Dexamethasone inhibits the effects of oestrogen on the pituitary gland in rats

The administration of glucocorticoids has been shown to be effective for the induction of ovulation in patients with anovulation. In the present study, we investigated the effects of a glucocorticoid on oestrogen-induced changes in the pituitary gland. A single ip injection of 10 micrograms oestradiol-17 beta (E2) in ovariectomized and adrenalectomized rats resulted in a significant increase in pituitary weight and progesterone receptor (PgR) concentration. In these animals, serum LH level was initially suppressed and restored to control level 24 h after E2 injection. However, 1 mg of dexamethasone (Dex) injected before E2, but not after E2 administration, completely inhibited both the increases in pituitary weight and PgR concentration. The restoration of serum LH level 24 h after E2 was also prevented. These antioestrogenic effects of Dex were blocked by ip administration of the synthetic antiglucocorticoid, RU486. Dex treatment alone did not have any effect on E2-induced changes in the dynamics of pituitary oestrogen receptor. Finally, E2-pellet implanted sc in ovariectomized and adrenalectomized rats for 7 days caused marked increases in pituitary weight and PgR concentration. A single ip injection of 250 micrograms clomiphene citrate (clomiphene) significantly reduced both the pituitary weight and PgR concentration in these animals, but 1 mg of Dex failed to have a similar effect. These results suggest that glucocorticoids antagonize E2 effects on the pituitary by a mechanism different from antioestrogens such as clomiphene. These antioestrogenic effects of glucocorticoid may be involved in induction of ovulation in anovulatory women.     

Ultrastructural evidence for oxytocin in the rat anterior pituitary gland

OT is synthetized in the hypothalamus. These neurons project to the posterior lobe of the pituitary and the external zone of the median eminence. In order to localize OT in the male rat anterior pituitary we have used immunocytochemistry on ultra-thin sections in target cell(s) obtained by cryoultramicrotomy. OT-like immunoreactivity was observed in lactotropes only. No immunoreactivity was observed in gonadotropes, somatotropes, corticotropes or thyrotropes. In lactotropes, immunoreactivity was localized at the plasma membrane level, in the cytoplasmic matrix and around the secretory granules, but not in the other organelles, and in the nucleus. No reaction was observed by using either non-immune serum or anti-OT serum incubated with OT. No modification of OT-like immunoreactivity was observed by using anti-OT serum incubated with heterologous peptides. These results 1) provide immunocytological evidence for the presence of OT in the anterior pituitary gland; 2) indicate the presence of this peptide in one particular cell type, and 3) support the hypothesis that OT could have a direct participation in the regulation of the PRL release.     

Estradiol positive feedback on the rat anterior pituitary gland in vitro

LH surges can be elicited by estradiol (E2). The mechanism could be either by induction of a surge of hypothalamic LH-RH or by directly increasing the responsiveness of the pituitary gland to hypothalamic LH-RH without an LH-RH surge. In order to study the positive feedback of E2 on the anterior pituitary gland, without any possible influence of an LH-RH surge, we utilized long-term in vitro superfusion of the anterior pituitary gland. The E2 concentration was 100 pg/ml and 10(-8) M LH-RH was pulsed every 30 min for 3 min. LH and FSH surge was detected about 21 h after the beginning of the E2 infusion in all three repeated experiments. No surge was detected in any other channels, i.e. control, E2 superfusion alone or LH-RH stimulation alone. This study demonstrates that in the rat the induction of the preovulatory LH peak can be accomplished by a direct effect of E2 upon the anterior pituitary gland, provided it is being stimulated by constant pulses of LH-RH.     

Evidence for neuromedin-B synthesis in the rat anterior pituitary gland.

