bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Early Genes in Estrogen Induced Phenotypic Conversion of Neuroblastoma Cells
Authors: Santagati Sabrina , Ma Zhi Qing , Ferrarini Concetta , Pollio Giuseppe , Maggi Adriana
Published: September 2006

Estrogens are known to modulate the growth rate and differentiation state of a number of cells. In uterine, as well as in mammary tumor cells, estrogen‐dependent proliferation and differentiation are correlated to a series of biochemical responses, including increased expression of proto‐oncogenes such as:, c‐fos, c‐jun and c‐myc. Since estrogens were shown to regulate the proliferation and the differentiation state of cells of nervous origin, the aim of the present study was to investigate whether these effects were associated to changes in the expression of early genes. In the model system utilized, the human cell line SK‐ER3, an increase in c‐fos mRNA and Fos protein without change of c‐jun and related genes mRNA concentration was observed after short term treatment with 17β‐estradiol (E2). A significant decrease of c‐fos, c‐jun and jun‐D proto‐oncogene mRNA levels were found after prolonged hormonal treatment. The exposure to the hormone did not determine any change in N‐myc expression. Since the three protooncogene mRNAs are rapidly induced following estrogen treatment in other cell systems and target tissues, it is concluded that the estrogen‐induced differentiation of neuroblastoma cells is correlated to a pattern of expression of early genes that might be peculiar for the activity of this hormone in neural cells.
Gonadotropin‐Releasing Hormone (GnRH) Neurons of the Developing Tectum of the Mouse
Authors: Wu T. J. , Gibson M. J. , Silverman A. J.
Published: September 2006

Using a modified immunocytochemistry protocol with enhanced sensitivity, we were able to visualize a population of neurons in the tectum of the developing mouse that contained GnRH‐like immunoreactivity. In microscopic studies conducted using 100 pm sections cut in sagittal or horizontal planes, 10–20 lightly‐stained neurons were first detected in the tectum at E 13.75 (morning of plug = E0.5). The number of immunostained cells increased exponentially reaching a peak at E 15.75 before decreasing in number. No positive neurons were seen in the tectum at PN20 or later. The GnRH cells were located medially along the dorsoventral axis of the tectum in a region of the brain distinct from that containing GnRH neurons that migrate into the CNS from the olfactory placode. To determine the nature of the immunoreactivity, two approaches were used. Analysis of tissue from an hpg mutant strongly supports the hypothesis that these cells make mammalian GnRH. lmmunocytochemical data suggest that although the precursor protein is synthesized, the cleaved and amidated decapeptide may be absent or be present at an undetectable level. Our results demonstrate that in addition to the GnRH neurons from the placade, a population of GnRH neurons exists in the mouse tectum. This population is developmentally regulated, appearing only during embryonic and early postnatal ages but not in the adult.
Regulation of Pituitary Vasopressin V1b Receptor mRNA during Stress in the Rat
Authors: Rabadan‐Diehl Cristina , Lolait Stephen J. , Aguilera Greti
Published: September 2006

Previous studies have shown a parallel relationship between pituitary vasopressin (VP) receptor content and responsiveness of the corticotroph during chronic stress. The regulation of pituitary VP receptors was further studied by analysis of V1b VP receptor mRNA levels in pituitaries of rats subjected to chronic immobilization, i.p. hypertonic saline injection (physical stress paradigms associated with increased pituitary responsiveness), and water deprivation, or to 2% saline in the drinking water (osmotic stress paradigms associated with decreased pituitary responsiveness). Northern blot hybridization with a 363 bp 32P‐labelled fragment of the rV1b receptor cDNA coding sequence revealed two bands of about 3.7 and 3.2 Kb, whereas a probe directed to the 5′ untranslated region recognized only the 3.7 Kb band. Repeated i.p. hypertonic saline injection, 3 times in 24 h at 8 h intervals, or daily for 8 days, increased the intensity of the 3.7 Kb band by 155 ± 17.5% (P<0.01) and 118 ± 14.6% (P<0.01), respectively, while the 3.2Kb band increased by 122 ± 39.3% (P<0.01) only after 3 times injection. Smaller increases of 39 ± 11 and 33 ± 9% (P<0.05) in the 3.7 Kb band were found after repeated immobilization 3 times in 24 h and 2 h for for 8 days respectively. In situ hybridization studies confirmed significant increases (P<0.05) in V1b receptor mRNA levels after 8 and 14 days repeated immobilization (63 ± 19% and 83 ± 10%) or i.p. hypertonic saline injection (110 ± 13% and 73 ± 20%). In response to acute stress, V1b receptor mRNA increased by 77 ± 5% (3.7 Kb band) after 4 h immobilization for 1 h, whereas both bands were reduced by 49 ± 5% and 45 ± 5%, 4 h after a single i.p. hypertonic saline injection. The decrease in V1b receptor mRNA following a single i.p. hypertonic saline injection was prevented by pretreatment with a V1 receptor antagonist, suggesting that increased VP secretion may account for this effect. In spite of the decrease in V1 b receptor mRNA following i.p. hypertonic saline injection, VP binding in pituitary membrane rich fractions, and VP‐stimulated inositol phosphate formation in quartered hemipituitaries were increased by 24 and 39%, respectively. V1b receptor mRNA levels were unchanged or decreased following prolonged osmotic stimulation.

