Dynamics of estrogen induction of glandular kallikrein in the rat anterior pituitary

Glandular kallikrein has recently been identified as an estrogen-induced protein of the rat anterior pituitary. This study examined the dynamics of the estrogen induction of anterior pituitary glandular kallikrein in the ovariectomized rat. The estrogen induction of uterine dry weight was also examined for purposed of comparison. 17β-Estradiol (0.1–100 μg/day) produced dose-dependent increases in anterior pituitary glandular kallikrein, with the highest dose producing a 60-fold increase. Time-course studies demonstrated that a lag phase of 2–3 days was required before these estrogen effects on glandular kallikrein became evident, and levels were still rising between 7 and 10 days of treatment. The dynamics of the estrogen induction of glandular kallikrein resembled the estrogen induction of uterine dry weight with regard to estrogen sensitivity and the presence of a lag phase before estrogen-induced increases. However, uterine dry weight responde more rapidly to estrogen than did anterior pituitary glandular kallikrein, and reached a plateau after 5 days of estrogen treatment.     

Development of the sex difference in glandular kallikrein and prolactin levels in the anterior pituitary of the rat

Glandular kallikrein is a major estrogen-induced and dopamine-repressed protein of the rat anterior pituitary that appears to originate from lactotrophs. This study examined the development of glandular kallikrein levels in the anterior pituitary in both female and male rats and compared it to anterior pituitary prolactin. In addition, the development of glandular kallikrein levels in the neurointermediate lobe of the pituitary and the kidney were also examined. During puberty, a dramatic surge in glandular kallikrein occurred in female anterior pituitaries (16- to 20-fold increase) and levels remained elevated thereafter. The dynamics of the increase were biphasic--glandular kallikrein increased between Day 30 and 45, plateaued between Days 45 and 55, and then increased again between Days 55 and 65. Female anterior pituitary prolactin increased 7- to 8-fold during puberty. The rise during puberty was biphasic and was generally synchronized with increases in glandular kallikrein. However, the initial rise was proportionately less than that of glandular kallikrein, and the secondary surge was more dramatic. In contrast to females, anterior pituitary glandular kallikrein remained at low levels in male rats; prolactin levels also remained unchanged through puberty and increased moderately thereafter. Glandular kallikrein in the female neurointermediate lobe remained unchanged through Day 55, almost doubled on Day 60, and returned to prepubertal levels by Day 65; males did not exhibit the transient surge in neurointermediate lobe levels. Starting at age 60 days, renal glandular kallikrein was found to be slightly higher (15-20%) in females than in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glandular kallikrein levels in the rat anterior pituitary during the estrous cycle and pregnancy

Glandular kallikrein (a trypsin-like serine protease) is a major estrogen-induced protein in the rat anterior pituitary, which appears to be associated with lactotropes. The present study examined glandular kallikrein levels in the anterior pituitary during the rat estrous cycle and pregnancy. After trypsin treatment of anterior pituitary homogenates (to activate latent forms of the enzyme), glandular kallikrein activity was measured by using the chromogenic substrate D-val-leu-arg-p-nitroanilide: 98-95% of the enzymatic activity was immunoprecipitable with glandular kallikrein antiserum. Glandular kallikrein levels did not change significantly during the various phases of the rat estrous cycle. However, a sharp decrease was observed starting on Day 15 of pregnancy and lasting through parturition; levels had almost returned to control values by Day 5 of lactation.     

Effect of 5 alpha-dihydrotestosterone and dexamethasone on estrogen receptors of the anterior pituitary and uterus.

The administration of 5 alpha-dihydrotestosterone (5 alpha-DHT) and dexamethasone has been shown to attenuate estrogen-induced prolactin release in the estrogen-primed rat. Therefore, the effect of these compounds was studied on anterior pituitary and uterine estrogen receptors. Injection of 0.8 mg/kg body weight of 5 alpha-DHT to ovariectomized adult rats treated with 2 micrograms estradiol/d for 4 days resulted in a significant decrease in occupied nuclear estrogen receptors of the anterior pituitary but not the uterus. Estrogen priming was essential for 5 alpha-DHT effect on occupied nuclear anterior pituitary estrogen receptors because this effect did not occur in ovariectomized vehicle-treated control animals. The administration of 1 mg/kg body weight of dexamethasone brought about a decrease in uterine but not anterior pituitary nuclear estradiol receptors. These results provide further evidence that the regulation of estrogen receptor dynamics is different in the anterior pituitary and the uterus and that different steroids can exert tissue-specific effects.     

