Pituitary Of Female Rats Research Articles

Exogenous orexin-A downregulates luteinizing hormone secretory activity in prepubertal female rats.

Orexin-A is a neuropeptide synthesized in the lateral hypothalamus. Orexin-A immunoreactive fibres overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Because data concerning the impact of orexin-A on the hypothalamo-pituitary axis activity are limited, we focused on the involvement of orexin-A and receptors of NPY in the modulation of LH release and LH subunit b (Lhb) mRNA expression in prepubertal female rats. Forty immature female Wistar rats were divided into 4 groups and received 2 intracerebroventricular (icv) microinjections of: 1 - artificial cerebrospinal fluid (CSF) (controls); 2 - CSF followed by orexin A; 3 - selective NPY receptor antagonist (BIBP) followed by CSF; 4 - BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected, and serum was stored at -20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at -80°C for RNA extraction. Real-time PCR amplification was carried out, and relative Lhb gene expression was calculated. In comparison to the CSF-treated controls with a mean LH serum concentration of 0.40 ± 0.02 ng/mL, the mean LH serum level was diminished both after orexin-A (0.27 ± 0.01 ng/mL) and after BIBP (0.30 ± 0.02 ng/mL) icv microinjections. In the presence of BIBP, orexin-A more effectively inhibited LH release (0.20 ± 0.01 ng/mL) when compared to the BIBP-treated group. Orexin-A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin-A, and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin-A and BIBP co-administration evoked a further reduction of Lhb gene transcriptional activity. Orexin-A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin-A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.

The ERα membrane pool modulates the proliferation of pituitary tumours.

The molecular mechanisms underlying the ERα nuclear/cytoplasmic pool that modulates pituitary cell proliferation have been widely described, but it is still not clear how ERα is targeted to the plasma membrane. The aim of this study was to analyse ERα palmitoylation and the plasma membrane ERα (mERα) pool, and their participation in E2-triggered membrane-initiated signalling in normal and pituitary tumour cell growth. Cell cultures were prepared from anterior pituitaries of female Wistar rats and tumour GH3 cells, and treated with 10 nM of oestradiol (E2). The basal expression of ERα was higher in tumour GH3 than in normal pituitary cells. Full-length palmitoylated ERα was observed in normal and pituitary tumour cells, demonstrating that E2 stimulation increased both, ERα in plasma membrane and ERα and caveolin-1 interaction after short-term treatment. In addition, the Dhhc7 and Dhhc21 palmitoylases were negatively regulated after sustained stimulation of E2 for 3 h. Although the uptake of BrdU into the nucleus in normal pituitary cells was not modified by E2, a significant increase in the GH3 tumoural cell, as well as ERK1/2 activation, with this effect being mimicked by PPT, a selective antagonist of ERα. These proliferative effects were blocked by ICI 182780 and the global inhibitor of palmitoylation. These findings indicate that ERα palmitoylation modulated the mERα pool and consequently the ERK1/2 pathway, thereby contributing to pituitary tumour cell proliferation. These results suggest that the plasma membrane ERα pool might be related to the proliferative behaviour of prolactinoma and may be a marker of pituitary tumour growth.

Histopathological and Immunohistochemical Changes Induced by BisphenolA in Reproductive Tissues of Female Rat

Background: Bisphenol A (BPA) is an environmental chemical that has beenwidely used in the manufacture of polycarbonate plastics andepoxy resins for many years. Due to its major applications inthe production of plastic food or beverage containers andthe coating of food cans, people of different ages are inevitably exposed to BPA in daily life. It is a contaminant with increasing exposure to it and exerts both toxic and estrogenic effects on mammalian cells. Aim of the work: the present study was designated to evaluate the histopathological and immunohistochemical effect of BPA on the histoarchitecture of pituitary ,adrenal,ovarian and uterine axis of female albino rats and the ameliorative effect of antiestrogen drug and stem enhance Experimental model and methods: 20 female albino rats weighing 100 – 120 g. were kept under observation for about 15 days before the onset of the experiment for adaptation, then the rats were classified into 4 groups 5 rats for each , the first group was left without any treatment for 30 days as negative control group , the second group was administered with 20 mg/kg.bw of BPA for 15 consecutive days as positive control, the third group administered with 20 mg/kg.bw of BPA for 15 consecutive days and then treated withantiestrogen drug as 0.1 mg/100gm.bw for 15 day, the fourth group administered with the same dose for the same period and the treated with stem enhance (4.5 mg/100.bw) for 15 days.All rats are scarified and organs were histologically examined after processing Results:the results showed thatPA has a histopathological effects on vital organs (pituitary, adrenal,ovary,oviduct and uterus) even for a short period with minimal ameliorative effect of antiestrogen drug and stem enhance.