These studies suggest that increased V1b receptor mRNA levels contribute to the VP receptor upregulation observed during repeated immobilization and i.p. hypertonic saline injection, whereas the lack of parallelism between V1b receptor mRNA and VP binding indicates that regulation of steady‐state levels of V1b receptor mRNA is not a primary determinant in the control of pituitary VP receptor concentration during stress.
ACTH‐Suppressive and Vasodilator Actions of Adrenomedullin in Conscious Sheep
Authors: Parkes David G. , May Clive N.
Published: September 2006

Adrenomedullin (ADM) is a 52 amino‐acid peptide which is a potent vasodilator in rats, and suppresses basal and CRF‐induced ACTH release from cultured pituitary cells. The present study examines the hemodynamic and hormonal actions of human ADM (1–52) infusion in conscious, chronically instrumented sheep. Five sheep were infused intravenously (IV) or intracerebroventricularly (ICV) with ADM at 100 pg/h for 60 min, and mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral conductance (TPC), coronary blood flow (CF), coronary conductance (CC), peak aortic flow (Fmax), and left ventricular dF/dt were monitored by a computer‐based data collection system every 2 min. Plasma concentrations of adrenocorticotropin (ACTH), arginine vasopressin (AVP) and renin were measured after 60 min of infusion. IV ADM produced a small fall in MAP of 3 ± 1 mmHg, associated with a reflex increase in HR of 14 ± 3 b/min. CO increased by 1.3 ± 0.3 1/min, whereas SV remained unchanged. TPC was markedly increased by 20 ± 3 ml/min/mmHg. Changes in CF were also seen with an increase of 10 ± 2ml/min, and CC increased in parallel by 0.15 ± 0.02 ml/min/mmHg. Fmax and dF/dt showed small increases of 2.1 ± 0.5 Vmin and 85 ± 20 1/min/sec respectively. Plasma concentrations of ACTH and cortisol were reduced by 58% and 55% respectively, whereas plasma renin concentration increased by 106%. There was no change in plasma levels of AVP. ICV infusion of ADM had no effect on any parameter measured. These data suggest that systemic ADM produces a sustained vasodilator action to lower blood pressure in sheep, and this is the first study to report the ACTH‐suppressor action of ADM in conscious animals. ADM may therefore be an important hormone involved in the regulation of pituitary/adrenal function, in addition to its cardiovascular and fluid regulatory actions in mammals.
Localization of [125I]IGF‐I Binding on the Ovine Pars Tuberalis
Authors: Williams Lynda M. , Kelly Denise , Hannah Lisa‐T. , Morgan Peter J.
Published: September 2006

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin‐stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF‐I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF‐I may influence the function of the ovine pars tuberalis the presence of receptors for IGF‐I has been investigated. Using in vitro autoradiography specific [125I]IGF‐I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [125I]IGF‐I with a mean dissociation constant (Kd) of 0.5 ± 0.1 nM (n=4). Competition studies revealed a rank order of potency of IGF‐I>IGF‐II> > >insulin, in displacing [125I]IGF‐I binding, indicative of a mixed population of IGF‐I and IGF‐II/rnannose‐6‐phosphate receptors and insulin‐like growth factor binding proteins (IGFBPs). Cross‐linking of [125I]IGF‐I to pars tuberalis membrane homogenates and analysis by SDS‐PAGE under reducing conditions confirmed the presence of both IGF‐I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the β‐sub‐unit of the IGF‐I receptor, was increased in the presence of IGF‐I, indicating the existence of functional IGF‐I receptors on the ovine pars tuberalis. In contrast in the rat [125I]IGF‐I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.
The Effects of Centrally Administered Porcine Relaxin on Drinking Behaviour in Male and Female Rats
Authors: Thornton Simon N. , Fitzsimons James T.
Published: September 2006

Of the reproductive hormones it has been suggested that relaxin may play an important role in the increased sodium appetite of pregnancy. ICV injection of porcine relaxin caused water‐replete male and female Wistar rats with access to water and 0.9% or 2.7% NaCl to drink on average about 3 to 8 ml of water within 1 h of injection. By 24 h the cumulative intake of water was no different from the control intake. The amounts of water drunk were similar after doses of 50, 100, 250 and 500 ng of relaxin. A dose of 5 ng was ineffective. Male rats generally drank more water than female rats after ICV injection of angiotensin or relaxin. Male SH rats which drink more water than male WKY rats in response to ICV angiotensin also drank more after ICV relaxin. Intakes of 0.9% or 2.7% NaCl were unaffected for up to 24 h after injection of relaxin, whereas angiotensin‐injected rats showed a significant increase in 0.9% NaCl 1 h after injection though this difference was no longer evident in the 24 h cumulative intake. Relaxin did not cause any increase in NaCl intake in SH rats. Insulin, which is similar in structure and molecular weight to relaxin, was without effect on drinking when doses comparable to dipsogenically effective doses of relaxin were injected ICV. In male Wistar rats treated with DOCA for 5–15 days, relaxin retained its weak stimulatory action on water intake but did not affect NaCl intake despite the increased baseline NaCl intake during DOCA. These results indicate that relaxin is a dipsogen in the rat but that it seems to have little short‐term effect on sodium appetite.
The Ovine Pars Tuberalis does not Appear to be Targeted by Melatonin to Modulate Luteinizing Hormone Secretion, but may be Important for Prolactin Release
Authors: Malpaux Benoît , Skinner Donal C. , Maurice Françoise
Published: September 2006