Cellular mechanisms of estrogen-and dopamine-induced control of glandular kallikrein in the anterior pituitary of the rat

Glandular kallikrein (GK, a trypsin-like serine protease) exhibits estrogen induction and dopamine repression in rat pituitary lactotrophs. Steroid induction may reflect primary actions to increase selectively the synthesis of specific proteins, or may be part of broad cellular responses secondary to steroid-induced phenotype transitions. This study examined the cellular mechanisms underlying estrogen and dopaminergic control of lactotroph GK using a quantified immunocytochemical approach. Pituitaries from ovariectomized rats exhibited little GK staining. Estradiol treatment for 10 days produced dose-dependent increases in pituitary mass, the percentage of lactotrophs (indicating lactotroph proliferation) and the percentage of GK-positive cells. Also, GK staining intensity was dependent upon estradiol dose, increasing 4-fold between 5 g and 50 g/48 h. Dopamine receptor blockade with haloperidol (2.5 mg/kg/24 h) elicited weak GK immunostaining in 46% of the lactotrophs in the absence of estradiol, and markedly potentiated GK staining intensity elicited with low but not high doses of estradiol. The results suggest that GK induction is a primary estrogen effect, and is not secondary to a phenotype transition: the induction is enhanced by estrogen-induced lactotroph proliferation. Dopaminergic systems strongly inhibit GK induction by low estradiol levels. This dopaminergic modulation may shift the induction of lactotroph GK to physiological events associated with high estradiol levels or low dopaminergic tone.     

Immunohistochemical demonstration of nitric oxide synthase (NOS) in the normal rat pituitary gland,estrogen-induced rat pituitary tumor and human pituitary adenomas

Nitric oxide synthase (NOS) immunoreactivity was examined in normal rat anterior pituitary glands, estrogen-induced rat pituitary tumors and human pituitary adenomas using a polyclonal antibody reacting with all three isoforms of NOS in both species. It was found that NOS immunorectivity in pituitary glandular cells is stronger in rat experimental pituitary tumors than in normal pituitaries. NOS immunoreactivity is also detectable in all but two human pituitary adenomas and seems to negatively correlate with microvascularization.     

Androgen receptor-mediated inhibition of estrogen-induced uterine peroxidase

Flutamide, an anti-androgen known to act through the androgen receptor, abolished the inhibitory action of testosterone on the induction of peroxidase in immature rat uteri without affecting inhibition produced by progesterone. The time course of the androgen effect on estrogen-induced uterine peroxidase, uterine weight and glucose 6-phosphate dehydrogenase activity was also determined together with the effect of flutamide on these steroid hormone-sensitive parameters. The possible mechanism of action of these compounds is discussed, particularly in the light of estrogen-induced eosinophilia. It is proposed that the observed interaction between testosterone and estradiol is mediated through their own specific receptors and not by illicit occupation of the estrogen receptor by the androgen. 5-Androstene-3 beta, 17 beta-diol (Adiol), an androgen known to exert estrogenic effects through the estrogen receptor, induced uterine peroxidase and was without significant effect on the action of estradiol, in contrast to testosterone.     

Antagonism of estrogen-induced prolactin release by progesterone

Previous work from our laboratory has shown that during the process of nuclear occupancy of the progesterone receptor complex (1-2 h), nuclear estradiol receptors of the anterior pituitary are depleted. The purpose of this study was to determine whether the depletion of nuclear estradiol receptors by progesterone had functional biological significance. The ovariectomized (26 days of age) immature rat was used as the model for analysis of this question. The ability of estradiol to release prolactin from the anterior pituitary was the function chosen to determine the biological significance of the progesterone and estradiol interactions. In response to estradiol exposure (2 micrograms/rat), prolactin release reached peak values from 8 h to 12 h and returned to control levels by 24 h. A second injection of estradiol 13 h after the initial injection stimulated a second increase in serum prolactin at 25 h. This model of two injections of estradiol 13 h apart served to provide adequate levels of anterior pituitary progesterone receptors and elevated serum prolactin levels upon which superimposed progestin modulation could be examined. A single injection of progesterone (0.8 mg/kg BW) 1 h before the second estradiol injection blocked the increase in serum prolactin. This action was a receptor-mediated event because progesterone had no effect without estrogen priming or when the progesterone antagonist RU486 was used. Finally, when the interval between the progesterone and second estradiol injection was extended to 4 h, a time period when progesterone does not deplete pituitary nuclear estrogen receptors, the estrogen-induced increase in serum prolactin was not blocked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dexamethasone suppresses estrogen action at the pituitary level without modulating estrogen receptor dynamics

