Stalk-median Eminence Research Articles

Effects of Chronic LHRH Treatment on Brain LHRH Content, Pituitary and Plasma LH and Ovarian Follicular Activity in the Anestrous Ewe1

Nine mature, anestrous ewes were used in the experiment. Osmotic minipumps (AIza Corp.) containing 1.7 mg synthetic LURU in 170 pl saline were implanted s.c. into 4 sheep. Each ewe received 4 minipumps, implanted 1 week apart. This treatment resulted in a relatively uniform level (54 ± 2 pg/mI, X ± SEM) of LHRH-like immunoreactivity in plasma during the 4 week period. After 4 weeks, the sheep were killed and the stalk-median eminence (SME), hypothalamus proper (HP), preoptic-suprachiasmatic area (P0-SC), a sample of cerebral cortex (C) and the pineal gland (PG) were removed from the brain, homogenized in acidified ethanol and assayed for LI-IRH. Chronic exposure to LHRU was without effect on the contents of LHRH in the SME, HP or P0-SC, which were 50 ± 5, 5.1 ± 0.6 and 4.8 ± 0.8 ng, respectively. LHRU was not detected in C. LHRH treatment did increase the content of LHRH in the PG (treated = 125 ± 24 pg, control = 80 ± 12 pg, P 2mm in diameter (P<0.01). The initial response of LU to the sustained administration of LHRU appears to be sufficient to induce ovulation and subsequent luteal function. The long term response, however, is not adequate to maintain ovarian follicular development.

Regional Brain Content of Luteinizing Hormone- Releasing Hormone in Sheep during the Estrous Cycle, Seasonal Anestrus, and after Ovariectomy*

Twenty-three sheep were killed during seasonal anestrus or on different days of the estrous cycle. The stalkmedian eminence (SME), hypothalamic tissue (HP) overlying the SME, and preoptic-suprachiasmatic tissue (PO-SC) were assayed for LHRH using RIA. LHRH contents of the SME during anestrus and days 3–8 of the estrous cycle were 35 ± 3 and 33 ± 5 n.g (χ ± SEM), respectively. Higher levels (59 ± 8 ng) were detected in proestrous-estrous ewes (days 1, 16, and 17), and intermediate contents were measured in ewes on days 10–l14. LHRH content of the HP (11 ± 1 ng) was not different during the reproductive stages examined, although it was correlated (r = 0.65) with that in the SME. In the PO-SC area, LHRH increased from 1.3 ± 0.2 ng on days 3–8 to 2.6 ± 0.4 ng by days 10–14, but by days 1, 16, and 17 it had fallen to 0.9 ± 0.2 ng. LHRH content of the PO-SC area of anestrous ewes was 2.1 ± 0.4 ng. Anterior pituitary content of LH was greater on days 1, 10–14, 16, and 17 than on days 3–8 of the estrous cycle or ...

Corticotrophin releasing factor may be modulated vasopressin

CORTICOTROPHIN RELEASING FACTOR (CRF) was the first of the postulated hypothalamic factors controlling the release of hormones from the pituitary gland to be demonstrated biologically1. However, it is extraordinary that after 24 years of research, techniques which have established the identity of the hypothalamic peptides involved in the release of thryotrophin, the gonadotrophins and growth hormone, have been unsuccessful when applied to the search for a factor responsible for adrenocorticotrophin (ACTH) release2,3. Vasopressin invariably releases ACTH from the anterior pituitary, but its partial agonistic properties in vitro4–6, its low potency in vivo7 and the isolation of active CRF fractions with little or no vasopressin activity2,3,8,9 have led to its rejection as a physiological CRF10,11. However, recent immunological and immunocytochemical findings have led to renewed interest in the role of vasopressin in ACTH release. High concentrations of immunoreactive vasopressin have been found in portal blood and a direct vasopressin-neurophysin containing axonal pathway to the hypophyseal portal system, which seems to be under the influence of the adrenal cortex, has been identified12,13. Using a sensitive releasing factor bioassay system, the perfused isolated anterior pituitary cell column14, and various immunological techniques, we have presented evidence to support the idea that vasopressin plays a major part in control of ACTH release6, and we now report the existence of hypothalamic factors with weak, labile CRF activity which potentiate the CRF activity of vasopressin to give it full agonistic properties indistinguishable from crude extracts of rat stalk median eminence (SME). Thus we propose that CRF is vasopressin modulated by other hypothalamic factor(s) released into the hypothalamo-hypophyseal portal system.