Antiapoptotic factor humanin is expressed in normal and tumoral pituitary cells and protects them from TNF-α-induced apoptosis.

Humanin (HN) is a 24-amino acid peptide with cytoprotective action in several cell types such as neurons and testicular germ cells. Rattin (HNr), a homologous peptide of HN expressed in several adult rat tissues, also has antiapoptotic action. In the present work, we demonstrated by immunocytochemical analysis and flow cytometry the expression of HNr in the anterior pituitary of female and male adult rats as well as in pituitary tumor GH3 cells. HNr was localized in lactotropes and somatotropes. The expression of HNr was lower in females than in males, and was inhibited by estrogens in pituitary cells from both ovariectomized female and orquidectomized male rats. However, the expression of HNr in pituitary tumor cells was not regulated by estrogens. We also evaluated HN action on the proapoptotic effect of TNF-α in anterior pituitary cells assessed by the TUNEL method. HN (5 µM) per se did not modify basal apoptosis of anterior pituitary cells but completely blocked the proapoptotic effect of TNF-α in total anterior pituitary cells, lactotropes and somatotropes from both female and male rats. Also, HN inhibited the apoptotic effect of TNF-α on pituitary tumor cells. In summary, our results demonstrate that HNr is present in the anterior pituitary gland, its expression showing sexual dimorphism, which suggests that gonadal steroids may be involved in the regulation of HNr expression in this gland. Antiapoptotic action of HN in anterior pituitary cells suggests that this peptide could be involved in the homeostasis of this gland. HNr is present and functional in GH3 cells, but it lacks regulation by estrogens, suggesting that HN could participate in the pathogenesis of pituitary tumors.

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic Gonadotropin

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic GonadotropinEstradiol and human chorionic gonadotropin (hCG) are very important in controlling the secretory activity of hormone producing cells in the female rat pituitary glands. The aim of the present study was to examine the morphometric parameters of immunohistochemically labeled ACTH cells in juvenile (16th day) female rat pituitaries after treatment with five doses of estradiol dipropionate (EDP) and two doses of hCG during the neonatal period of life. The controls were treated on the same schedule with an equivalent volume of vehicle. All animals were sacrificed 24 h after the last treatment. ACTH-producing cells were studied using the peroxidase-antiperoxidase immunohistochemical procedure. The absolute and relative pituitary weights were increased (p<0.05) only in the EDP treated group by 120.0% and by 121.1% respectively, in comparison with the controls. In this group, the volume of ACTH cells, volume of their nuclei and volume density were significantly decreased (p<0.05) by 6.4%, 33.3% and 46.2% respectively, compared to the corresponding controls. After treatment with hCG, there were no significant (p>0.05) changes neither in the volume of ACTH cells nor in the volume of their nuclei, in comparison with the controls. On the basis of the results obtained in our study, it can be concluded that EDP, injected into female rats during the neonatal period of life, has an inhibitory effect on the immunohistomorphometric parameters of ACTH cells, but such an effect is not clearly expressed after treatment with hCG.

Effects of genistein on gonadotropic cells in immature female rats

The effects of genistein on pituitary gonadotropic cells of immature female rats were examined and compared to actions of the synthetic estrogen, 17α-ethynylestradiol. Immature female rats received 50mg/kg/bw of genistein in dimethylsulfoxide (DMSO) subcutaneously (s.c.) daily for 3 days at 18, 19 and 20 days of age. A second group was injected with 1μg/kg of 17α-ethynylestradiol in olive oil in the same schedule. The genistein control group received DMSO only, while 17α-ethynylestradiol controls were given sterile olive oil only. Changes in cell number per mm2, cell volume and volume density of follicle-stimulating (FSH) and luteinizing (LH) immunolabeled cells were evaluated by morphometry and stereology. Genistein induced significant increases in the number of FSH cells (by 21%) and LH cells (by 20%) per mm2 compared to corresponding controls. Volumes of FSH and LH cells were significantly increased by 19.7% and 20% and their volume densities by 20% and 20.2%, respectively. Estradiol markedly affected gonadotropes in the same manner, but to a greater extent. It can be concluded that genistein acted as an estrogenic agonist in the pituitaries of immature female rats, and as such, stimulated gonadotropic cells.