The pineal hormone, melatonin, transduces photoperiodic information to the neuroendocrine axis of seasonally breeding mammals to regulate reproduction. It is not known where or how melatonin achieves this effect, but the recent identification of the pars tuberalis (PT) as the area with the highest density of melatonin binding sites suggests that this pituitary subdivision may be an important target for the actions of this indoleamine on luteinizing hormone (LH) and prolactin release. The present study was designed to test this hypothesis. Ovariectomized oestradiol‐implanted ewes were exposed to inhibitory long days for 85 days and then received melatonin micro‐implants (Day 0) in the mediobasal hypothalamus (MBH; n = 7) or PT (Melatonin‐PT; n = 5). The effect of these micro‐implants was compared to ewes receiving empty micro‐implants in the PT (Sham‐PT; n = 5). For LH, bi‐weekly jugular blood samples were collected and for prolactin, samples were collected every 20 min for 5 h, with the first hour discarded, on Days ‐4, 26 and 69. Melatonin implanted in the MBH stimulated LH secretion in 3 ewes by Day 46±0 after implantation, and one ewe by Day 67 after implantation. In contrast, no Melatonin‐PT or Sham‐PT ewes exhibited an increase in LH secretion by the end of the study (Day 70). A subsequent experiment, in which the Sham‐PT ewes were implanted with melatonin both subcutaneously and in the PT showed that the micro‐implants did not impair the ability of the ovine reproductive neuroendocrine axis to respond to melatonin. For prolactin, both the Melatonin‐PT ewes and MBH ewes (which had displayed an increase in LH secretion) exhibited a significant decrease in prolactin secretion by Day 26 and this decrease persisted until the end of the study. This result suggests that melatonin may have two sites of action on prolactin secretion; the MBH and PT, or, if melatonin diffused from the MBH to the PT, or vice versa, that only one of these sites is important. In contrast, these data suggest that the PT is not an important target for the action of melatonin on LH secretion.
Effects of Monoclonal Antibodies to Specific Epitopes of Rat Interleukin‐1 Beta (IL‐Iβ) on IL‐1β‐Induced ACTH, Corticosterone and IL‐6 Responses in Rats
Authors: Schotanus Karel , Meloen Rob H. , Puijk Wouter C. , Berkenbosch Frank , Binnekade Rob , Tilders Fred J. H.
Published: September 2006

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL‐1β and found that MoAbs binding to the aminoacid sequences 66–85 and 123–143 of mature rIL‐1β inhibited the binding of rIL‐1β to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL‐1 interfere with the biological effects of rIL‐1β in adult male rats in vivo. Administration of rIL‐1β (1 or 5 μg/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL‐6 in a time‐ (0.5–4 h) and dose‐dependent manner. Because 2 h after 5μg/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL‐1β was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37°C or at 4°C. Plasma ACTH, CORT and IL‐6 responses to these mixtures are compared to those obtained after preincubation of rIL‐1β with a non‐IL‐1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66–85 domain of rIL‐1β, reduced the ACTH and IL‐6 responses by 48 and 45% respectively. In contrast, a MoAb to the 123–143 domain (SILK 5) and SILK 16, which binds to the 106–124 domain did not affect any of the IL‐1 induced responses, whereas a MoAb directed to domain 78–97 of rIL‐1β (SILK 20) enhanced the ACTH and CORT responses by 51 and 41% respectively, but not the IL‐6 response.

These observations lead us to conclude that the domain 66–85, harbours sequences that are important for receptor binding and for the biological actions of rIL‐1β in the rat and that this biologically active domain is located at the ‘closed side’ rather than at the ‘open side’ of the IL‐1β molecule where domains involved in receptor binding and biological activity are considered to be located.
Neuropeptide Y (NPY) May Integrate Responses of Hypothalamic Feeding Systems and the Hypothalamo‐Pituitary‐Adrenal Axis
Authors: Hanson E. Simon , Dallman Mary F.
Published: September 2006