3H]Spiroperidol (Spiperone) Binding to a Putative Dopamine Receptor in Sheep and Steer Pituitary and Stalk Median Eminence*

The dopamine antagonist [3H]spiroperidol (spiperone) binds with high affinity to one class of sites in the sheep and steer anterior pituitary, posterior pituitary, and stalk median eminence. The specific binding in the anterior pituitary of the sheep satisfies all criteria studied for a receptor, including high affinity binding (dissociation constant = 0.85 nM for sheep and 0.38 nM for steer), saturability (sites = 343 fmol/mg protein for sheep and 210 fmol/mg for steer), temperature dependence, and reversibility. The rank order of potency of various agonists to compete with [3H]spiroperidol for the sites in the sheep anterior pituitary is consistent with interactions at a dopamine receptor (i.e. bromergocryptine > apomorphine > dopamine > norepinephrine ≥ epinephrine). Furthermore, this potency series parallels that seen for the inhibition of PRL secretion from the anterior pituitary in vitro. Dopamine metabolites, β-endorphin, TRH, and the catecholestrogen methoxyestrone did not compete for [3H]spiroper...

Somatostatin secretion from the rat neurohypophysis and stalk median eminence (SME) in vitro: Calcium-dependent release by high potassium and electrical stimulation

Within the central nervous system, immunoreactive somatostatin (SRIF) has been localized to fibers in the external zone of the median eminence, neuronal cell bodies in the anterior hypothalamus, axons in the posterior pituitary lobe, and spinal ganglia and fibers in the spinal cord. 1 This extensive brain distribution, the synaptosomal localization, and the effects of SRIF on behavior and on the spontaneous electrical activity of single neuron units in many parts of the brain suggest that the peptide may serve as a central neurotransmitter or a modulator of neuronal function. 2 Although these observations imply an important role for SRIF in brain function, little is currently known about the factors controlling the secretion of SRIF from nerve cells. Since the neurohypophysis and stalk median eminence (SME) region of the hypothalamus contain a high concentration of SRIF and can be readily isolated, we have used them as models for studying the release of the peptide from nerve terminals in vitro. 3

Characterization of Rat Stalk Median Eminence Vasopressin and Its Involvement in Adrenocorticotropin Release

The perfused isolated rat anterior pituitary cell column was used as a bioassay for corticotropinreleasing factor (CRF). The system responded to arginine vasopressin (AVP) in a dose-dependent manner with a minimum effective dose of 10-10 M. Log doseresponse curves for AVP and crude stalk median eminence extract (SME) were statistically significantly nonparallel (P < 0.05) and similar results were found for lysine vasopressin, arginine vasotocin (AVT), oxytocin, and human posterior lobe extract. Vasopressin and its analogs had no effect on LH release. Using RIA for AVP, SME was found to contain 10.83 ng (7.7–14.3 ng, n = 12) AVP, which could account for no more than 30% of its CRF activity. The CRF activity of both SME and AVP was totally quenched by incubation overnight with vasopressin antisera. Partial quenching was obtained with higher dilutions of the antisera. Vasopressin-stimulated ACTH release was affected in a similar way to SME-stimulated ACTH release by the presence of corticosterone (0.2 μg/ml)...

Perfused Rat Isolated Anterior Pituitary Cell Column as Bioassay for Factor(s) Controlling Release of Adrenocorticotropin: Validation of a Technique

A suspension of isolated rat anterior pituitary cells, with viability of 90% was mixed with 0.5 g preswollen Bio-Gel P2 and packed into a column. ACTH in the fractions collected from the perfused column was measured by immunoassay and bioassay with well correlated results (r = 0.84,P = 0.01). Cells were stimulated with 2–4-min pulses or crude stalk median eminence extract (SME) and responses were recorded as total ACTH released in excess of background. A log doseresponse curve was obtained over the range 0.0025–0.1 SME/ml (one SME = extract of 3–4 mg tissue/ml), giving a highly significant regression of response on dose (P < 0.01) with an index of precision of 0.164 ± 0.007 (mean ± SEM, n = 8). LH, as measured by immunoassay, was also released in response to SME. No significant ACTH release was seen with a cortex extract. ACTH elease was stimulated by 56 mM potassium ions; SMEstimulated ACTH release was dependent on calcium and magnesium ions and was inhibited by the presence of corticosterone (0.2 μg/ml)...