Neuromedin-B shows a widespread distribution throughout mammalian neural and peripheral tissues and may be involved in the modulation of a variety of physiological processes. The highest concentrations of neuromedin-B are found in the anterior pituitary gland, suggesting that it may be of physiological importance within this tissue. To examine this hypothesis, we have used Northern blotting to demonstrate that neuromedin-B is present in the anterior pituitary as a result of local synthesis and have examined the effects of endocrine manipulations on its mRNA and immunoreactive peptide content. In thyroidectomized male rats, neuromedin-B content was decreased (104.1 +/- 5.8 vs. control, 390.9 +/- 23.3 fmol/gland; P less than 0.01) as was its mRNA (7 +/- 1.3% vs. 100%; P less than 0.014), while treatment of intact animals with T4 produced no effect on either peptide content or mRNA. Adrenalectomized male rats showed a significant increase in both neuromedin-B content (313.8 +/- 11.8 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.05) and mRNA (377 +/- 27% vs. 100%; P less than 0.014), while dexamethasone treatment increased peptide content (347.8 +/- 32 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.01) without any effect on mRNA levels. In female rats, ovariectomy decreased neuromedin-B content (132.4 +/- 13.3 vs. control, 335.0 +/- 37.2 fmol/gland; P less than 0.01) and mRNA levels (6 +/- 2% vs. 100%; P less than 0.014) while estrogen treatment of both ovariectomized and intact rats produced large increases in neuromedin-B (887.8 +/- 114.4 and 1328 +/- 175 fmol/gland, respectively, vs. control, 335.0 +/- 37.2 fmol/gland; both P less than 0.01) and its mRNA (246 +/- 18% and 378 +/- 31%, respectively, vs. 100%; both P less than 0.014). In castrated male rats, no significant alteration in peptide content was observed, and treatment of both castrated and intact male rats with testosterone was similarly without effect. These results demonstrate that 1) neuromedin-B is locally synthesized within the rat anterior pituitary gland; and 2) the local production of neuromedin-B is influenced by endocrine status, which is consistent with an autocrine/paracrine role for this peptide in this tissue.     

Microtubules are mobilized in the lactating rat anterior pituitary gland during suckling

Microtubules in the lactating rat anterior pituitary gland were depolymerized into their constituent tubulin dimers by exposure of the pituitary to 4 C. The tubulin then was quantified with the [3H]colchicine binding assay, which was adapted for use with individual rat pituitary glands. The binding of [3H]colchicine to the tubulin fraction contained in high speed supernatants of lactating rat pituitary glands proved to be specific and saturable, and was pH, temperature, and time dependent. The amount of [3H]colchicine bound was linear over the range of protein concentrations tested (2-22 micrograms). To determine whether suckling affected the levels of microtubules in the anterior pituitary, tubulin levels were measured in groups of lactating rats after they were suckled (six pups per litter) for 0, 5, 10, 15, and 30 min following 4-5 h of nonsuckling. The tubulin concentration in the anterior pituitary progressively increased from 4 to 22 mumol during the 30 min of suckling; the increase was statistically significant (P less than 0.05) by the 10th min. Plasma PRL levels analyzed from trunk blood rose from 5 to 75 ng/ml during the 30-min suckling period. These results indicate that a mobilization of microtubules occurs in the anterior pituitary at the same time that PRL is being transformed and released into the circulation.     

Androgen receptors in the rat brain, anterior pituitary gland and ventral prostate gland: effects of orchidectomy and ageing.

Available high-affinity binding sites for 5 alpha-dihydrotestosterone (DHT) were measured in cytosols obtained from the amygdala, hypothalamus, anterior pituitary gland and ventral prostate gland of 12-week-old rats at various times after orchidectomy, and in the corresponding tissues of 18-month-old male rats. It is suggested that the lower affinity of the DHT binding reaction in brain and ventral prostatic cytosols after orchidectomy or ageing respectively, may explain, at least in part, the changes in the responsiveness of the tissues to androgens.     

