Effect of androgens on hypothalamic pro-opiomelanocortin

Testosterone and estradiol have been shown to affect the hypothalamic content of several pro-opiomelanocortin (POMC)-derived peptides in castrated male and female rats, respectively. It was unclear, however, whether the effects of testosterone on hypothalamic POMC were due to conversion by aromatization to estradiol or whether there were independent androgen actions on hypothalamic POMC. In order to answer this question, the effect of treatment with the nonaromitizable androgen 5-alpha-dihydrotestosterone (DHT) on the concentration of beta-endorphin (beta-EP) in the medial basal hypothalamus (MBH) was studied in castrated male rats and compared to the effect of treatment with testosterone or estradiol. The concentrations of two other POMC-derived peptides, corticotropin-like intermediate lobe peptide (CLIP) and alpha-MSH were measured as well. Adult male rats were castrated and received either no treatment or treatment with subcutaneously implanted silastic capsules, containing either DHT, testosterone or estradiol, designed to produce steroid levels in a physiological range. After 4 weeks the mean concentration of beta-EP in the MBH of the untreated castrated rats was 1,640 +/- 56 fmol/mg protein. This was reduced significantly to 1,184 +/- 74 fmol/mg protein after DHT treatment (p less than 0.001). Similar reductions to 1,340 +/- 95 and 1,130 +/- 85 fmol/mg protein were noted after testosterone and estradiol treatment, respectively. The mean CLIP concentration of 1,870 +/- 73 fmol/mg protein in the untreated animals fell to 1,390 +/- 95 after DHT (p less than 0.001) compared to 1,520 +/- 105 and 1,260 +/- 101 after testosterone and estradiol treatment, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic effect of orchiectomy on pro-opiomelanocortin gene expression in the hypothalamus

Previous studies have shown that the hypothalamic concentration of pro-opiomelanocortin (POMC) mRNA and several POMC-derived peptides increases in the rat 4 weeks after orchiectomy and that this increase can be prevented by testosterone replacement. In this study, we have examined the short-term effects of orchiectomy on POMC gene expression in the medial basal hypothalamus (MBH). Adult male rats were studied at various time points between 1 day and 4 weeks after orchiectomy and compared to sham-orchiectomized rats. The MBH was homogenized and, after an aliquot was removed for beta-endorphin (beta-EP) radioimmunoassay, the RNA was isolated and the amount of POMC mRNA was measured using a solution hybridization S1 nuclease protection assay. In the first experiment, POMC mRNA was significantly higher 4 weeks after orchiectomy compared to that of the intact controls: 1.34 +/- 0.14 vs. 0.86 +/- 0.04 pg/micrograms RNA (p less than 0.01). Three days after orchiectomy, POMC mRNA was somewhat lower, 0.71 +/- 0.06 pg/micrograms RNA, but not significantly different from the controls. In a second experiment, POMC mRNA levels were measured 1, 2, 3, 4 and 7 days after orchiectomy. At 1 and 2 days after orchiectomy, POMC mRNA was lower than the controls: 0.51 +/- 0.02 and 0.52 +/- 0.06 vs. 0.70 +/- 0.09 pg/micrograms RNA. Levels then steadily increased to 0.61 +/- 0.04, 0.70 +/- 0.09 and 0.78 +/- 0.11 pg/micrograms RNA at 3, 4 and 7 days after orchiectomy, respectively. The mean level at 1-2 days after orchiectomy was significantly less than the controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive correlation between proopiomelanocortin and tyrosine hydroxylase mRNA levels in the mediobasohypothalamus of ovariectomized rats: response to estradiol replacement and withdrawal.

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.     

Regulation of beta-endorphin, corticotropin-like intermediate lobe peptide, and alpha-melanotropin-stimulating hormone in the hypothalamus by testosterone