Iodine concentration and signs of apoptosis in the thyroid and pituitary of female rats after different single doses of potassium iodide

The aim of this work was to study the content of iodine as well as the expression of caspase 8 and caspase 32 in the thyroid and anterior pituitary in rats after a single dose of iodide. A total of 49 inbred rat females weighing 250-300 g at the stage of diestrus and/or metestrus were used. Pituitaries and thyroids were dissected from 15 control rats and from the groups of 8 rats each given potassium iodide by gavage in doses of 1, 4, 8 and 25 μg/100 g at 48 h before sacrifice. In two rats of each group the level of iodine in thyroids and pituitaries was estimated in terms of weight percent of iodide in dry tissue (wt % I-2/dry tissue) using the wavelength dispersive spectrometry (WDS) quantitative analysis. The expression of caspase 8 and caspase 32 in thyroids and pituitaries in terms of the percentage of positive immunostained area (% PA) was measured by streptavidin-biotin method using specific polyclonal antibodies. RESULTS; In the thyroids, iodine concentration increased after 1 μg/100 g, but decreased after 8 and 25 μg/100 g, while that in the pituitaries significantly increased after all doses of iodide with the peak after 8 mg/100 g. After the same iodide dose also the peak of caspase 32 and caspase 8 appeared in the pituitary. However, in the thyroid only increased caspase 32 was found together with a decrease of iodine concentration. Several interrelations between iodine in the thyroid and pituitary were found. In addition, the signs of apoptosis appeared directly related to the concentration of iodine in the pituitary, but inversely related to iodine concentration in the thyroid.

17β-oestradiol acts as a negative modulator of insulin-induced lactotroph cell proliferation through oestrogen receptor alpha, via nitric oxide/guanylyl cyclase/cGMP

17β-oestradiol interacts with growth factors to modulate lactotroph cell population. However, contribution of isoforms of the oestrogen receptor in these activities is not fully understood. In the present study, we have established participation of α and β oestrogen receptors in effects of 17β-oestradiol on lactotroph proliferation induced by insulin and shown involvement of the NO/sGC/cGMP pathway. Cell cultures were prepared from anterior pituitaries of female rats to evaluate lactotroph cell proliferation using bromodeoxyuridine (BrdUrd) detection, protein expression by western blotting and cGMP by enzyme immunoassay. In serum-free conditions, 17β-oestradiol and α and β oestrogen receptor agonists (PPT and DPN) failed to increase numbers of lactotroph cells undergoing mitosis. Co-incubation of 17β-oestradiol/insulin and PPT/insulin significantly decreased lactotroph mitogenic activity promoted by insulin alone. Both ICI 182780 and NOS inhibitors (L-NMMA and L-NAME) induced reversal of the anti-proliferative effect promoted by 17β-oestradiol/insulin and PPT/insulin. Moreover, 17β-oestradiol, PPT and insulin increased sGC α1 protein expression and inhibited β1, whereas co-incubation of 17β-oestradiol/insulin or PPT/insulin induced increases of the two isoforms α1 and β1. 17β-oestradiol and insulin reduced cGMP production, while 17β-oestradiol/insulin co-incubation increased this cyclic nucleotide. Our results suggest that 17β-oestradiol is capable of arresting lactotroph proliferation induced by insulin through ER α with participation of the signalling NO/sGC/cGMP pathway.

Sex differences in adrenocortical structure and function. XXV. Quantitative analysis of ACTH-immunoreactive cells in the anterior pituitary of gonadectomized and gonadal hormone replaced male and female rats.

Coupled immunocytochemical and stereologic studies of ACTH-immunoreactive cells were performed on anterior pituitaries of control male and female rats and on gonadectomized and testosterone or estradiol replaced animals. The average area and average volume of studied cells as well as their number were similar in anterior pituitary glands of control male and female rats. Orchiectomy increased average area of ACTH-immunoreactive cells, an effect reversed by testosterone while neither ovariectomy nor estradiol replacement had an effect on the area of studied cells. Average volume of these cells was similar in all experimental groups studied. Orchiectomy lowered the total number of ACTH-immunoreactive cells in the gland, an effect reversed by testosterone. Also ovariectomy lowered their number while after estradiol they were more numerous, even in comparison with control females. There is no significant correlation between the size and total number of ACTH-immunoreactive cells of the rat anterior pituitary gland and the size of the adrenal gland in both, male and female control, gonadectomized and testosterone or estradiol replaced rats.