Neuropeptide Y (NPY) is a powerful stimulus to food intake in the rat. Exogenous NPY given into the third ventricle or into the paraventricular nucleus (PVN) of the hypothalamus stimulates both food consumption as well as the hypothalamus‐pituitary‐adrenal (HPA) axis. Presumably NPY activates the adrenocortical system through direct stimulation of CRF containing cells in the PVN. Food intake is also a major regulator of adrenocortical activation. Rhythms in HPA axis activity follow rhythms in food consumption, and rats that have been food deprived overnight have inhibited HPA axis responses to restraint stress and corticosteroid feedback the following morning. To investigate the interaction of NPY with both feeding and HPA axis activation three sets of experiments were performed: Animals fed ad lib were injected icv with NPY (2.5 μg) and allowed access to food or not post injection; animals were fasted overnight prior to NPY injection; finally, dose response experiments were performed to examine the relative sensitivities of feeding and HPA axis activation to exogenous NPY. Ad lib fed animals allowed access to food after NPY injection had slightly greater ACTH responses to NPY while glucocorticoid and insulin responses were not significantly different from ad lib fed animals not allowed access to food post injection. Animals allowed to eat post injection had significantly decreased food consumption the night following injection, however, total 24 h food consumption was not different between these animals and those given food 8 h post NPY injection. In overnight fasted animals NPY injections produced ACTH responses of equal magnitude to those in ad lib fed animals. Insulin responses to NPY were significantly elevated compared to CSF controls in overnight fasted animals. Dose response studies revealed that the adrenocortical system responds to icv NPY with at least as great sensitivity as feeding systems. NPY is discussed as a potential integrator of feeding and responsiveness in the HPA axis.
Evidence that Hypothalamic Neuropeptide Y Gene Expression Increases Before the Onset of the Preovulatory LH Surge*
Authors: Sahu Abhiram , Crowley William R. , Kalra Satya P.
Published: September 2006

Neuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid‐primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h. This secondary activation of NPY gene expression occurred with a slow, two‐fold increase in serum LH at 1500 h, followed by a rapid ascension to peak levels at 1800 h and was associated with an increase at 1400 h of serum progesterone levels which reached their peak at 1800 h. These results demonstrate that a dynamic, biphasic augmentation in hypothalamic NPY gene expression occurs selectively on proestrus, and that the first incremental response is observed some time before the onset of preovulatory LH hypersecretion. Because preproNPY mRNA levels at 1000 h on proestrus were similar to the low levels seen on the preceding diestrous II phase, a neural timing mechanism, and not changes in ovarian hormone levels during this phase may be responsible for the increase in NPY gene expression after 1000 h of proestrus. Because of our previous observations that progesterone can rapidly augment preproNPY mRNA in the MBH and because a rise in serum progesterone occurs hours before the onset of the LH surge, we suggest that the secondary rise in preproNPY mRNA is facilitated by this antecedent increase in serum progesterone. Cumulatively, these results are in accord with the thesis that activation of hypothalamic NPY gene expression is one of the key early neural events initiated by the neural clock that times the preovulatory LHRH and LH surges.
Neuropeptide‐E‐1 Antagonizes the Action of Melanin‐Concentrating Hormone on Stress‐Induced Release of Adrenocorticotropin in the Rat
Authors: Bluet‐Pajot Marie‐Thérèse , Presse Françoise , Voko Zoltan , Hoeger Carl , Mounier Françoise , Epelbaum Jacques , Nahon Jean‐Louis
Published: September 2006

The physiological role of melanin‐concentrating hormone (MCH) in mammals is still very elusive, but this peptide might participate in the central control of the hypothalamopituitary adrenal (HPA) axis during adaptation to stress. Cloning and sequencing of the rat MCH (rMCH) cDNA revealed the existence of additional peptides encoded into the MCH precursor. Among these peptides, neuropeptide (N) glutamic acid (E) isoleucine (I) arnide (NEI) is co‐processed and secreted with MCH in rat hypothalamus. In the present work we examined: (1) The pattern of rMCH mRNA expression during the light and dark conditions in the rat hypothalamus and (2) The effect of intracerebroventricular (ICV) injections of rMCH and NEI in the control of basal or ether stress‐modified release of corticotropin (ACTH), prolactin (PRL) and growth hormone (GH) secretion in vivo in light‐on and light‐off conditions. Our data indicate that rMCH mRNA levels do not change during the light‐on period, but increase after the onset of darkness. Either alone or co‐administered, rMCH and NEI do not modify basal secretion of GH and PRL at any time tested nor do they alter ether stress‐induced changes in these two hormonal secretions. At the end of the light on period corresponding to the peak of the circadian rhythm in ACTH, administration of rMCH but not NEI leads to a decrease in ACTH levels while MCH is not effective during the light off period of the cycle (i.e. when basal ACTH levels are already low). Using a moderate ether induced stress, ACTH levels are only stimulated during the dark phase of the cycle. rMCH (63 or 210 nmoles) prevents the rise in ACTH release while NEI alone does not modify the stress response. Co‐administration of both peptides before stress results in an abolition of the rMCH induced inhibition of ACTH plasma levels. Taken together, these data indicate that rMCH may act as a central corticotropin inhibitory factor involved in the circadian rhythmicity of plasma ACTH levels and that NEI antagonizes its action.
Hypothalamic Mediation of Reduced GH Secretion in Diabetic Rats: Evidence for Reduced Cholinergic Inhibition of Somatostatin Release
Authors: Ismail Ikram , Lewis Mary , Peters John R. , Scanlon Maurice F.
Published: September 2006