Ovarian steroids have previously been shown to regulate the hypothalamic content of beta-endorphin (beta EP) and its release into hypophyseal portal blood. Although the hypothalamic content of beta EP in cycling female rats was unchanged by ovariectomy, chronic treatment of ovariectomized rats with estradiol lowered hypothalamic beta EP levels. In this study, the hypothalamic content of beta EP was compared in male and cycling female rats, and the effects of orchiectomy and testosterone replacement on hypothalamic beta EP were examined. The beta EP content of the medial basal hypothalamus (MBH) was significantly higher in female rats compared to that in males of either the same weight (175-200 g) or the same age (65 days; P less than 0.025). When male rats were studied 4 weeks after castration, the beta-EP content of the MBH increased from a value of 2100 +/- 103 fmol in the controls to 2680 +/- 126 fmol (P less than 0.005). The hypothalamic beta EP content in the castrated males was similar to that in the intact females (2700 +/- 158 fmol). The increase in hypothalamic beta EP induced by castration was blocked by testosterone replacement. When orchiectomized animals were treated for 4 weeks with Silastic capsules filled with testosterone, there was a significant fall in the hypothalamic content of beta EP compared to that in the unreplaced animals. beta EP fell from 3180 +/- 115 to 2033 +/- 53 fmol in the MBH (P less than 0.001), from 1693 +/- 122 to 934 +/- 80 fmol in the anterior hypothalamus (P less than 0.001), and from 148 +/- 26 to 90.3 +/- 11 fmol in the median eminence (P less than 0.05). Testosterone replacement was also associated with a significant decline in the hypothalamic content of corticotropin-like intermediate lobe peptide and alpha MSH. Corticotropin-like intermediate lobe peptide fell from 2400 +/- 53 to 1560 +/- 84 fmol in the MBH (P less than 0.001) and from 1200 +/- 74 to 805 +/- 94 fmol in the anterior hypothalamus (P less than 0.01). alpha MSH fell from 1660 +/- 162 to 884 +/- 75 fmol in the MBH (P less than 0.001) and from 823 +/- 106 to 544 +/- 92 fmol in the anterior hypothalamus (P less than 0.05). Thus, testosterone, as well as estradiol, affects the hypothalamic content of several proopiomelanocortin-derived peptides. The effect on brain peptide content, however, depends on whether the steroids are secreted relatively constantly, as in the male, or fluctuate, as in the cycling female.     

Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol

We have previously demonstrated that neurons in the rostral arcuate nucleus expressing the messenger RNA (mRNA) for pro-opiomelanocortin (POMC) are responsive to modulation by physiological levels of testosterone. It is uncertain, however, whether testosterone's action is mediated through direct activation of androgen receptors or through aromatization to estradiol and subsequent binding to estrogen receptors. We examined this question by evaluating the effectiveness of estradiol and dihydrotestosterone (DHT), a nonaromatizable androgen, in reversing the castration-induced diminution of POMC mRNA in the arcuate nucleus. Using in situ hybridization, we measured POMC mRNA content within arcuate neurons of intact, castrated, castrated testosterone-replaced, castrated estradiol-replaced, and castrated DHT-replaced male rats. Adult male rats were castrated and implanted (s.c.) with a Silastic capsule filled to one of the following specifications: crystalline testosterone (30 mm; n = 4); 17 beta-estradiol (E2) diluted 1:1 with cholesterol (5 mm; n = 4); DHT (40 mm; n = 4); or empty (30 mm; n = 4). Control, sham-operated animals (n = 4) were left intact. Analysis of the results showed that following castration, POMC mRNA content was significantly reduced in cells of the arcuate nucleus (intact: 152 +/- 3 grains/cell vs. castrate: 110 +/- 3 grains/cell). Replacement with physiological levels of testosterone prevented the decline of POMC mRNA levels (castrated testosterone-replaced: 143 +/- 6 grains/cell), as did replacement with physiological levels of estrogen (castrated estrogen-replaced: 149 +/- 8 grains/cell). Treatment with DHT failed to prevent the postcastration decline in POMC mRNA content (castrated DHT-treated: 118 +/- 4 grains/cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-melanocyte-stimulating hormone antagonizes the neuroendocrine effects of corticotropin-releasing factor and interleukin-1 alpha in the primate.