Prolactin Released in vitro from the Pituitary of Lactating, Pregnant, and Steroid-Treated Female or Male Rats Stimulates Prolactin Secretion from Pituitary Lactotropes of Male Rats

We have previously shown that soluble factor(s) in conditioned media (CM) from the central and peripheral regions of the anterior pituitary (AP) gland of lactating rats promoted the in vitro dose-related release of prolactin (PRL) from pituitary glands of male rats. In the present experiments we sought to determine whether CM from rats in different physiological states provoked similar effects (like those of lactating rats), and the nature of the factors, whether 23K PRL or other variants of the hormone, were responsible for these effects. Stimulatory effects were induced by CM from pregnant females and steroid-treated castrated males or females, but not from untreated castrated rats, intact males, or by a PRL standard. More potent effects occurred with CM from APs of early- than from mid- or late-lactating rats, and from rats unsuckled for 8 or 16 h than from those unsuckled for 32 h. With respect to the nature of factor(s) responsible for these effects, immunoprecipitation of PRL from the CM of lactating females and of steroid-treated, castrated males eliminated, whereas dephosphorylation or deglycosylation of CM of lactating rats greatly increased its effects upon PRL release. Also, electrophoretic analysis and Western blotting of the CM proteins under native and denaturing conditions revealed a variety of PRL variants, ranging from 14 to <90 kDa, in CM from lactating rats, and the main effects on PRL release were provoked by the 23- to 46-kDa PRL variants. These results indicate that specific effects upon male rat lactotropes may be exerted by PRL variants released from APs of lactating and non-lactating rats.

The Effect of Castration and Androgen Replacement on Rat Pituitary Follicle Stimulating Hormone Investigated by Reversed-Phase High Performance Liquid Chromatography

Rat follicle stimulating hormone (rFSH) was analysed in extracts of rat anterior pituitary using a newly developed separation method of reversed-phase high performance liquid chromatography (RP-HPLC) and post-column detection of immunoreactivity (rFSH-IR) by radioimmunoassay (RIA) or immunoprecipitation with a specific anti-rFSH antibody. Good separation of rFSH was achieved on a Waters Radial-Peak 8MBC18 cartridge with a linear (60 min) gradient from 10-60% acetonitrile at constant 0.1% trifluoroacetic acid concentration. The elution profiles of radioiodinated purified rFSH [(125I)rFSH] as well as extracts of adult male and female rat pituitaries contained two main immunoreactive components (peaks 1 and 2) which eluted at 16-17 min and 21 min respectively. In the (125I)rFSH elution profile, the peaks of immunobinding corresponded to two major peaks of radioactivity. The male pituitary showed greater binding to anti-rFSH-serum of peak 1 and 2 eluate compared to female pituitary. At day 20 after orchidectomy, the amount of immunoreactive rFSH (rFSH-IR) increased in both peaks (by 30% in peak 1 and 180% in peak 2); however, the rFSH-IR level in peak 2 appeared to be more variable compared with that in peak 1. Subcutaneous implantation of 5 mg or 15 mg of dihydrotestosterone (DHT) at the time of orchidectomy prevented the post-castration elevation of rFSH-IR at 10 and 20 days after surgery. The precise nature and physiological significance of the two elution peaks are not clear at this time; however, only peak 2 showed immunoreactivity with anti-rLH-alpha-subunit antibody (AFP 87713681) and the elution time of peak 2 coincided with that of (125I) rLH-alpha subunit (AFP-7264 B).(ABSTRACT TRUNCATED AT 250 WORDS)

KiSS‐1 and GPR54 Genes are Co‐Expressed in Rat Gonadotrophs and Differentially Regulated In Vivo by Oestradiol and Gonadotrophin‐Releasing Hormone