The effects of testosterone and oestrogen on gonadectomised and intact male rat anterior pituitary mitotic and apoptotic activity

We have used a direct, non-immunochemical and highly accurate method to quantify the effects of testosterone and oestrogen on mitotic and apoptotic activity in the young, male rat anterior pituitary in vivo. Surgical gonadectomy resulted in a 3-fold increase in mitotic activity by the fourth post-operative day, which returned gradually to levels seen in intact animals over the subsequent 3-4 weeks. Both a single dose of Sustanon, a mixture of long-acting testosterone esters in arachis oil, and the same dose divided over 7 days (starting 6 days after gonadectomy), initially suppressed mitotic activity to levels seen in intact animals, but was associated after 48-96 h with a wave of increased mitotic activity. The latter was blocked by co-administration of Sustanon with the non-steroidal aromatase inhibitor letrozole and was not seen when the non-aromatisable androgen dihydrotestosterone was substituted for Sustanon. Oestrogen alone in gonadectomised and intact rats produced a marked increase in mitosis as expected. With the exception of a transient increase in response to a single high-dose injection of Sustanon in gonadectomised animals, apoptotic activity was unaffected by all of the above. This study suggests that pituitary mitotic activity is tonically inhibited by gonadal hormone production (at least in the short term) in adult male rats. The study also suggests that supraphysiological testosterone treatment -- while unable to reduce anterior pituitary mitotic activity in untreated, intact animals --suppresses the early increase in mitotic activity induced by gonadectomy. Oestrogen, either exogenous or generated locally by aromatisation, stimulates anterior pituitary mitotic activity in a time-dependent manner.     

Adrenocorticotrophic hormone in the anterior and neurointermediate lobes of the fetal rat pituitary gland

The concentration of ACTH in the pars distalis and pars intermedia of the fetal rat hypophysis from days 17-21 of pregnancy was measured with a specific radioimmunoassay and a bioassay using isolated adrenal cells from adult rats. In both lobes of the pituitary gland, a significant correlation was observed between immunoreactive and bioreactive values, expressed as pg equivalents synthetic human 1-39 ACTH per microgram protein. In the pars distalis, ACTH concentrations increased steadily from days 17-20 and then remained unchanged to term. At this time they were tenfold higher than on day 17. In the neurointermediate lobe, ACTH was detected only from day 18; the concentration of ACTH increasing between days 18 and 19. At each of the stages of pregnancy examined, the concentration of ACTH in the pars distalis was greater than that in the pars intermedia. These data have demonstrated that ACTH is present in both anterior and neurointermediate lobes of the fetal rat hypophysis, that the functional differentiation of the pars distalis takes place earlier than that of the pars intermedia, and that the concentrations of corticotrophin in the pars distalis and in the pars intermedia have different patterns of development as gestation progresses.     

Effect of calcium on adenylate cyclase of rat anterior pituitary gland

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Matrix metalloproteinase-9 expression in folliculostellate cells of rat anterior pituitary gland

Folliculostellate (FS) cells of the anterior pituitary gland express a variety of regulatory molecules. Using transgenic rats that express green fluorescent protein specifically in FS cells, we recently demonstrated that FS cells in vitro showed marked changes in motility, proliferation, and that formation of cellular interconnections in the presence of laminin, a component of the extracellular matrix, closely resembled those observed in vivo. These findings suggested that FS cells express matrix metalloproteinase-9 (MMP-9), which assists their function on laminin. In the present study, we investigate MMP-9 expression in rat anterior pituitary gland and examine its role in motility and proliferation of FS cells on laminin. Immunohistochemistry, RT-PCR, immunoblotting, and gelatin zymography were performed to assess MMP-9 expression in the anterior pituitary gland and cultured FS cells. Real-time RT-PCR was used to quantify MMP-9 expression in cultured FS cells under different conditions and treatments. MMP-9 expression was inhibited by pharmacological inhibitor or downregulated by siRNA and time-lapse images were acquired. A 5-bromo-2'-deoxyuridine assay was performed to analyze the proliferation of FS cells. Our results showed that MMP-9 was expressed in FS cells, that this expression was upregulated by laminin, and that laminin induced MMP-9 secretion by FS cells. MMP-9 inhibition and downregulation did not impair FS motility; however, it did impair the capacity of FS cells to form interconnections and it significantly inhibited proliferation of FS cells on laminin. We conclude that MMP-9 is necessary in FS cell interconnection and proliferation in the presence of laminin.     