The Goto‐Kakizaki (GK) rat is a new model of diabetes mellitus and in this study we have characterized the diabetic and growth hormone (GH) secretory status of male GK rats at 6 and 16 weeks of age. We have also investigated the role of endogenous somatostatin (SS) and cholinergic manipulation on the GH responses to GH‐releasing hormone (GHRH). GK rats were non‐obese with significant fasting hyperglycaemia, hyperinsulinaemia and absent insulin responses to IV glucose. The GH response to GHRH was reduced at 16 weeks compared with normal, age‐matched Wistar rats but no differences were observed at 6 weeks. Pretreatment of older rats (16 weeks) with anti‐somatostatin antibodies (SS‐Ab) significantly increased GH responses to GHRH in both normal and GK groups. Cholinergic augmentation with pyridostigmine (PD) reversed the blunted GH responses to GHRH in older GK rats but had no effect in the normal or young (6 weeks) GK rats. These results indicate that SS release mediates the blunted GH response to GHRH in GK rats and that reduced hypothalamic cholinergic signalling to the somatostatinergic neurone may mediate the increase in SS release. This view is supported by the results from in vitro studies in which cholinergic muscarinic blockade with pirenzepine (PIR) caused dose‐related stimulation of SS release from normal rat hypothalami but was without effect on GK rat hypothalami. The cause of this alteration in hypothalamic function is, at present, unknown.
Circadian Rhythms: Peering into the Molecular Clockwork
Authors: Hastings Michael
Published: September 2006

Recent developments in our understanding of the molecular basis of circadian rhythms look set to provide one of the most elegant demonstrations of the relationship between the activity of individual genes and the execution of biologically relevant and complex patterns of behaviour. At the same time the lid is being opened on one of the classic ‘black boxes’ of biology, the circadian clock.
Effect of Lactation on Hypothalamic Preproenkephalin Gene Expression
Authors: Ottinger M. A. , Rosewell K. L. , Weiland N. G. , Margaretten K. T. , Wise P. M.
Published: September 2006

Enkephalin appears to modulate several aspects of reproductive function in female rats. The purpose of this study was to determine if lactation influences preproenkephalin gene expression in one or more hypothalamic nuclei known to be involved in maternal or reproductive behavior and prolactin secretion. Lactating rats were killed on day 3 (LAC 3) or day 10 (LAC 10) of lactation. Controls consisted of regular 4‐day cycling rats that were killed on diestrous day 1, with 9 to 12 females per group. We used in situ hybridization histochemistry to assess preproenkephalin gene expression in individual cells in the medial preoptic nucleus, anterior, medial and posterior arcuate nucleus, magnocellular and parvocellular aspects of paraventricular nucleus, and ventromedial nucleus. Preproenkephalin mRNA in the anterior arcuate nucleus increased to reach significance (P < 0.05) at day 10 of lactation. Levels in the medial arcuate nucleus increased significantly (P < 0.001) by day 3 of lactation (LAC 3) and remained elevated on day 10 (LAC 10). No significant differences between lactating and control rats were detected in preproenkephalin mRNA levels in the posterior arcuate nucleus, medial preoptic nucleus or in the ventromedial nucleus. Substantial levels of preproenkephalin mRNA were found in the paraventricular nucleus, particularly in a limited region of the magnocellular portion. However, these levels did not change with lactation. These data provide evidence for differential regulation of the preproenkephalin gene during lactation. This change may contribute to. lactational hyperprolactinemia and suppressed GnRH secretion, leading to reproductive acyclicity.
Melatonin Receptors Couple Through a Cholera Toxin‐Sensitive Mechanism to Inhibit Cyclic AMP in the Ovine Pituitary
Authors: Morgan Peter J. , Barrett Perry , Hazlerigg David , Milligan Graeme , Lawson Wilfred , MacLean Alison , Davidson Gary
Published: September 2006

The nature of melatonin receptor‐G‐protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins. ADP‐ribosylation of ovine PT membrane proteins using 32P‐NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins. Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX. Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for 125I‐MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%). A CTX‐sensitive component also mediates the inhibition of forskolin‐stimulated cyclic AMP accumulation as pre‐treatment of PT cells with CTX (5μg/ml) for 16 h blocked this response.

Gsα is a major substrate for ADP‐ribosylation by CTX, and 16 h pre‐treatment of PT cells with CTX (5μg/ml) caused a down‐regulation of Gsα. Northern analysis showed only one major transcript of Gsα of about 2 kb, which would encompass all of the known splice variants of the Gs gene. Screening of a cDNA library from ovine PT for Gs‐related genes and sequencing of clones, combined with RT‐PCR of PT mRNA, revealed no novel products. On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the Gs class of G‐protein. These results indicate that a distinct CTX‐sensitive mechanism mediates the inhibition of cyclic AMP by the melatonin receptor, which involves either a novel a‐sub‐unit of a heterotrimeric G‐protein or a protein which associates and functionally modulates the activity of the melatonin receptor/G‐protein complex.
Increased Fos Expression in Preoptic Calcitonin Gene‐Related Peptide (CGRP) Neurones following Mating but not the Luteinizing Hormone Surge in Female Rats
Authors: Herbison Allan E. , King Ian S. , Tan Keith K. C. , Dye Sandra
Published: September 2006