Distribution of CRF Activity and Immunoreactive ACTH within the Hypothalamic-Neurophypophyseal Complex in Various Species

SummaryUsing an in vitro CRF assay employing cultured rat adenohypophyseal cells from adrenalectomized female donors and ACTH measurement by radioimmunoassay, the distribution of corticotrophinreleasing factor (CRF) activity and immunoreactive ACTH within the hypothalamic-neurohypophyseal complex and cerebral cortex was compared in various mammalian species (rat, sheep, pig, dog, cattle and man). For all species studied the highest concentration of CRF activity was always in the hypophyseal stalk. ACTH concentration was higher in the pars nervosa of the posterior pituitary lobe than in the stalk or hypothalamic median eminence in rat, sheep, cattle, and man, whereas in the pig and dog ACTH concentration was higher in the stalk than in the pars nervosa or median eminence.

Multiphasic effect of porcine stalk-median eminence extract on growth hormone release from perifused rat pituitaries.

The present experiments describe the use of an in vitro perifusion system to observe the time course of the GH response to extracts of porcine stalkmedian eminence (pSME). It was presumed that both releasing and inhibiting activities were present in the pSME and that the characterization of a response profile would provide more information than measurement of the net response alone. Release of 3H-labeled growth hormone ([3H]GH) from perifused rat pituitaries preincubated in [3H]leucine was measured by specific immunoprecipitation. A methanol extract of pSME elicited a complex [3H]GH release response: initial stimulation of release was followed by inhibition and then by a rebound release, the latter occurring after termination of the pSME pulse. When pituitary explants were simultaneously exposed to pulses of pSME during continuous, maximal somatostatin (SRIF) inhibition, a blunted initial stimulatory response was observed and the rebound release phase was abolished. Continuous exposure to pSME produced only a brief response in both the SRIF-inhibited (blunted) and the uninhibited explant. These data suggest that extracts of pSME contain principles which both stimulate and inhibit rGH release. The pSME inhibitory effect is indistinguishable from that of SRIF because 1) continuous exposure to pSME does not further inhibit [3H]GH release from pituitary explants already maximally inhibited by SRIF, and 2) the post pSME rebound does not occur in the presence of maximal inhibitory concentrations of SRIF. Maximal SRIF inhibition does not prevent the early stimulation of rGH release by pSME. The perifusion-immunoprecipitation system is a discriminating technique which can demonstrate the simultaneous presence of two hypothalamic factors with opposing effects upon rGH secretion. (Endocrinology102: 1520, 1978)

Hypothalamic and Brain Thyrotropin-Releasing Hormone Content and Pituitary-Thyroid Function in Histidine-Deficient Rats

Because starvation is known to depress pituitary-thyroid function, and the essential amino acid histidine is a precursor of thyrotropin releasing hormone (TRH, pyroglutamyl-histidyl-prolinamide) we tested the hypothesis that histidine deficiency would decrease the secretion of TRH, and influence its concentration in extrahypothalamic brain. Rats given an histidine-deficient diet ad libitum for 4 days lost weight compared with controls (−25 ±3 vs. +15 ± 4g; P < 0.001), showed a reduction in serum histidine (4.8 ± 0.9 vs. 11.6 ± 0.6 μg/ml; P < 0.01) and brain histidine (2.4 ± 0.6 vs. 4.7 ± 0.7 μg/g; P < 0.05), and a lowered serum thyroxine (T4) level (4.5 ± 0.4 vs. 5.7 ± 0.4 μg/dl; P < 0.05), with non-significant reductions in serum thyroid stimulating hormone (TSH) and triiodothyronine (T3). However, there were no significant changes in the immunoreactive TRH levels in brain, hypothalamus and stalk median eminence (SME). In a second experiment lasting 9 days in which the controls were pair-fed, with both g...