Immunohistochemical localization of kallikrein within the prolactin-producing cells of the rat anterior pituitary gland

The localization of tissue kallikrein in the pituitary gland of rats was investigated by an immunohistochemical technique using antiserum against rat urinary kallikrein. Kallikrein-positive cells were detected in the anterior lobe of the pituitary of both male and female rats, but were not observed in the posterior lobe of the pituitary in either sex. The kallikrein-positive cells in the anterior pituitary of female rats in oestrus were found to correspond to the prolactin-producing cells, whereas the cells producing GH, LH and ACTH were negative for kallikrein. It is possible, therefore, that the tissue kallikrein may be involved in the production of prolactin and not that of the other anterior pituitary hormones, such as GH, LH, FSH, ACTH and TSH.     

Long-term negative feedback effects of oestrogen and progesterone on the pituitary gland of the long-term ovariectomized ewe

The effects of long-term treatment with physiological doses of oestradiol or oestradiol plus progesterone on plasma gonadotrophin levels and pituitary content of LH and gonadotrophin-releasing hormone (GnRH) receptors were studied in ovariectomized-hypothalamo-pituitary disconnected ewes given 250 ng pulses of GnRH every 2 h (i.v.). A pilot experiment showed that 3 cm long Silastic implants (s.c.) reduced both LH pulse frequency and pulse amplitude in long-term (greater than 6 months) ovariectomized ewes. The main experiment was conducted over 3 weeks in ovariectomized-hypothalamo-pituitary disconnected ewes that had received pulsatile GnRH replacement for 1 week after pituitary surgery. Group 1 (n = 5) received GnRH pulses alone throughout the study. Group 2 (n = 6) received oestradiol in week 2 and oestradiol plus progesterone in week 3 and in group 3 (n = 6) the steroid treatments were reversed. Oestradiol reduced (P less than 0.05) the mean (+/- S.E.M.) amplitude of LH in pulses in group 2 (from 8.2 +/- 1.6 to 5.0 +/- 0.5 micrograms/l) and group 3 (from 11.6 +/- 1.2 to 9.3 +/- 1.0 micrograms 1): an additional effect of progesterone was seen in group 2 but not group 3. The amplitudes of the LH pulses did not change in the control ewes. Plasma concentrations of FSH were reduced by approximately 50% by the oestradiol treatments with no additional effects of progesterone. There was no effect of steroidal treatment on pituitary content of LH or pituitary levels of GnRH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between unbound oestrogen receptors in the uterus and anterior pituitary gland after implantation of oestradiol-17 beta into rats

This paper describes the effect of oestradiol-17 beta implants on unbound cytosolic and nuclear oestrogen receptors in the uterus and anterior pituitary gland of ovariectomized adult rats. Rats were ovariectomized and implanted 1 week later with oil or oestradiol-17 beta/oil solution in silicone elastomer capsules. In the latter animals the capsules either remained in situ until decapitation (subgroup 1) or were removed 48 h after implantation (subgroup 2). Implants of oestradiol-17 beta caused a significant depletion in both forms of oestrogen receptor in the uterus and anterior pituitary gland of rats in subgroup 1. In subgroup 2, however, there was an almost complete return to control concentrations of uterine cytosolic receptors and anterior pituitary cytosolic and nuclear receptors. Concentrations of uterine nuclear oestrogen receptors showed only a partial recovery. These data suggest that both forms of oestrogen receptor constitute an integrated population of oestrogen receptors, without dependence on intracellular location, and that in the presence of oestradiol these receptors are bound more quickly than they are synthesized. The results also indicate the existence of a dynamic equilibrium of unbound receptors between the cytosolic and nuclear compartments for both target organs, but show that in the absence of oestradiol this equilibrium is restored in the anterior pituitary sooner than in the uterus.     

Immunocytochemical localization of angiotensin immunoreactivity in gonadotropes and lactotropes of the rat anterior pituitary gland

Utilizing an immunocytochemical technique wherein two different antigens can be visualized on the same tissue section, angiotensin-like immunoreactivity was found in rat adenohypophyseal cells that showed staining with antisera to hLH, hFSH or human prolactin. Angiotensin-like immunoreactivity was not found in cells that showed staining with antisera to hTSH, C-terminal ACTH or hGH. The nature and possible physiologic significance of the angiotensin-like immunoreactivity is discussed.     