The administration of glucocorticoid combined with antiestrogen such as clomiphene has been shown to be effective for the induction of ovulation in patients with anovulation. The present study was undertaken to examine the effects of glucocorticoid on estrogen-induced changes in the pituitary gland. A single intraperitoneal (i.p.) administration of 10 micrograms estradiol-17 beta (E2) in ovariectomized and adrenalectomized rats resulted in a significant stimulation of pituitaries with regard to wet tissue weight and progesterone receptor content. An i.p. administration of 1 mg dexamethasone in these animals had no effects on both the values. However, the E2-induced increases in pituitary weight and progesterone receptor content were significantly inhibited by pretreatment with 1 mg of dexamethasone. The pretreatment with dexamethasone, on the other hand, had no significant effect on the dynamics of pituitary estrogen receptor induced by the injection of E2, i.e. the degree of nuclear translocation, occupancy and cytoplasmic receptor replenishment. The inhibitory effect of dexamethasone, therefore, does not seem to be mediated through estrogen receptor system in the pituitary. These results suggest that dexamethasone acts directly on the pituitary gland to suppress the action of E2, and which may be involved in the process of induction of ovulation by glucocorticoid-clomiphene treatment.     

Depletion and release of prolactin from rat pituitaries and pituitary tumors in vitro

Depletion of pituitary prolactin (PRL) and PRL release into culture medium were simultaneously examined over a 3.5- to 4.0-hr incubation period from anterior pituitary fragments obtained from Fischer-344 or Wistar-Furth female rats treated with estrogen for 5 days, in pituitary tumors induced by 8 weeks of diethylstilbestrol (DES) treatment in Fischer-344 rats and in MtTW15 pituitary tumors transplanted subcutaneously in Wistar-Furth rats for 4 weeks. Our objective was to determine if the event known as transformation, which we define as a loss in the tissue PRL content without a corresponding and equivalent increase in the medium PRL content, occurs in rat pituitary tumors. Our results indicated that transformation did not occur in vitro in rat anterior pituitary tumors induced in Fischer-344 rats by DES treatment but was present in pituitaries from Fischer-344 rats treated for 5 days with estrogen, which served as controls. We also observed in vitro transformation in the anterior pituitary of Wistar-Furth rats treated with estrogen for 5 days (controls) and in the pituitaries of Wistar-Furth rats inoculated with the MtTW15 tumor for 4 weeks, but not in the MtTW15 tumor itself. Although transformation was present in both Fischer-344 and Wistar-Furth rats treated acutely with estrogen the timing of the transformation was delayed 1-2 hr in the Fischer-344 rats compared with Wistar-Furth females. We concluded that transformation does not precede release of prolactin in rat pituitary tumors and that in normal pituitaries the mechanisms of transformation are induced differently between the strains of rats examined.     

Stimulation of 17 beta-hydroxy steroid dehydrogenase in the rat anterior pituitary gland by progesterone

Anterior pituitary gland and hypothalamic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity was measured in the immature castrated estradiol primed rat to determine if differences in enzyme activity could explain the progesterone induced reduction of bound estradiol nuclear receptors of the anterior pituitary gland but not the hypothalamus. Higher levels of 17 beta-HSD activity were found in the anterior pituitary gland as compared to the hypothalamus. The enzyme activity in the anterior pituitary gland was stimulated by progesterone administered either in combination with estradiol for 4 days or as a single injection following 4 days of estradiol priming. No progesterone effects were found on hypothalamic 17 beta-HSD. Under the experimental conditions used, progesterone administration did not alter uterine 17 beta-HSD. An increase in anterior pituitary gland and uterine 17 beta-HSD was also induced by estrogen administration.     

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

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

The regulation of adenohypophyseal prolactin secretion: effect of triiodothyronine and methylene blue on estrogenized rat adenohypophysis

Estrogens and thyroid hormones contribute importantly to cell proliferation and tumor transformation in the pituitary gland. We found that methylene blue antagonized estrogen-promoted adenohypophyseal enlargement and the enhancement of prolactin secretion. The purpose of the present article is to provide a review about neurotransmitters and their receptors involved in estrogen-induced anterior pituitary growth and in the antagonistic effects of triiodothyronine (T3) and methylene blue (MB). Central dopaminergic and noradrenergic systems are the most important factors regulating pituitary growth and function. Recently nitric oxide (NO) was added to the list of the neurotransmitters and neuropeptides involved in the control of the anterior pituitary secretion. Our data suggest that estrogen-induced anterior pituitary growth is associated with decreased synthesis and metabolism of central catecholamines, reduction of adenohypophyseal beta-adrenergic receptors and increase of dopamine DA-2 receptors. We found that the treatment with T3 or MB prevented both estrogen-induced catecholaminergic inhibition and dopamine DA-2 receptor increment in the anterior pituitary. In contrast to T3, MB given alone also slightly decreased the anterior pituitary weight. Serum levels and anterior pituitary content of prolactin were increased after treatment with estradiol benzoate (EB), whereas T3 or MB partially attenuated prolactin hypersecretion after estrogen administration. This is in accord with the attenuation of EB-induced inhibition of dopaminergic system by T3 and MB. MB given in combination with EB also partially attenuated EB-promoted rise of adenohypohyseal NO synthase activity which plays an important role in the regulation of prolactin secretion. Further studies on central catecholaminergic systems, pituitary receptors, the nitrergic system and mechanisms of intracellular signal transduction are necessary for better understanding of pituitary tumor transformation and possibly for the discovery of new approaches towards treating patients with these diseases.     