The functional relationship between sexually dimorphic neural populations and sex differences in reproductive functioning is unclear. The present study has investigated the function of the sexually dimorphic, estrogen‐receptive, calcitonin gene‐related peptide (CGRP) neurones in the female preoptic area by examining patterns of Fos immunoreactivity within these cells in relation to the luteinizing hormone surge and lordosis behaviour. In the first experiment, ovariectomized rats were treated with estradiol alone or estradiol plus progesterone to induce the luteinizing hormone surge. The percentage of CGRP neurones with Fos‐positive nuclei was not different in estradiol alone (18 ± 4%) and estradiol/progesterone‐treated (24 ± 3%) rats although the number of Fos‐immunoreactive cells in the medial preoptic nucleus was increased 2‐fold (P<0.01) in estrogen/progesterone‐treated rats and 40 ± 5% of luteinizing hormone‐releasing hormone neurones were found to express Fos in this group. In the second experiment, ovariectomized rats were treated with estradiol and progesterone and either, mated with a single male or placed in an empty cage, for 30 min. The number of Fos‐immunoreactive cells in the medial preoptic nucleus was increased 4‐fold in mated rats (P<0.01) and the percentage of CGRP neurones with Fos‐positive nuclei increased from 24 ± 3% to 38 ± 2% (P<0.01) in mated animals. No differences were detected in the number of luteinizing hormone‐releasing hormone neurones with Fos‐positive nuclei in mated and non‐mated animals.

These results suggest that a sub‐population of CGRP neurones in the medial preoptic nucleus may express Fos on a constitutive basis in steroid‐treated animals and that, while not altered in relation to the luteinizing hormone surge, Fos expression by these cells is increased following mating. Although the precise role of these CGRP neurones has yet to be ascertained, the present experiments provide direct evidence of a functional relationship between a specific sexually dimorphic neural population and a component of sexually differentiated reproductive functioning.
A 5‐Day Estradiol Therapy, in Amounts Reproducing Concentrations of the Early‐Mid Follicular Phase, Prevents the Activation of the Hypothalamo‐Pituitary‐Adrenal Axis by Interleukin‐1α in the Ovariectomized Rhesus Monkey
Authors: Xia‐Zhang Linna , Xiao Ennian , Ferin Michel
Published: September 2006

In a previous report, we have shown that intracerebroventricular (icv) administration of the cytokine interleukin‐1a (IL‐1α) in the ovariectomized (OVX) rhesus monkey results in the acute activation of the hypothalamo‐pituitary‐adrenal (HPA) axis and the inhibition of LH and FSH secretion. Here, we compare the cortisol response to IL‐1α administration in OVX monkeys and in OVX animals replaced with estradiol (E) to reproduce E concentrations typical of the early‐mid follicular phase. Cortisol, LH and FSH were measured after an icv infusion of physiological saline or IL‐1α (2.1 or 4.2 μg/30 min) in both groups. E‐containing capsules were implanted sc 5 days prior to the experiment. In OVX, E concentrations were <5 pg/ml. Cortisol concentrations decreased throughout the afternoon after saline infusion (to 49.7 ± 5.1% of baseline at 5 h; n = 7), but increased significantly after IL‐1α to 158.3 ± 13.8% (n = 7). In OVXE, cortisol also declined after saline (to 76.4 ± 16.2%; n = 5). There were 2 types of response to IL‐1α: in grp 1 (mean E 18.0 ± 0.7 pg/ml), the cortisol response was similar to that in OVX (160.8 ± 17.0%; n = 5), while in grp 2 (E: 30.7 ± 3.1 pg/ml), the cortisol response was absent (66.6 ± 7.2% of baseline at 5 h; NS vs saline in OVXE; n = 7). The cortisol response to IL‐1α was restored in 2 monkeys when E was increased to >100 pg/ml, confirming our previous observations. While saline infusion did not affect LH (102.3 ± 10.2% of baseline at 5 h) or FSH (102.5 ± 4.4%) secretion in OVX monkeys, there was a significant decrease in both hormones after IL‐1α (LH: 33.3±3.7%, FSH: 66.2 ± 6.5%; P<0.05 vs saline). This effect was lessened in OVXE animals: By 5 h, areas under the LH curve were 62.8 ± 10.9% of baseline in grp 1 and 85.3 ± 7.9% in grp 2 (NS vs saline), while those under the FSH curve were 84.0 ± 6.5% in grp 1 and 77.7 ± 4.3% in grp 2 (NS vs saline).

The data demonstrate a striking effect of a 5‐day estradiol treatment in preventing the HPA axis response to the cytokine IL‐1α in the OVX monkey. This action, however, occurs only within restricted estradiol concentrations that reproduce E levels typical of the early‐mid follicular phase of the menstrual cycle. While the precise mechanism through which estradiol exerts this action remains to be investigated, the results may have clinical relevance to the issue of estrogen replacement therapy in physiology and pathology.
Induction of Cellular Follicle‐Stimulating Hormone in the Hamster Adenohypophysis Requires Intermittent Stimulation by Luteinizing Hormone Releasing Hormone
Authors: Woller Michael J. , Campbell Gary T. , Blake Charles A.
Published: September 2006