Role of nitric oxide in the metabolism of arachidonic acid in the rat anterior pituitary gland

Nitric oxide (NO) affects cyclooxygenase (COX) and lipooxygenase (LOX) activities in several tissues. The aim of this study was to investigate the effect of NO on the AA metabolism in the anterior pituitary. LOX and COX products from anterior pituitaries of Wistar male rats were determined by [14C]-AA radioconversion method. Sodium nitroprusside (NP, 0.5 mM) and DETA NONOate (1 mM), NO donors, decreased 5-hydroxy-5,8,11,14-eicosatetraenoic acid (5-HETE) synthesis (P<0.05), effects that were reversed by hemoglobin. L-arginine also inhibited LOX activity. To the contrary, the inhibition of NO synthase by L-NAME (0.5 mM) or aminoguanidine (0.5 mM) increased 5-HETE production (P<0.05). COX activity was slightly stimulated by NP and L-arginine. However, DETA NONOate induced a stimulation of the synthesis of all prostanoids (P<0.05), this effect being reversed by hemoglobin. Neither NOS inhibitors nor hemoglobin modified basal prostanoids synthesis. These results indicate that NO inhibits LOX activity and stimulates COX activity in the anterior pituitary gland. The inhibition of LOX by NO may be another mechanism involved in the effects of NO on hormone release in the anterior pituitary.     

Ornithine decarboxylase activity and polyamines in the anterior pituitary gland during the rat oestrous cycle

The biosynthesis of polyamines, an ubiquitous group of amines shown to be essential for normal cellular growth and differentiation, was studied in the rat anterior pituitary gland during the different stages of the oestrous cycle. The activity of ornithine decarboxylase (ODC), which catalyses the rate-limiting step in the biosynthesis of polyamines, was low during oestrus, metoestrus and dioestrus. However, a marked transitory rise in ODC activity was found in the pituitary gland on the evening of pro-oestrus. The rise in ODC activity was accompanied by an increase in the pituitary content of the polyamines putrescine and spermidine. Ovariectomy did not significantly change the basal ODC activity in the pituitary gland. Oestrogen treatment of ovariectomized rats resulted in a marked stimulation of pituitary polyamine biosynthesis. The largest effects were observed when oestrogen was given as two injections 72 h apart, which gave rise to levels of ODC activity comparable to those observed on the evening of pro-oestrus. The increase in polyamine synthesis in the anterior pituitary gland during pro-oestrus appeared not to be related to the preovulatory secretion of LH or prolactin, since neither LH-releasing hormone nor thyrotrophin-releasing hormone (which induces a secretion of prolactin) affected pituitary ODC activity. The observed biosynthesis of polyamines may be associated with the cellular proliferation which occurs in the anterior pituitary gland at oestrus.     

Insulin-induced hypoglycemia increases proopiomelanocortin messenger ribonucleic acid levels in rat anterior pituitary gland

To study the effect of acute stress on ACTH secretion and synthesis in rat pituitary and hypothalamus, ACTH content and POMC mRNA levels (measured by use of Northern blot analysis) in these tissues as well as the levels of ACTH in plasma and those of CRF in the hypothalamus were determined after insulin-induced hypoglycemia. Plasma ACTH levels increased at 30 and 60 min. ACTH levels in the anterior pituitary lobe (AP) decreased at 30 min, and then returned to control levels at 60 min. No change was seen in the intermediate-posterior pituitary (IP) or the hypothalamus after insulin injection. CRF levels decreased at 30 and 60 min, then returned to control levels at 90 min in the medial basal hypothalamus, including the median eminence. Hybridization with a cDNA probe revealed a single size class of POMC mRNA in AP, IP, and hypothalamus, and the size of POMC mRNA in these tissues did not change during the experimental period. POMC mRNA levels in AP increased at 60 min and reached a peak at 120 min, but those in IP and hypothalamus did not change. These results suggest that 1) insulin-induced hypoglycemia stimulates both secretion and synthesis of ACTH (at least by increasing POMC mRNA levels) in the AP, and 2) the levels of ACTH and POMC mRNA in the IP and hypothalamus are not affected by insulin-induced hypoglycemia.