Kisspeptin, the product derived from KiSS-1, and its cognate receptor, GPR54, both exert a role in the neuroendocrine control of reproduction by regulating gonadotrophin-releasing hormone (GnRH) secretion. In the present study, we demonstrate, using dual immunofluorescence with specific antibodies, that the KiSS-1 and GPR54 genes are both expressed in rat gonadotrophs. All luteinising hormone beta-immunoreactive (LH beta-ir) cells were stained by the KiSS-1 antibody but some kisspeptin-ir cells were not LH beta positive; thus, we cannot exclude the possibility that kisspeptins are expressed in other pituitary cells. All GPR54-ir are co-localised with LH beta cells, but only a subset of LH beta cells are stained with the GPR54 antibody. Using the real-time reverse transcription-polymerase chain reaction (RT-PCR), we found that the expression of KiSS-1 and GPR54 is differentially regulated by steroids. In the female, KiSS-1 mRNA levels dramatically decreased following ovariectomy (OVX), and this decrease was prevented by administration of 17beta-oestradiol (E(2)), but not by administration of GnRH antagonist or agonist. Administration of E(2) in OVX rats receiving either GnRH antagonist or agonist clearly shows that E(2) acts directly on the pituitary to positively control KiSS-1 expression. In OVX rats, administration of the selective oestrogen receptor (ER)alpha ligand propylpyrazoletriol, but not the selective ER beta ligand diarylpropionitrile, mimics this effect. By contrast, our study shows that GPR54 expression is positively regulated by GnRH and negatively controlled by chronic exposure to E(2). In summary, our data document for the first time that, in the female rat pituitary, KiSS-1 expression is up-regulated by oestradiol, similarly to that seen in the anteroventral periventricular nucleus of the hypothalamus. Conversely, GPR54 is up-regulated by GnRH, which exclusively targets gonadotrophs.

Transition of Leptin Receptor Expression during Pubertal Development in Female Rat Pituitary

Leptin is thought to play important roles in pubertal development and reproductive function in the female. Leptin receptor is expressed in various tissues including the pituitary and hypothalamus. We investigated the transition of leptin receptor (Ob-R) expression in female rat pituitary during pubertal development. Real-time RT-PCR was performed for long-form leptin receptor (Ob-Rb) and short-form leptin receptor (Ob-Ra) in the pituitary and cerebrum of the rats aged 4 (juvenile), 6 (pubertal), and 8 weeks (adult). Double immunohistochemical colocalization of Ob-R and luteinizing hormone (LH) was performed in pituitaries from 4-week-old female rats. Serum leptin concentrations of 4-, 6-, and 8-week-old rats were measured by radioimmunoassay. In the pituitary, expression of Ob-Rb mRNA in 4-week-old rats (1.00 +/- 0.16) was significantly higher than in 8-week-old rats (0.61 +/- 0.07, p<0.05), although expression of Ob-Ra mRNA did not differ among 4-, 6-, and 8-week-old rats. In cerebrum, Ob-Ra and Ob-Rb mRNA expressions did not differ significantly among 4-, 6-, and 8-week-old rats. Intense staining of Ob-R and colocalization of Ob-R and LH were seen in 4-week-old rat pituitary. On the other hand, serum concentrations of leptin in 6- and 8-week-old rats were significantly higher than those in 4-week-old rats (p<0.05, p<0.01, respectively). In conclusion, since the pituitary gonadotroph is a potential target of leptin, the juvenile rat pituitary might prepare for a subsequent increase of serum leptin concentration by expressing Ob-Rb.

Estradiol suppresses NMU mRNA expression during sexual maturation in the female rat pituitary

Neuromedin U (NMU) suppresses food intake and gonadotropin secretion. However, the developmental transition of NMU expression in the pituitary gland and the regulation of NMU expression are unclear. The objective of this study was to examine the transition of the expression of NMU mRNA in the pituitary glands of female rats from the juvenile period to the mature period of development. Furthermore, factors such as estradiol, insulin, leptin, and inhibin A, whose expressions change throughout puberty and which affect gonadotropin secretion in pituitary cell culture, were examined. In the pituitary gland, the expression of NMU mRNA was significantly lower in 8-week-old rats than in 4- and 6-week-old rats. In the pituitary cell culture, the expressions of NMU mRNA in the estradiol- and insulin-treated groups were significantly lower than in the control group. These results suggest that the expression of NMU mRNA in the female rat pituitary is reduced as the rats develop from the pubertal to the mature period. Additionally, the reduction of NMU expression in the pituitary may be related to the increases in serum estradiol and insulin levels that occur during the pubertal period, which may negate NMU suppression of gonadotropin secretion.

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.