Tissue-specific regulation of rat estrogen receptor mRNAs

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.     

Effects of dexamethasone and indomethacin on estrogen-induced uterine growth

Certain aspects of estrogen-induced uterine growth are reminiscent of an inflammatory response. Dexamethasone (DEX) and indomethacin (IND), two anti-inflammatory agents that interfere with arachidonic acid metabolism, were examined with respect to their effects on several growth-associated responses of the uterus to estrogen. Ovariectomized rats were given a s.c. injection of either DEX (2 mg) or IND (8 mg) immediately prior to receiving a s.c. injection of estradiol (10 ωg). At 4 hr, DEX inhibited estrogen-stimulated uterine wet weight and ornithine decarboxylase (ODC) activity by 100% and 48%, respectively. At 24 hr, ³H-leucine incorporation into protein was inhibited 44% and ³H-thymidine incorporation into DNA was depressed 83%. Estrogen-stimulated increases in uterine protein/DNA ratio and epithelial microvilli density at 24 hr were not inhibited by DEX. IND inhibited estrogen-stimulated wet weight by 64% and ³H-thymidine incorporation into DNA by 42%, yet did not inhibit the increases in ODC activity, ³H-leucine incorporation into protein or protein/DNA ratio. These results suggest that the inflammation-like component of estrogen-induced uterine growth is mediated, at least in part, by arachidonic acid metabolites and is directed primarily toward stimulating cell division, and not cell growth.     

Estrogen-dependent growth of a rat pituitary tumor involves,but does not require,a high level of vascular endothelial growth factor

Long-term (10-week) treatment of Fischer 344 (F344) rats with the synthetic estrogen diethylstilbestrol (DES) increases the level of vascular endothelial growth factor (VEGF) in the pituitary. This is concurrent with the development of a large tumor of the pituitary of F344 rats. A role for VEGF in estrogen-dependent pituitary tumor growth is also supported by the fact that pituitary VEGF level is not increased by estrogen treatment in rats of the tumor-resistant Brown Norway (BN) strain. However, VEGF is not increased by estrogen treatment in an F(1) hybrid of F344 and BN, even though F(1) hybrid rats do form pituitary tumors in response to estrogen. Quantitative trait locus (QLT) mapping reveals that control of estrogen-dependent VEGF expression is linked to the Edpm5 QTL, which was previously identified as a QTL for estrogen-dependent pituitary tumor growth. In contrast, the QTL Edpm2-1 and Edpm9-2, which have been shown to each have a significant effect on estrogen-dependent pituitary mass of a magnitude similar to Edpm5, do not have any effect on VEGF level. Taken together, our results support the association of VEGF expression with growth of the estrogen-induced rat pituitary tumor, as has been reported by others, but they also indicate that there is significant pathways of growth regulation that are independent of high-level VEGF expression.     

Depression of estrogen-induced uterine peroxidase in alloxan-diabetic rats

The influence of alloxan diabetes on reproductive function and the estradiol-stimulated increase in uterine peroxidase was investigated. Alloxan monohydrate in a dose of 75 mg/kg body weight effectively produced permanent diabetes. In adult rats, 20 days of diabetes resulted in cessation of the estrous cycle and a significant reduction in the gain of body weight, the weights of anterior pituitary gland, ovary, uterus, the level of serum progesterone and the activity of the estradiol-stimulated uterine peroxidase (P less than 0.05). After 10 days of insulin treatment, the ovarian weight, the estrous cycle and the level of ovarian hormones were restored to normal whereas the uterine weight and the estradiol-stimulated uterine peroxidase activity were only partially recovered. Persistent depression of the uterine response in the insulin-treated diabetic rats to both endogenous and exogenous ovarian hormone stimulation suggests that the uterus was directly affected by diabetes. The direct effect of diabetes upon the uterus was further demonstrated in the ovariectomized immature rat in which diabetes depressed the stimulatory action of estradiol on both uterine weight and uterine peroxidase activity.