alpha-Melanocyte stimulating hormone (alpha-MSH), a peptide derived from POMC has previously been shown to antagonize the action of exogenously administered beta-endorphin (beta-EP) on pituitary PRL and LH release in the primate. In this study, we have tested the ability of alpha-MSH to block some of the acute pituitary effects of CRF and interleukin-1 alpha (IL-1 alpha), effects which are thought in part to result from the release of endogenous beta-EP. Experiments were performed in ovariectomized rhesus monkeys bearing a chronically implanted lateral ventricular cannula for peptide infusion. Peripheral blood samples for LH, cortisol, and PRL RIA were obtained at 15-min intervals during a 3-h control period when saline was infused into the ventricle, followed by a 5-h experimental period. CRF (15 micrograms/h) infused alone for 5 h caused a significant suppression of pulsatile LH release; by the fifth hour, LH secretion was reduced to 32.5 +/- 2.4% of the control saline infusion. The CRF-induced suppression of LH was prevented by coinfusion of alpha-MSH (60 micrograms/h); by the fifth hour LH was 89.0 +/- 3.6% of the control (P less than 0.05 vs. CRF alone). alpha-MSH also prevented the CRF-induced decrease in LH pulse frequency (P less than 0.05). IL-1 alpha (4.2 micrograms) was infused alone for 30 min or in combination with alpha-MSH (120 micrograms/h for 2 h). After IL-1 alpha alone, LH decreased to 30.1 +/- 2.4% of baseline at 5 h. This decrease was prevented by alpha-MSH; by 5 h LH was 101 +/- 5.1% of baseline (P less than 0.005 vs. IL-1 alpha alone). IL-1 alpha did not affect LH pulse frequency but pulse amplitude was reduced; this reduction was prevented by alpha-MSH (P less than 0.05). IL-1 alpha also stimulated PRL release. PRL rose from a mean baseline of 3.5 +/- 0.3 ng/ml to a peak of 13.8 +/- 2.7 ng/ml; after coinfusion of alpha-MSH the mean peak PRL response was only 4.4 +/- 1.5 ng/ml (P less than 0.001 vs. IL-1 alpha alone). After CRF infusion, cortisol increased to 136 +/- 7.9% of the mean morning baseline concentration. This increase was not prevented by alpha-MSH coinfusion; after CRF plus alpha-MSH, cortisol increased to 121 +/- 6.0% of baseline. In contrast, alpha-MSH prevented the IL-1 alpha-induced increase in cortisol: 167 +/- 15.5% vs. 91.7 +/- 8.3% (P less than 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)     

Concentrations of immunoreactive luteinizing hormone releasing hormone in discrete brain regions of the cockerel: effects of castration and testosterone replacement therapy

The validity of using a radioimmunoassay employing an antiserum raised against synthetic luteinizing hormone releasing hormone (LH-RH) for the quantification of luteinizing hormone releasing factor (LH-RF) in birds was investigated. Extracts of avian hypothalamus yielded displacement curves which were parallel to that of the synthetic LH-RH standard and the immunoreactive potencies of a number of extracts assayed concurrently using two different anti-LH-RH sera were found to be similar. Moreover, after chromatography of cockerel hypothalamic extract on carboxymethyl-cellulose, immunoreactive and biologically active LH-RF were found in the same eluate fractions. Immunoreactive LH-RH was shown to be widely distributed in cockerel hypothalamus with the highest concentrations present in the mediobasal hypothalamus (MBH; 6.55 +/- 1.86 pg/microgram protein, n = 6) and medial preoptic region (POR; 0.95 +/- 0.07 pg/microgram protein, n = 6). The postcastration rise in plasma LH in the cockerel was accompanied by significant (P less than 0.05) increases in the concentration of LH-RH in five hypothalamic areas including the POR; testosterone replacement therapy completely reversed these effects. Although castration raised the mean concentration of LH-RH in four other hypothalamic areas including the MBH, these differences were not significant. However, testosterone replacement therapy depressed LH-RH in all four regions to levels significantly (P less than 0.05) less than those in castrated cockerels. These findings constitute the first direct evidence that the negative feedback action of testosterone on LH secretion in the cockerel is mediated, at least in part, by an action on hypothalamic LH-RF-producing neurones.     

Post-translational processing of pro-opiomelanocortin in the Brattleboro (di/di) rat pituitary