Increased Caloric Intake on a Fat‐Rich Diet: Role of Ovarian Steroids and Galanin in the Medial Preoptic and Paraventricular Nuclei and Anterior Pituitary of Female Rats

Previous studies in male rats have demonstrated that the orexigenic peptide galanin (GAL), in neurones of the anterior parvocellular region of the paraventricular nucleus (aPVN) projecting to the median eminence (ME), is stimulated by consumption of a high-fat diet and may have a role in the hyperphagia induced by fat. In addition to confirming this relationship in female rats and distinguishing the aPVN-ME from other hypothalamic areas, the present study identified two additional extra-hypothalamic sites where GAL is stimulated by dietary fat in females but not males. These sites were the medial preoptic nucleus (MPN), located immediately rostral to the aPVN, and the anterior pituitary (AP). The involvement of ovarian steroids, oestradiol (E(2)) and progesterone (PROG), in this phenomenon was suggested by an observed increase in circulating levels of these hormones and GAL in MPN and AP with fat consumption and an attenuation of this effect on GAL in ovariectomised (OVX) rats. Furthermore, in the same four areas affected by dietary fat, levels of GAL mRNA and peptide immunoreactivity were stimulated by E(2) and further by PROG replacement in E(2)-primed OVX rats and were higher in females compared to males. Because both GAL and PROG stimulate feeding, their increase on a fat-rich diet may have functional consequences in females, possibly contributing to the increased caloric intake induced by dietary fat. This is supported by the findings that PROG administration in E(2)-primed OVX rats reverses the inhibitory effect of E(2) on total caloric intake while increasing voluntary fat ingestion, and that female rats with higher GAL exhibit increased preference for fat compared to males. Thus, ovarian steroids may function together with GAL in a neurocircuit, involving the MPN, aPVN, ME and AP, which coordinate feeding behaviour with reproductive function to promote consumption of a fat-rich diet at times of increased energy demand.

Direct Pituitary Effects of Kisspeptin: Activation of Gonadotrophs and Somatotrophs and Stimulation of Luteinising Hormone and Growth Hormone Secretion

Recent, compelling evidence indicates that kisspeptins, the products of KiSS-1 gene, and their receptor GPR54, represent key elements in the neuroendocrine control of reproduction, and that they act primarily by regulating gonadotrophin-releasing hormone (GnRH) secretion at the hypothalamus. Conversely, and despite earlier reports showing GPR54 expression in the pituitary, the potential physiological roles of kisspeptins at this gland have remained elusive. To clarify this issue, cultures of rat pituitary cells were used to evaluate expression of KiSS-1 and GPR54, and to monitor the ability of kisspeptin-10 to stimulate Ca(2+) responses in gonadotrophs and to elicit luteinising hormone (LH) secretion in vitro. The results obtained show that both GPR54 and KiSS-1 are expressed in the pituitary of peripubertal male and female rats. Moreover, kisspeptin-10 induced a rise in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) in approximately 10% of male rat pituitary cells. Intriguingly, kisspeptin-responsive cells included not only gonadotrophs, in which a 62.8 +/- 16.0%[Ca(2+)](i) rise was observed, but also somatotrophs, wherein kisspeptin induced a 60.3 +/- 5.5%[Ca(2+)](i) increase. Accordingly, challenge of dispersed pituitary cells with increasing kisspeptin-10 concentrations induced dose-related LH and growth hormone (GH) secretory responses, which were nevertheless of lower magnitude than those evoked by the primary regulators GnRH and GH-releasing hormone, respectively. In particular, 10(-8) M kisspeptin caused maximal increases in LH release (218.7 +/- 23.6% and 180.4 +/- 7.2% in male and female rat pituitary cells, respectively), and also stimulated maximally GH secretion (181.9 +/- 14.9% and 260.2 +/- 15.9% in male and female rat pituitary cells, respectively). Additionally, moderate summation of kisspeptin- and GnRH-induced LH responses was observed after short-term incubation of male rat pituitary cells. In conclusion, our results provide unequivocal evidence that kisspeptins exert direct pituitary effects in peripubertal male and female rats and suggest a possible autocrine/paracrine mode of action. The precise relevance and underlying mechanisms of this potential new actions of kisspeptins (i.e. the direct modulation of gonadotrophic and somatotrophic axis at the pituitary) deserve further analysis.