Failure of stalk-median eminence (SME) extract to suppress serum prolactin levels in rats with hypothalamic islands

Failure of stalk-median eminence (SME) extract to suppress serum prolactin levels in rats with hypothalamic islands

Control of Bioactive Corticotropin Release from the Neuro-Intermediate Lobe of the Rat Pituitaryin Vitro

The purpose of this study was to identify agents capable of regulating the release of biologically active ACTH from the isolated neuro-intermediate lobe of the rat pituitary. Agents found to be potent secretagogues included acetylcholine (100 mug/ml), hypothalamic stalk-median eminence extract (0.33 eq), arginine antidiuretic hormone (100 mU/ml) and serotonin (100 mug/ml). Lower doses of arginine antidiuretic hormone (5.5 mU/ml) and serotonin (2 mug/ml) were ineffective. Dopamine 2 and 5 mug/ml) inhibited the release of biologically active ACTH whereas norepinephrine (5 mug/ml) did not. Dexamethasone (0.25 mug/ml) did not alter the basal or stimulated release of ACTH from the isolated neuro-intermediate lobe in contrast to its effect on ACTH release from the isolated anterior pituitary. Similarly, the tripeptide, prolyl-leucyl-glycinamide, which has been reported by some to inhibit MSH release, had no effect on either the basal or stimulated release of ACTH. The data suggest that regulation of ACTH release from the neuro-intermediate lobe in vivo may involve both stimulatory (acetylcholine) and inhibitory (dopamine) inputs.

Absence of Immunoreactive Luteinizing Hormone-Releasing Hormone in Ovine Cerebrospinal Fluid Collected from the Third Ventricle

SummaryCerebrospinal fluid (CSF) was collected repetitively from the third ventricle of three ovariectomized sheep. Immuno-reactive LHRH could not be detected (<100 pg/ml) in any of 70 samples of CSF collected from untreated, estradiol-treated, and LHRH-treated ewes. The LHRH im-munoassay accurately measured the hormone in extracts of ovine stalk-median eminence tissue. Furthermore, no immunoreactive LHRH was lost when synthetic LHRH was added to freshly collected CSF and stored in the manner used for the attempted measurement of endogenous LHRH. Intravenous injection of LHRH increased plasma concentrations of the hormone to 420 pg/ml but LHRH could not be detected (<100 pg/ ml) in CSF collected concurrently.

Involvement of intracellular calcium in hormone secretion from rat pituitary cells

Cells were dissociated from normal rat pituitaries by a combination of mechanical agitation and enzymatic action, seeded into culture flasks and grown in monolayer culture. When such cells were exposed to an extract of the hypothalamic stalk-median eminence area (HSME) a dosedependent secretion of ACTH was observed. A 2-h exposure to Ca-free media significantly reduced the HSME-stimulated release of ACTH but the measured levels were still greater than the unstimulated controls. When the 45Ca 2+ uptake into the cultured cells was measured both control and HSME-stimulated cells yielded identical results (60–80 nmol Ca/mg cell protein). Upon removal of the calcium associated with the surface coat it was found that HSME actually decreased the cellular uptake of calcium. Since variations in uptake can result from changes in influx or efflux as well as from variations in pool size or turnover times of calcium exchange with intracellular compartments, a series of isotope washout experiments were performed. Neither HSME nor theophylline affected the rate constant of calcium efflux from what is believed to be the cytosol pool to the extracellular media. Both agents, however, prompted a shift of intracellular calcium into a more tightly bound compartment. The data suggest that the calcium required for pituitary hormone secretion is derived primarily from an intracellular rather than extracellular origin. It may be that, via the action of cyclic AMP, such calcium can be mobilized from intracellular stores and shifted to a more tightly bound compartment where it can participate in the intracellular processes associated with secretion.

Effect of the dopa decarboxylase inhibitor MK-486 on L-dopa-induced inhibition of prolactin secretion: evidence for CNS participation in the L-dopa effects.