In homozygous (di/di) Brattleboro rats, pro-opiomelanocortin (POMC) synthesis and processing, and the release of its products, is by definition not under the control of hypothalamic arginine vasopressin (AVP) in contrast with normal rats or heterozygote (di/+) littermates. To explore the role of AVP on these parameters, we have compared homozygotes and heterozygotes in terms of POMC mRNA levels, pituitary content of immunoreactive (ir)-ACTH, ir-beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH) and ir-N-acetyl-EP (NacEP), plasma levels of ir-ACTH and ir-beta-EP, and RP-HPLC profiles of the various forms of ir-alpha-MSH and ir-NacEP. Homozygous rats had immeasurably low levels of hypothalamic ir-AVP, in contrast with their heterozygote littermates; hypothalamic ir-corticotropin releasing factor did not differ between strains. No difference between-strains was seen in levels of POMC mRNA; elevated levels of all pituitary peptides, except ir-ACTH, were found in di/di rats; plasma levels of both ir-ACTH and ir-beta-EP were lower in di/di rats; pituitary ir-alpha-MSH RP-HPLC profiles were similar in both strains, but those for ir-NacEP showed a striking increase in Nac alpha-EP in di/di rats. We interpret these data as evidence for decreased degradation/retarded release of POMC products from the di/di anterior pituitary, for increased processing of POMC products to shorter forms in both anterior pituitary and neuro-intermediate lobe, and thus for a role of AVP in the processing of POMC, as well as POMC synthesis and ACTH/beta-EP release.     

Effect of gonadectomy and reposition of gonadal steroids on alpha-melanocyte-stimulating hormone concentration in discrete hypothalamic areas during a 24-hour period

Hypothalamic alpha-melanocyte-stimulating hormone (alpha-MSH) was measured by radioimmunoassay in males after orchidectomy and after orchidectomy plus testosterone replacement, and in females after ovariectomy and after ovariectomy plus estradiol or progesterone, or estradiol and progesterone (EP) replacement. Gonadectomy inverted the diurnal rhythm of the alpha-MSH content observed in intact males in the medial basal hypothalamus (MBH) and preoptic hypothalamic area (POA), and produced a notable decrease of alpha-MSH total content in the three regions studied (MBH, POA and dorsolateral hypothalamus, DLH). The addition of testosterone restored the rhythm of the intact males and increased alpha-MSH content in MBH and POA. No diurnal variations in alpha-MSH content were observed in ovariectomized females. A circadian rhythm similar to that of proestrus was observed in MBH after estradiol or EP replacement, and in POA after the addition of any steroid. In DLH the injection of estradiol produced variations through the day, but they are somehow different from those described for proestrus. Treatment with progesterone significantly decreased alpha-MSH content in MBH and DLH, but not in POA. In this region an increase in alpha-MSH content was noticed after EP replacement. We conclude that gonadal steroids can alter the content of hypothalamic alpha-MSH and influence the diurnal variations of the peptide. This may be important in the modulation of several types of behavior or in the neuroendocrine control of gonadotropin release in females, where alpha-MSH seems to modulate the release of luteinizing hormone and prolactin.     

Further studies on the effects of testosterone on hypothalamic LH-RH and serum LH levels: castration-induced delayed response

We have previously reported that luteinizing hormone releasing hormone (LH-RH) levels in the medial basal hypothalamus (MBH) of adult male rats castrated for 2 weeks can be raised by testosterone (T) or estradiol (E2) treatment for 3-4 days. The present study was undertaken to determine the time after castration when the hypothalamus becomes refractory to this feedback action and whether prolongation of exposure to steroids would reinstate the hypothalamic LH-RH response. Treatment with T (Silastic capsules, s.c.) for 4 days, when commenced either immediately or 14 days after castration, significantly raised the MBH LH-RH levels. A 4-day regimen of E2 or dihydrotestosterone, on the other hand, evoked similar increase when commenced up to 21-28 days after castration. Further delay in institution of the 4-day treatment rendered the three steroids completely ineffective. When the effects of prolongation of treatment were tested in long-term castrated rats (56 days), it was found that T required 14 days as compared to 7 days of exposure with E2 or dihydrotestosterone to produce an equivalent MBH LH-RH response. Furthermore, the MBH LH-RH and serum LH responses were not always correlated, since increments in the MBH LH-RH were observed when serum LH levels were suppressed by physiological levels of T or dihydrotestosterone and also when they were unchanged with low levels of T or E2. These studies show that long-term castration causes a dichotomy in responsiveness of the hypothalamo-pituitary axis to steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen decreases rat hypothalamic proopiomelanocortin messenger ribonucleic acid levels

