by their different specificities and developmental patterns in whole brain and brain regions. The presence and properties of these receptors in the brain of neonatal mice are discussed with respect to their possible role in sexual differentiation of the brain.

Effects of nafoxidine on the luteinizing hormone surge: temporal distribution of estrogen receptors and induction of cytoplasmic progestin receptors in the hypothalamus-preoptic area, pituitary, and uterus of the immature rat.

The antiestrogen nafoxidine 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride; 2 mg/rat), blocked LH surges induced by Silastic implants containing estradiol (E2) in oil (150 micrograms/ml) in the immature female rat and had no stimulatory effect on gonadotropin secretion by itself. Furthermore, the administration of progesterone (P) to rats primed for 24 h with nafoxidine alone or nafoxidine plus E2 did not lead to premature and enhanced LH surges (facilitation) as it does in E2-primed animals. Pituitary LH content was depleted by 30-50% after E2-induced or P-facilitated LH surges, but was unchanged after the administration of nafoxidine or nafoxidine plus E2. To investigate the cellular mechanisms involved, levels of total cytoplasmic and nuclear estrogen receptors and cytoplasmic progestin receptors in the hypothalamus-preoptic area (HPOA), pituitary, and uterus were measured by [3H]E2 or [3H]R5020 (3H-labeled 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) exchange assays at various times after the administration of E2 and /or nafoxidine. The ability of nafoxidine to bind in vitro to cytoplasmic or nuclear estrogen receptors was, respectively, 4% and 2% (HPOA), 5% and 6% (pituitary), and 1% and 2% (uterus) relative to E2 (100%). After the injection of nafoxidine or the insertion of E2 implants, cytoplasmic estrogen receptors were depleted with a similar time course and remained depressed for at least 48 h in both the HPOA and pituitary. In the uterus, the antiestrogen prevented the replenishment of cytoplasmic estrogen receptors observed in response to E2. Nuclear estrogen receptor levels in the HPOA and pituitary peaked 1 h after E2 and subsequently declined to a plateau from 24-48 h (at 2-4 times the levels found in untreated rats). After nafoxidine, accumulation of these receptors in the nucleus was more gradual and prolonged. Absolute levels of estrogen receptors translocated to the nucleus by E2 or nafoxidine were comparable. E2 treatment led to a substantial induction of cytoplasmic progestin receptors (approximately 2-fold in the HPOA, approximately 4-fold in the pituitary, and approximately 7-fold in the uterus after 48 h), and this process was considerably inhibited by nafoxidine. These results support the notions that antagonism by nafoxidine of estrogen action may be due to a defective association of the antiestrogen-receptor complex with nuclear sites, ad progestin receptor induction may be a prerequisite for the facilitation of gonadotropin surges by P.

Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors.

The present study examined the question of whether modulation of estradiol-induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28-day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor.

Comparison of the physicochemical properties of uterine nuclear estrogen receptors bound to estradiol or 4-hydroxytamoxifen.

We have compared the physicochemical properties of rat uterine nuclear estrogen receptors (ER) labeled with (E2) or the high affinity antiestrogen 4-hydroxytamoxifen [OH Tam; 1-[4-(2-dimethylaminoethoxy)phenyl]-1-(4-hydroxyphenyl) 2-phenyl-but-1-(Z)ene]. Nuclear ER labeled in vivo with either ligand had sedimentation rates of 4.5-5S and mol wt (calculated from sedimentation coefficients and Stokes radii) of about 80,000. The 3S and 4S forms labeled by in vitro exchange with [3H]E2 or [3H]OH Tam, respectively, had mol wt of 31,000 and 50,000. Based on apparent Kd values, numbers of binding sites per uterus, and dissociation kinetics, the ligand-binding sites of the nuclear E2- and OH Tam-ER complexes could not be distinguished. An appreciable proportion (25-75%) of 4.5-5S estrogen- or antiestrogen-ER complexes were retained on DNA-cellulose columns and were eluted with 0.21 M KCl, while the 3S and 4S forms had lost the DNA-binding site. Nuclear ER bound to E2 or OH Tam were differentially sensitive to proteolysis with trypsin or alpha-chymotrypsin. Both in vivo and in vitro labeled [3H]OH Tam-ER complexes sedimented as discrete species (S20,w = 3.8-3.9; mol wt, approximately 40,000) after trypsin treatment (50 micrograms/ml; 1 h at 23 C); under the same conditions, peaks of [3H]E2 were obliterated. After digestion with alpha-chymotrypsin (10 micrograms/ml; 1 h at 23 C), nuclear ER labeled in vivo with [3H]E2 or [3H]OH Tam sedimented at 2.9S (mol wt, 29,000) or 4.0S (mol wt, 47,000), respectively; at 50 micrograms/ml, [3H]E2-ER complexes were barely discernible, while binding of [3H]OH Tam was only partially decreased. Nuclear ER labeled with the nonsteroidal estrogen [3H]diethylstilbestrol resembled [3H]E2-ER complexes in sensitivity to proteolysis. These results suggest that nuclear estrogen- and antiestrogen-ER complexes may differ in conformation such that they are differentially susceptible to degradation. This may influence their interactions with chromatin or specific DNA sequences as well as their release from nuclear binding sites.