The present studies were performed in order to determine whether the inhibition of prolactin (Prl) secretion which follows the systemic administration of L-dopa and its conversion to dopamine (DA) is mediated by central or by peripheral mechanisms. The effects of pretreatment with the dopa-decarboxylase inhibitor MK-486 (L-α-methyldopa-hydrazide) on the L-dopa and DA inhibition of Prl secretion and on dopa-decarboxylase activity in anterior pituitary, hypothalamus, cerebral cortex and adrenal medulla were evaluated in rats using a dose reported to cause peripheral but not central dopa-decarboxylase inhibition. I.v. infusions of DA, 1 and 10 µg/kg/min, and of L-dopa, 0.1 and 1 mg/kg/min for 15 min, suppressed plasma Prl levels as well as the Prl release response to 0.2 porcine stalk median eminence equivalents (pSME). I.p. administration of MK-486, 50 mg/kg, 30 min before the infusion of L-dopa did not alter either basal or pSME-stimulated Prl secretion or the inhibitory effect of L-dopa. Dopa-decarboxylase activity in the anterior pituitary and the adrenal medulla was completely inhibited by MK-486, while hypothalamic and cerebral cortex dopa-decarboxylase activity was reduced 85 and 84%, respectively. Since dopa conversion to DA could not have occurred peripherally in the presence of MK-486, the results indicate that the residual dopa-decarboxylase activity within the central nervous system (CNS) was capable of permitting sufficient conversion of L-dopa to DA to inhibit Prl secretion. These results provide evidence that L-dopa is capable of exerting its suppressive action on Prl secretion when its decarboxylation to DA is restricted to CNS sites.

Effects of Hypothalamic Extract and Corticosterone on Incorporation of &lt;sup&gt;14&lt;/sup&gt;C-Phenylalanine into Newly Synthesized ACTH-Like Protein &lt;i&gt;in vitr&lt;/i&gt;&lt;i&gt;o&lt;/i&gt;

The purpose of the present experiments was firstly, to examine the efficacy of the oxycellulose-Amberlite CG-50 extraction procedure for the isolation of rat pituitary ACTH and secondly, to study the effects of both hypothalamic extract and corticosterone on the synthesis of rat pituitary ACTH. Briefly, 'cold' rat pituitary ACTH was monitored by bioassay in each step of the isolation procedure as a function of corticosterone stimulation in incubated adrenal slices. Labeled ACTH was isolated from rat pituitaries previously incubated in vitro and obtained from adrenalectomized animals and cortisone acetate treated animals to provide further data to substantiate that the labeled isolated protein contained ACTH. Cold ACTH was detected in two purified fractions (oxycellulose and Amberlite CG-50). Adrenalectomy stimulated and cortisone acetate suppressed the incorporation of 14C-phenylalaine into the ACTH-like protein in these two fractions. A continuously increasing uptake of the label into ACTH-like protein in the isolated fractions occurred with time providing evidence that de novo synthesis had taken place. Corticosterone inhibited ACTH synthesis at both 10(-4) and 10(-6) M, whereas, hypothalamic stalk median eminence (HSME) caused a significant, but not dose-related, increase in ACTH synthesis with 3/5, 1, and 2 HSME equivalents added.

Dynamics of luteinizing hormone release after intravenous administration of crude stalk-median eminence extract or synthetic gonadotropin-releasing hormone.

SummaryThe comparative ability of crude acidic extracts of stalk-median eminence (SME) and of synthetic Gn-RH to induce the release of LH in ewes was examined. Intravenous injections of 4 SME equiv...

Dissociation of prolactin-releasing activity from thyrotropin-releasing hormone in porcine stalk median eminence.

The prolactin (PRL)-releasing activity of porcine stalk median eminence (pSME) was characterized by an in vivo bioassay and concomitant radioi-munoassay of plasma PRL and thyrotropin (TSH) levels. Methanol extracts of pSME stimulated PRL release in 3-day estrogen-primed rats when administered by the intracarotid route in doses ranging from 0.1 to 2.0 pSME equivalents. Synthetic thyrotropin-releasing hormone (TRH) stimulated the release of PRL and TSH in the dose range of 10 to 300 ng. PRL release was greater in response to a maximally effective dose of pSME than the release elicited by a maximal dose of TRH, and pSME administered together with a greater than mazimally effective dose of TRH caused additional PRL but not TSH secretion. Lysine vasopressin and prostaglandin E1 and E2 stimulated PRL release only at doses several orders of magnitude greater than the dose present in pSME. Somatostatin inhibited the release of TSH but not that of PRL whether the stimulus employed was pSME or TRH. The effective inhibitory dose of somatostatin was also significantly greater than the reported hypothalamic content. When pSME was subjected to incubation with plasma, a treatment reported to inactivate TRH, TSH-releasing activity was destroyed to a greater extent than was PRL-releasing activity. When pSME was adsorbed onto charcoal, the supernatant solution was devoid of TRH, as determined by complete removal of a [3H]TRH marker, yet substantial PRL-releasing activity was retained. TSH-releasing activity eluted from the charcoal with methanol was considerably greater than that expected on the basis of the recovery of [3H]TRH, suggesting the presence in the crude extract of a TSH-release inhibitor or of a TSH-releasing factor other than TRH. Based on the above evidence, we conclude that crude pSME contains PRL-releasing substance(s) distinct from the tripeptide TRH.