We investigated the effectiveness of continuous vs intermittent LHRH stimulation of the neonatal female anterior pituitary gland on inducing cellular FSH immunoreactivity in the Golden Syrian hamster. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized‐ovariectomized adult hosts with a catheter implanted in the external jugular vein. In experiment 1, vehicle or LHRH (6 ng/h) was infused continuously or LHRH was pulsed at 1 h (6 ng) or 12 h (72 ng) intervals through the catheters for 8 days. Hamsters were decapitated for collection of trunk blood shortly after the end of treatment, and grafts were prepared for immunocytochemical staining for LH and FSH. Anterior pituitary glands removed from neonatal (day 1) and day 9 female pups also were stained for LH and FSH. The mean percentage of adenohypophysial cells staining for LH increased from 11% in neonatal pups to mean percentages (24‐28%) that were similar in day 9 pups and in all groups with grafts. The mean percentage of adenohypophysial cells staining for FSH increased from 1% in neonatal pups to percentages (16–21%) that were similar in day 9 pups and in grafts in hosts administered 6 or 72 ng LHRH pulses. By contrast, the mean percentage of FSH cells did not increase in grafts in hosts administered vehicle or LHRH by continuous infusion. Serum LH concentration was low in hosts given vehicle or LHRH by continuous infusion but elevated in hosts given 72 ng LHRH pulses and in all but one host given 6 ng LHRH pulses. Serum FSH concentration was detectable in some of the hosts given 6 or 72 ng LHRH pulses and non‐detectable in the rest of the hosts. In experiment 2, the mean percentage of adenohypophysial cells staining for FSH did not increase in grafts in hosts infused with LHRH continuously at 72 ng/h compared to that observed in hosts pulsed with vehicle every h for 8 days (1.4 vs 2.0%). The data demonstrate that intermittent but not continuous stimulation of neonatal female adenohypophysial cells by LHRH is effective in inducing cellular FSH immunoreactivity. The data also strongly suggest that LHRH release is episodic during the first week after birth in the female hamster. To our knowledge, these results are the first to provide evidence suggesting that the episodic LHRH release system is functioning to exert an important biological effect in a neonatal mammal and that the mechanism for down‐regulation of LHRH receptors in gonadotrophs can be activated this early in life.
Regulation of Corticotropin‐Releasing Factor Secretion and Synthesis in the Human Neuroblastoma Clones‐ BE(2)‐M17 and BE(2)‐C
Authors: Kasckow John W. , Han Jin‐Hee , Parkes David G. , Mulchahey Jeffrey J. , Owens Michael J. , Risby Emile D. , Fisher Jennifer , Nemeroff Charles B.
Published: September 2006

The BE(2)‐M17 and BE(2)‐C human neuroblastoma cell lines have been shown to synthesize and secrete corticotropin‐releasing factor (CRF) following retinoic acid treatment. It has been demonstrated that CRF secretion and intracellular synthesis increases in response to forskolin treatment. In this report, we have further characterized these cells in response to protein kinase C activators, dexamethasone, interleukin—1x, as well as various neurotransmitters and peptides. Nanomolar concentrations of the phorbol ester—phorbol 12 myristate 13—acetate (TPA), increased intracellular CRF content in both cell lines while increasing secretion only in the BE(2)‐M17 cell. Nanomolar concentrations of dexamethasone were not able to alter basal levels of secretion and content in either cell type. However, in the BE(2)‐Ml7 cell but not the BE(2)‐C cell, the same concentrations of dexamethasone added to 30 μM forskolin augmented levels of CRF secretion and content. Likewise, the same augmented response in CRF secretion and content was seen only in the BE(2)‐M17 cell line when nanomolar concentrations of dexamethasone were added to 20 nM TPA. Furthermore, only in the BE(2)‐M17 cell line were micromolar levels of the biogenic amine serotonin able to increase levels of CRF secretion and content. No effects on CRF in both cell lines were demonstrable with picomolar levels of interleukin‐10: as well as micromolar levels of acetylcholine, norepinephrine, arginine‐vasopressin, oxytocin, and angiotensin‐II. The potential usefulness of these cells as models of central nervous system or placental CRF‐containing neurons is discussed.
S–100 Antigen‐Positive Folliculostellate Cells are not the Source of IL‐6 Gene Expression in Human Pituitary Adenomas
Authors: Ueta Yoichi , Levy Andrew , Chowdrey Hardial S. , Lightman Stafford L.
Published: September 2006

We have investigated the expression of IL‐6 in a random selection of 27 human pituitary adenomas, comprising 8 somatotroph, 5 corticotroph, 3 mammotroph and 11 endocrinologically inactive adenomas, using a 35S‐labelled 1.1kb riboprobe complementary to human IL‐6. Positive and negative IL‐6 transcript controls were generated from the IL‐6‐secreting human bladder carcinoma cell line T24/83. Tissue from a malignant melanoma was used as a positive S‐100 immunocytochemical control tissue. Of the 27 human pituitary adenomas examined by in situ hybridization, 7 (26%) contained IL‐6 transcripts: these were 3 of 5 corticotroph adenomas, 2 of 8 somatotrophinomas and 2 of 11 endocrinologically inactive adenomas. In each case, IL‐6 transcript‐positive cells constituted less than 1% of the total pituitary tissue mass examined. Alternate wax embedded 3 μm thick sections from 5 of the 7 IL‐6 transcript positive tumours were examined immunocytochemically for S‐100 antigen, or by in situ hybridization for IL‐6 transcripts. Immunocytochemistry for S‐100 antigen was completely negative in 3 of the 5 tumours and in the remaining 2, there was no evidence of IL‐6 transcripts and S–100 antigen co‐localization in any of the sections examined. This suggests that in pituitary adenomas, cells other than classical folliculostellate cells are responsible for IL‐6 production.
Sexually Dimorphic Expression of Androgen Receptor Immunoreactivity by Somatostatin Neurones in Rat Hypothalamic Periventricular Nucleus and Bed Nucleus of the Stria Terminalis
Authors: Herbison Allan E.
Published: September 2006