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin.

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen and estrogen receptors in brain cytosol from male, female, and testicular feminized (tfm/y hermaphrodite) mice.

Specific binding of [3H] 5alpha-dihydrotestosterone (DHT) and [3H] estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm/y male-female), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT- and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4-5 S in gradients containing 0.5M KCl. KD's (mean +/- SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y male-female are 1.1 +/- 0.4, 0.9 +/- 0.4, and 0.8 +/- 0.1 X 10(-9)M, respectively. DHT binding activity in brain cytosol from tfm/y male-female mice is reduced to about 20-30% of that from their normal littermates, as is the case for tfm/y male-female kidney cytosol. The residual androgen receptor in tfm/y male-female brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y male-female mice. KD's (mean +/- SE) for estradiol binding are 1.6 +/- 0.5 X 10(-10)M for castrated males, 2.4 +/- 0.4 X 10(-10)M for females, and 1.8 +/- 0.4 X 10(-10)M for tfm/y male-female. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the Ki is 8-9 X 10(-9)M. The decrease in the number of DHT binding sites in the brain of tfm/y male-female mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol.

The chronic intracerebroventricular infusion of interleukin-1 beta alters the activity of the hypothalamic-pituitary-gonadal axis of cycling rats. I. Effect on LHRH and gonadotropin biosynthesis and secretion.

We have previously reported that the acute injection of interleukin-1 beta (IL-1 beta) into the brain ventricles of intact female rats promptly decreases LHRH release and inhibits gene expression of this peptide in the medial preoptic area (MPOA). The present studies were therefore designed to determine whether continuous exposure to the cytokine would disrupt the estrous cycle. IL-1 beta was injected intracerebroventricularly for 4-6 days at a rate of 4 ng/h. Daily vaginal smears were obtained to follow the cycle; pituitary LH and FSH secretion were measured at regular intervals. Steady state levels of LH and FSH messenger RNA (mRNA) in the pituitary, and LHRH gene expression in the MPOA, were measured at the end of the treatment. Infusion of IL-1 beta caused a total disruption of the estrous cycle, characterized by persistent smears indicative of the diestrus stage. When compared to animals treated with the vehicle, rats infused with IL-1 beta showed a significant decrease in circulating LH concentrations, which was accompanied by lowered mRNA levels in the pituitary. This statistical difference (P < 0.01) persisted even when treated rats were compared to control in a similar stage of the cycle (i.e. diestrus). Plasma FSH levels remained low at all times after IL-1 beta infusion but showed the expected cyclic changes in control animals. At the end of treatment, LHRH gene expression was also markedly suppressed in LHRH neurons distributed between the rostral preoptic area/organum vasculosum of the lamina terminalis and the MPOA of these animals. These results indicate that prolonged infusion of IL-1 beta into brain ventricles disrupts the estrous cycle, an event accompanied by decreased biosynthesis/release of LHRH and gonadotropins. We report in a related study that IL-1-treated rats also show increased plasma progesterone levels. However, it is improbable that this change was responsible for the interruption of the cycle described here; indeed we have previously observed that the central administration of IL-1 beta to intact rats resulted in an immediate blockade of the spontaneous activity of LHRH perikarya during the afternoon of proestrus and significantly decreased LHRH mRNA levels in gonadectomized animals. Taken together, these data suggest that the primary effect of IL-1 beta is at the level of LHRH perikarya, and that the resulting interruption of the cycle is caused by altered LHRH neuronal activity and blunted gonadotropin secretion.