Ovariectomy and estrogen (E) or E plus progesterone treatment has previously been shown to alter both hypothalamic content and portal plasma levels of beta-endorphin. To determine if these changes were accompanied by changes in beta-endorphin synthesis, we used a RNA dot blot method to quantify proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus-median eminence region of rats. Animals were bilaterally ovariectomized, implanted with Silastic capsules containing E or oil, and killed 1 or 3 days after implantation. Total nucleic acid was isolated from dissections of the arcuate-median eminence by proteinase-K/sodium dodecyl sulfate/phenol extraction, and POMC mRNA was quantified by dot blot analysis. Although 1 day of E treatment had no effect on hypothalamic POMC mRNA levels, 3 days of E treatment caused a significant reduction of approximately 40% of POMC mRNA levels relative to oil controls in two replicate experiments. These results suggest that the decreases in hypothalamic POMC peptide levels after E administration reported previously may be due to a decrease in POMC peptide biosynthesis resulting from a decrease in hypothalamic POMC mRNA.     

Effect of steroid hormones and antihormones on hypothalamic beta-endorphin concentrations in intact and castrated female rats

The aim of the present study was to evaluate the effects of estrogens and androgens on hypothalamic beta-endorphin (beta-EP) concentrations. Intact or castrated female rats were chronically (2 weeks) treated with estrogen (estradiol benzoate) and/or antiestrogens (clomiphene, cyclophenil or epimestrol), and with androgens (dihydrotestosterone or dehydroepiandrosterone sulphate) and/or antiandrogen (cyproterone acetate). A group of rats treated with vehicle were studied as comparison. The beta-EP concentrations were measured by radioimmunoassay on acidic extracts of rat hypothalami. The administration of clomiphene and cyclophenil significantly reduced hypothalamic beta-EP concentrations in intact rats, while both drugs or estradiol benzoate increased the peptide concentration in castrated rats. Both intact and castrated rats treated with epimestrol showed hypothalamic beta-EP concentrations higher than vehicle treated rats. The estradiol-induced increase of beta-EP was not changed by the concomitant administration of antiestrogens. The administration of dihydrotestosterone significantly decreased beta-EP concentrations in both intact and castrated female rats, while the treatment with dehydroepiandrosterone sulphate only slightly decreased beta-EP levels in intact female rats. The cyproterone acetate-chronically treated rats showed higher beta-EP concentrations than vehicle-treated rats and these changes were reversed by the concomitant addition of dihydrotestosterone or dehydroepiandrosterone sulphate. These results showed that estrogens play a positive role while androgens negatively influence the hypothalamic beta-EP concentrations in female rats, supporting the view that central beta-EP might be a target of gonadal steroid feedback signals.     

In vivo hypothalamic release of thyrotropin-releasing hormone after electrical stimulation of the paraventricular area: comparison between push-pull perfusion technique and collection of hypophysial portal blood

Unilateral electrical stimulation for 15 min of the paraventricular area of anesthetized rats induced a 2- to 3- fold increase in plasma TSH levels and caused an increased release of TRH into hypophysial stalk blood from 217 +/- 25 to 530 +/- 90 pg/15 min (n = 6). This experimental model was then used to determine the in vivo hypothalamic release of TRH by push-pull perfusion of either the mediobasal hypothalamus (MBH) or anterior pituitary (AP). Before stimulation, TRH release per 15 min was 4.2 +/- 0.7 pg from the MBH (n = 18) and 3.5 +/- 0.3 pg from the AP (n = 13). Unilateral electrical stimulation of the paraventricular area led to higher plasma TSH levels in 27 of 31 rats, and levels during stimulation increased from 0.89 +/- 0.04 to 1.86 +/- 0.10 ng/ml (n = 31). No significant increase in TRH in the perfusates was observed when push-pull perfusion was done in the MBH contralateral to the site of stimulation (n = 6). However, TRH release increased 2- to 3-fold during the perfusion of the MBH ipsilateral to the site of stimulation (15.4 +/- 4.3 pg/15 min; n = 13). In conclusion, push-pull perfusion of the MBH or AP can be used to estimate hypothalamic TRH release. However, the output of TRH by push-pull perfusion is low and varies considerably between individual rats. Thus, the practical value of push-pull perfusion for measurement of in vivo TRH release seems limited.     