Aromatase Gene Expression and Regulation in the Female Rat Pituitary

Aromatase cytochrome P450, the key enzyme of estrogen biosynthesis from androgens, is encoded by CYP19. Its structure shows some peculiarities: exons II to X encode the protein, while multiple alternative exons I encode unique 5'-untranslated regions of the aromatase mRNA transcripts. Immunohistochemistry studies in the rat have shown that pituitary aromatase expression is sex-dependent and varies across the estrous cycle, suggesting that estrogens might be involved in the regulation of aromatase activity and might act locally as a paracrine or autocrine factor in the pituitary. In the present study, we used RT-PCR to characterize aromatase transcripts and real-time PCR to quantify the expression of the total aromatase mRNA at the different stages of the estrous cycle and from an ovariectomy and estradiol replacement model. We identified the two previously described aromatase transcripts with a specific 5'untranslated region of the brain 1f and the gonadal PII transcripts. Total aromatase mRNA expression in the pituitary varied significantly during the estrous cycle, with the highest level occurring on the day of metestrus. After ovariectomy, we observed an increase of aromatase mRNA levels, and this effect was completely prevented by estradiol administration. These results suggest that pituitary aromatase mRNA expression is downregulated by estrogens.

Cloning, Expression and Regulation of Chicken Ovalbumin Upstream Promoter Transcription Factors (COUP-TFII and EAR-2) in the Rat Anterior Pituitary Gland

Chicken ovalbumin upstream promoter transcription factors (COUP-TF)-II (NR2F2) and EAR-2 (NR2F6) are structurally related orphan members of the nuclear receptors superfamily. There are growing evidences that these factors play important roles during processes of differentiation and proliferation of several tissues. To better understand their role in the differentiated adult rat pituitary gland, we cloned COUP-TFII and EAR-2 cDNAs from an anterior pituitary cDNA library. Subsequently, we raised and characterized specific antibodies to the N-terminal domain of both nuclear receptors. We next examined their cellular and subcellular distribution in the pituitary gland and determined their regulation during pregnancy. COUP-TFII and EAR-2 pituitary genes display, respectively, 90 and 100% homologies with their human and mouse homologues. Cellular expression of both nuclear receptors was mainly detected in the lactotropes of male and female rats, with a prominent distribution in the nuclear compartment for EAR-2, and interestingly both proteins were significantly upregulated in pituitaries of pregnant vs. cycling female rats. Thus, our results have characterized cloning of rat pituitary COUP-TFII and EAR-2 genes, demonstrated that they are both specifically expressed in lactotropes, and strongly suggested that they may play an important role in modulating prolactin (PRL) gene expression during pregnancy.

Adenohypophyseal and hypothalamic GABA B receptor subunits are downregulated by estradiol in adult female rats

Gamma-aminobutyric acid (GABA) participates in neuroendocrine regulation. Since steroid hormones have been shown to modulate the GABAergic system, here we evaluated the effect of chronic in vivo estradiol administration on GABA B receptor (GABA BR) expression. GABA B1 and GABA B2 subunits were analyzed by Western Blot and RT-PCR, in hypothalami and anterior pituitaries of adult female rats: a) treated for 1 week with estradiol–valerate (a single dose of 100 μg /kg: E1), b) implanted with a 10 mg pellet of estradiol–benzoate for 5 weeks (E5) or c) on proestrous (P), d) ovariectomized (OVX). Pituitary GABA BR levels were correlated to a biological effect: baclofen, a GABA BR agonist, action on intracellular calcium titers ([Ca 2+] i) in pituitary cells. E5 pituitaries showed a significant decrease in the expression of GABA B1 and GABA B2 mRNAs compared to P. The GABA B1a splice variant of GABA B1 was always more abundant than GABA B1b in this tissue. Similar to the pituitary, hypothalamic GABA B1 and GABA B2 mRNAs decreased in E5; this was confirmed at the protein level. In the hypothalamus GABA B1b was the main variant expressed in P rats, and was the one significantly sensitive to estradiol-induced decrease, as determined by Western Blots. Castration did not modify GABA BR expression with regards to P in either tissue. In P pituitary cells baclofen induced a decrease in [Ca 2+] i, in contrast this effect was lost in E5 cells. We conclude that chronic estradiol treatment negatively regulates the expression of the GABA BR subunits in the pituitary and the hypothalamus. This effect is coupled to a loss of baclofen action on intracellular calcium in pituitary cells.