Gonadal steroids exert important regulatory actions on the hypothalamic neurones regulating growth hormone secretion and are believed to play a role in generating its sexually dimorphic pattern of secretion. Recent evidence indicates that estrogen actions on one of these neural populations, the periventricular somatostatin (SOM) neurones, are likely to be indirect as they do not possess nuclear estrogen receptors in either sex although androgen receptors (ARs) have been reported within these cells in male rats. The present study has used double‐labelling immunocytochemistry procedures to examine whether sex differences exist in AR expression by SOM neurones located in the periventricular nucleus and bed nucleus of the stria terminalis (BNST). Within the hypothalamus, SOM‐immunoreactive neurones were found concentrated in the periventricular nucleus while both anterior and posterior divisions of the BNST contained scattered populations of SOM cells. Cells immunoreactive for the AR were detected in all of these areas. Although the intensity of AR cell nuclei staining was equivalent in males and females in regions such as the lateral septum, the intensity of AR staining in many individual cells of the periventricular nucleus and posterior BNST of the female was reduced when compared with the male. Double‐labelling experiments revealed that approximately 40% of periventricular SOM neurones expressed AR immunoreactivity in the male compared with significantly (P<0.01) fewer cells in the female (∼7%). In the BNST, double‐labelled cells were only detected within the principle encapsulated, interfascicular and transverse nuclei of its posterior division. Approximately 60% of SOM cells in these nuclei expressed AR immunoreactivity in the male while significantly (P<0.01) fewer did so in the female (∼25%). These results indicate that substantial sex differences exist in AR expression by SOM neurones in both the periventricular nucleus and BNST. Such differences in AR expression by periventricular SOM cells may contribute to their sexually dimorphic nature and, consequently, sex differences in growth hormone secretion.
Species Differences in Estrogen Receptor and Progesterone Receptor‐mRNA Expression in the Brain of Sexual and Unisexual Whiptail Lizards
Authors: Young Larry J. , Nag Pratip K. , Crews David
Published: September 2006

Circulating concentrations of gonadal steroid hormones and reproductive behavior in female vertebrates vary as a function of ovarian state. Steroids secreted by the ovary, specifically estrogen and progesterone, influence the expression of behaviors associated with reproduction by interacting with intracellular sex steroid receptors located in specific regions of the brain. Using in situ hybridization, we analyzed estrogen receptor and progesterone receptor messenger RNA expression in several brain regions of ovariectomized, vitellogenlc, and postovulatory individuals from two species of whiptail lizards (Cnemidophorus uniparens and C. inornatus). Although these species are genetically very similar, they differ in two aspects of their reproductive biology: (i) the unisexual C. uniparens alternate between expressing female‐typical and male‐like pseudosexual behaviors while female C. inornatus normally express only female receptive behavior, and (ii) circulating estradiol concentrations in reproductively active female C. uniparens are approximately five‐fold lower than in reproductively active female C. inornatus. We found that the regulation of sex steroid receptor gene expression was region specific, with receptor‐mRNA expression being increased, unchanged, or decreased during vitellogenesis depending on the area. Furthermore, Several species differences in the amount of sex steroid receptor‐mRNA were found that may be relevant to the species differences in circulating estrogen concentrations and sexual behavior.
Glial Ensheathment of GnRH Neurons in Pubertal Female Rhesus Macaques
Authors: Witkin Joan W. , O'Sullivan Honor , Ferin Michel
Published: September 2006

During the period of development, prior to full sexual maturity, gonadotropin hormone‐releasing hormone (GnRH) neurons are fully capable of synthesizing and processing the GnRH decapeptide. Nonetheless, the secretion of the hormone is not adequate to stimulate adult patterns of gonadotropin release. The present study was undertaken to examine ultrastructural characteristics of the GnRH neuron and its relationship to its environment in early‐ midpubertal female rhesus monkey. These neurons bore all the ultrastructural immunocytochemical characteristics of those in mature animals, but quantitative morphometrics revealed that they were extensively apposed by glial processes. Such ensheathment was described earlier in ovariectomized adult animals and was found to be reversible by administration of gonadal steroids. The density of synaptic input to GnRH neurons in the pubertal animals did not differ significantly from that of adult intact or ovariectomized animals from a previous study. Chemical identification will be required to determine whether there are age or hormonal differences in the innervation of these neurons.

These results provide anatomical evidence in support of indications from other studies that the ovarian steroidal milieu affects GnRH‐glial relationships. Further testing will be required to determine whether the attainment of sexual maturity in the female rhesus macaque is dependent upon a reduction in glial ensheathment of GnRH neurons.