Variations in hypothalamic luteinizing hormone releasing hormone content and release in vitro and plasma concentrations of luteinizing hormone and testosterone in developing cockerels

An in-vitro superfusion system was used to study age-dependent changes in the functional activity of LH releasing hormone (LHRH) neurones terminating in the mediobasal hypothalamus (MBH) of the cockerel. Fragments of MBH tissue were obtained from cockerels killed at 8, 12, 16 and 20 weeks of age and both the rate of release of LHRH in vitro and the residual content of LHRH were determined by radioimmunoassay. Blood was collected from a similar group of cockerels of the same age for determination of plasma LH and testosterone concentrations. Superfused cockerel MBH showed both basal and depolarization-induced release of LHRH and the calcium-dependency of the release process was demonstrated. The viability of MBH in vitro was indicated by the observation that the rate of CO2 production by the tissue remained constant during a 3-h period of superfusion. Both the basal rate of release of LHRH (7.24 +/- 0.63 pg/2MBH per h; mean +/- S.E.M., n = 4) and the residual content of LHRH (1.10 +/- 0.24 ng/2MBH) were lowest in the 8-week-old cockerel and increased progressively to reach levels three (P less than 0.001) and 13 (P less than 0.001) times greater, respectively, by 20 weeks of age. Concentrations of LH and testosterone in plasma did not increase significantly until 16 weeks of age when the respective values were 5.86 +/- 0.37 micrograms/l and 1.88 +/- 0.31 nmol/l (n = 11). Whereas plasma testosterone increased further to 5.76 +/- 0.42 nmol/l (n equal 11) at 20 weeks of age, plasma LH fell significantly (P less than 0.02) to a concentration of 4.08 +/- 0.41 micrograms/l (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiestrogen action in the medial basal hypothalamus and pituitary of immature female rats: insights concerning relationships among estrogen, dopamine, and prolactin

We have examined the effects of nonsteroidal antiestrogens (AEs) and estradiol (E) on dopamine (DA) levels and turnover rates in the medial basal hypothalamus (MBH) and on serum and pituitary PRL to gain insight into DA-PRL-E/AE interrelationships. In 21-day-old female rats, E was found to increase MBH DA levels and turnover and serum PRL concentrations in a time- and concentration-dependent manner. Changes were observed by 1 day, and after 3 days of E treatment (1 microgram/day), MBH DA levels increased 2-fold (to 1300 pg/mg tissue), and DA turnover rates increased 5-fold (to 1170 pg/mg tissue . h). The AEs tamoxifen, monohydroxytamoxifen, CI628, and LY117018 (50 micrograms/day for 3 days) weakly stimulated uterine weight gain and significantly suppressed the uterotropic action of E. The AEs LY117018, monohydroxytamoxifen, CI628, and tamoxifen competed with E for binding to the MBH estrogen receptor and displayed relative binding affinities of 190%, 185%, 6.7%, and 1.4%, with E set at 100%; these affinities are similar to those found for uterine estrogen receptors. The AEs increased DA turnover rates only 2-fold, and they antagonized the E-induced 5-fold increase in DA turnover rates very successfully. In animals treated with bromocriptine, E and AE failed to increase the low serum PRL levels, yet they evoked significant (approximately 2-fold) increases in DA turnover rates and nearly 2-fold increases in MBH DA content. Hence, a part of the actions of E and AE on MBH DA appears to be exerted independently of changes in circulating PRL and may occur by direct action of these compounds on the estrogen receptor system present in the MBH. In addition, these studies reveal that AEs behave as partial estrogen agonists/antagonists in terms of their effects on MBH DA turnover.     

Chromatographic characteristics of pro-opiomelanocortin-derived peptides from the rat transplantable tumour 7315a

Adrenocorticotrophin (ACTH) and other pro-opiomelanocortin (POMC)-derived peptides produced by the 7315a corticomammotrophic tumour have been poorly studied although they elicit profound hypertrophy and hyperplasia in the adrenal glands of recipient Buffalo rats. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides by four radioimmunoassay (RIA) systems: ACTH, alpha-MSH, beta-lipotrophin (beta-LPH)/endorphin and N-terminal POMC (N-POMC). Chromatograms were compared with those of pars distalis extracts from normal Buffalo rats. All four RIA systems detected immunoreactive material in tumour extracts. ACTH, beta-LPH/endorphin and N-POMC were present in approximately equimolar amounts (ACTH content 93.40 +/- 5.27 (S.E.M.) pmol/g) whereas alpha-MSH was present in smaller amounts (2.83 +/- 0.13 pmol/g). Total peptide content correlated well with tumour weight. ACTH immunoreactivity (IR) in Sephadex chromatograms was located in a large 20,000 mol. wt peak, an ACTH(1-39) peak and a smaller peak coinciding with ACTH(1-24). The latter two peaks showed biological activity consistent with ACTH(1-39) and an ACTH (1-24)-like peptide respectively. The beta-LPH/endorphin RIA revealed a peak eluting at approximately 20,000 mol. wt which could not be ascribed to any known POMC peptide containing the endorphin sequence. A beta-LPH-like peak, a beta-endorphin-like peak and a smaller-sized peak, which contained the bulk of the beta-LPH/endorphin IR, were detected; the low molecular weight peak probably representing alpha- or gamma-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroidal regulation of hypothalamic neuropeptide Y release and gene expression.