Developmental changes in testicular inhibin and androgen-binding protein during sexual maturation in the cynomolgus monkey, Macaca fascicularis.

Inhibin was measured by RIA in testicular extracts and plasma of cynomolgus monkeys during four stages of sexual maturation. Immunoactive inhibin levels were compared to those of another Sertoli cell secreted protein, androgen-binding protein (ABP). ABP steroid-binding (bioactive) activity was measured in testes and epididymal segments using the radiolabeled ligand [3H]dihydrotestosterone (DHT). Testicular immunoreactive inhibin concentrations were maximal in late prepubertal monkeys, 2.5-3.5 yr old, while the total testicular content of inhibin progressively increased with age into adulthood. Bioactive testicular ABP concentrations were maximal during the pubertal period of the cynomolgus monkey (3.5-4.0 yr old), while the total ABP content of the testes also increased with sexual maturation. Mean (+/- SE) plasma concentrations of inhibin and testosterone (T) in adults, 6-8 yr old (17.72 +/- 3.5 microliters inhibin equivalents/ml and 7.07 +/- 2.45 ng/ml T, respectively), were significantly higher (P less than 0.05 and P less than 0.001, respectively) than those in early prepubertal, juvenile monkeys, aged 1.5-2.5 yr (5.85 +/- 2.1 microliters inhibin equivalents/ml and 0.27 +/- 0.02 ng/ml T). The increased plasma levels of inhibin and T in adults were associated, respectively, with the increased inhibin and androgen contents of the testes in these same animals. The developmental changes in testicular steady state mRNA concentrations for the inhibin alpha-, beta A-, and beta B-subunits as well as ABP were examined during sexual maturation by Northern blot analysis using heterologous human cDNA probes. Densitometric analysis of the autoradiograms revealed that the inhibin alpha-subunit mRNA concentrations were higher than those of inhibin-beta A and -beta B and ABP mRNA during all stages of pubertal development. Although the relative concentrations of each inhibin subunit mRNA were decreased in the adult animals relative to those in the juvenile monkeys, the total amount of steady state mRNA for the subunits was greater than that in the immature animals. A similar situation existed for the ABP mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.

Effects of castration on luteinizing hormone and follicle-stimulating hormone secretion by pituitary cells from male rats.

Because the role of the pituitary in the testicular control of gonadotropin secretion remains controversial, we examined the effects of castration on the release of LH and FSH under basal conditions and in response to GnRH stimulation by dispersed pituitary cells in monolayer culture as well as by cells perifused with pulses of GnRH. These effects were compared to changes in LH beta, FHS beta, and alpha-subunit mRNA levels determined by Northern blot analysis. Pituitary cells were prepared from 7-week-old intact rats and rats orchidectomized 2 weeks previously. Castration increased basal FSH secretion from monolayer cultures, interpulse FSH release from perifused pituitary cells, FSH beta mRNA concentrations and serum FSH levels each approximately 2-fold, whereas pituitary FSH contents were similar in cells from intact and castrated rats. Pituitary LH content rose 3-fold, LH beta mRNA rose 5.6-fold, and basal LH secretion increased 6-fold, but serum LH levels increased 22-fold. Thus, the change in FSH synthesis inferred from the increase in FSH beta mRNA was proportional to the increase in FSH secretion both in vitro and in vivo. Whereas the basal release of LH in vitro was also proportional to the change in LH beta mRNA, secretion of LH in vivo exceeded these changes, underscoring the importance of increased GnRH to the serum LH castration response. Castration resulted in an increase in the sum of FSH content and secretion during 10 days in culture in the absence of GnRH, indicating ongoing FSH synthesis. Total LH declined in cells from intact rats, and this decline was prevented by castration; this effect may be due to a castration-related decrease in intracellular LH degradation or increased LH synthesis in the absence of GnRH. Castration also augmented the GnRH-stimulated release of LH and FSH from monolayer cultures 4.5- and 1.8-fold, respectively, and increased the amplitude of GnRH-stimulated LH and FSH pulses 5- and 2-fold in experiments with perifused pituitary cells. The EC50 for GnRH was unaffected by castration.(ABSTRACT TRUNCATED AT 400 WORDS)