Observations on the untreated progeny of hypothyroid male rats

The untreated progeny (F 1) of hypothyroid male rats that were either radiothyroidectomized (Tx) or had the neo-T 4 syndrome (an endocrine disorder produced by large doses of thyroxine (T 4) injected during the neonatal period) were studied. The mother rats were all normal. The fathers never had any contact with their progeny. Unexpectedly, the progeny usually showed delayed eye opening, decreased weaning weights, and increased final body weight. The thyroid glands from F 1 offspring of both Tx and neo-T 4 fathers were enlarged significantly in all but F 1 males of Tx fathers. The F 1 of Tx fathers had significantly smaller uteri, both absolutely and relatively. The ovaries were significantly larger, whereas the testes were significantly smaller. Pituitary TSH, stalk-median eminence (SME) TSH, and serum TSH were all normal with the exception of an increase in SME TSH in F 1 males born of neo-T 4 fathers. The response to thyrotropin releasing hormone (TRH) stimulation of the F 1 adult progeny of neo-T 4 fathers was significantly blunted in the males, whereas the response was normal in the offspring of Tx fathers. The mechanisms by which hypothyroid fathers caused changes in their progeny is not known.

Effect of oral contraceptive on the pituitary and plasma L.H. levels and on the hypothalamic LH-RH level (author's transl)

The effects of the oral contraceptive on the pituitary content and plasma level of LH and the hypothalamic LH-RH level were investigated in 16 adult female rabbits. The oral contraceptive preparation, Sophia-C (Norethindrone 2mg + Mestranol 0.1 mg), as administered orally by a stomach tube each day for 7 days in 8 adult female rabbits. At the end of the treatment, the rabbits were bled from the abdominal aorta into heparinized syringes and the plasma was separated. The stalk median eminences were excised. All the materials were stored in -80 degrees C until assayed. Plasma level and the pituitary content of LH and the hypothalamic LH-RH were measured by radioimmunoassay. All the dose response curved were drawn using logitlog transformation. Radioimmunoassay procedures for LH was described in detail elsewhere. Purified rabbit LH for iodination (125-I) and strandard were prepared by T. Makino and R.O. Greep, at Research Laboratories for Human Reproduction, Harvard Medical School, Boston, U.S.A. The starting B/T was 25% at the final dilution of the antibody of 1/20,000. Minimal detectable quantity was about 40 pg/tube. The 50% intercepts were approximately 460 pg/tube. Radioimmunoassay procedures for LH-RH were performed according to the method described by Arimura et al. Antiserum against synthetic LH-RH was kindly supplied to us by Drs. A. Arimura and A.V. Schally, New Orleans, U'S.A. The synthetic LH-RH was kindly supplied to us by Dr. N. Yanaihara. The starting B/T ratio was 29% at the final dilution of the antibody of I/17,500. Minimal detectable quantity was about 40pg/tube and the 50% intercepts were 150pg/tube. It has been assumed that oral contraceptive drugs exert their action by blocking the hypothalamic LH-RH, resulting in a depression of the plasma level of LH, because plasma level of LH returned to the normal level when LH-RH was administered intravenously even while oral contraceptive steroids were given continuously. However, these findings concerning the site of action of the drugs furnished only indirect evidences. The possibility of a direct inhibitary effect of these steroids on the anterior pituitary cannot be ruled out. Administration of 2mg of Norethindrone and 0.1mg of Mestranol significantly depressed both the pituitary content and the plasma level of LH. On the other hand, they significantly increased the hypothalamic LH-RH level. Those findings strongly suggested the direct action of this drug on the anterior pituitary in the female adult rabbits. Detailed mechanisms on how the hypothalamic LH-RH was increased were not known in this experiment, further investigations are now in progress.