Neuropeptide Y (NPY) readily stimulates the release of hypothalamic LHRH and pituitary LH release in intact and gonadal steroid-primed gonadectomized rats. We have now tested the hypothesis that the release and synthesis of hypothalamic NPY may be regulated by gonadal steroids. To measure the effects of gonadal hormones on NPY release, a permanent push-pull cannula was implanted in the anterior pituitary (AP) of sham castrated (controls) or castrated (CAST) male rats, and 1 week later, the AP was perfused with artificial cerebrospinal fluid over a 3-4 h period. NPY concentrations in the perfusates collected at 10-min intervals were measured by RIAs. The NPY release pattern in the AP was episodic in both intact and CAST rats, and the frequency of NPY episodes was similar in two groups. However, the amount of NPY detected in the AP of CAST rats was significantly less than that of intact rats because the mean rate of release and the amplitude of NPY episodes in the perfusates of CAST rats were significantly reduced. This observation of attenuated hypothalamic NPY output in vivo and previous evidence of decreased hypothalamic NPY contents after CAST implied that the synthesis of hypothalamic NPY may be regulated by testicular secretions. Therefore, the effects of testosterone (T)-replacement on preproNPY messenger RNA (mRNA) in the medial basal hypothalamus (MBH) was evaluated. Rats were CAST and received either empty or T-filled Silastic capsules sc. Two weeks later, the level of perproNPY mRNA in the MBH was determined by solution hybridization/ribonuclease protection assay using a complementary RNA probe complementary to the rat NPY precursor mRNA. We observed that the levels of preproNPY mRNA were 2-fold higher in the MBH of T-replaced CAST as compared to control CAST rats. These findings are consistent with the hypothesis that gonadal steroids enhance the neurosecretory activity of hypothalamic NPYergic neurons, and for the first time reveal a coupling between the level of gene expression and the secretion of a neuropeptide involved in the regulation of hypothalamic LHRH and pituitary LH release.     

A model for combined morphological and functional investigations on the isolated mediobasal rat hypothalamus

We have developed a model for combined morphological and functional in vitro studies of the isolated mediobasal hypothalamus (MBH) by considering two prerequisites: (1) the tissue must be well preserved, free of morphological artefacts and functionally unimpaired until the end of the in vitro incubation, and (2) the tissue must be processed for morphology in optimal conditions. To test our model we have studied some aspects of the luteinizing hormone-releasing hormone (LHRH) system in 4-month-old male Sprague-Dawley rats. After decapitation the MBH was isolated and put in a flask containing 0.5 ml Hepes-buffered Locke's medium gassed by 5 ml/min of O2/CO2 (95%/5%) and shaken in a water bath at 37 degrees C. After a 10-min washing, the medium was changed twice at an interval of 20 min. After the in vitro incubation the tissue was satisfactorily preserved as judged by light- and electron-microscopic analysis. LHRH, somatostatin and thyrotropin-releasing hormone could be demonstrated by alkaline phosphatase or peroxidase-antiperoxidase immunohistochemistry on semithin sections and by immunogold technique on thin sections. The LHRH secretion was close to basal values after 30 min of incubation (22.1 +/- 4.8 pg/MBH) and then remained constant for another period of 20 min (17.6 +/- 2.6 pg/MBH). During the second 20 min of incubation LHRH secretion increased in presence of 61.6 mM K+ (110.7 +/- 8.7 pg/MBH). Thus the isolated hypothalamus was excitable until the end of the in vitro incubation. We conclude that this model can be successfully used for combined morphological and functional studies.