Pituitary Hormone Gene Expression in Male Golden Hamsters: Interactions Between Photoperiod and Testosterone

Daylength regulates neuroendocrine function in male golden hamsters. Exposure to short days triggers gonadal regression and decreases serum luteinizing hormone (LH), prolactin and testosterone concentrations. Inhibitory photoperiods also amplify the negative feedback actions of androgens upon gonadotropin secretion. To examine whether these changes arise from altered adenohypophyseal gene expression, we measured the abundance of the messenger ribonucleic acids (mRNAs) encoding β-LH, prolactin and proopiomelanocortin in anterior pituitaries of male golden hamsters which were either left intact, castrated, castrated and implanted with testosterone, or pinealectomized and maintained in either long (14 h light/10 h dark) or short (5 h light/19 h dark) days. Short days caused testicular atrophy in intact male hamsters and reduced serum LH in intact and castrated, testosterone-replaced hamsters. The relative abundance of β-LH mRNA was suppressed by exposure to short days only in castrated hamsters. Serum prolactin was decreased by short days regardless of circulating testosterone concentrations. Prolactin mRNA abundance was decreased by short days in all pineal-intact groups. Castration reduced proopiomelanocortin mRNA abundance in long days and testosterone replacement reversed this effect. In the presence of testosterone, photoperiod influenced serum LH concentrations without altering hypophyseal abundance of β-LH mRNA. In contrast, photoperiodic influences on prolactin secretion were correlated with alterations in steady-state mRNA abundance.     

Influence of Testosterone on LHRH Release,LHRH mRNA and Proopiomelanocortin mRNA in Male Sheep1

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated +T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P < 0.01) mean LHRH and LH and decreased (P < 0.01) LHRH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron, but decreased (P < 0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P= 0.057) silver grain area per LHRH neuron without significantly affecting (P > 0.10) LHRH cell numbers while reducing (P < 0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day;n=4). Blood samples were collected at 10 min intervals for 4h prior to and during the final 4 h of infusion. Infusion of T decreased (P < 0.01) mean LH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P < 0.01) silver grain area per POMC neuron without affecting (P > 0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.     

Patterns of amygdaloid and arcuate electroencephalograms associated with puberty in the female rat

At puberty the female rat changes from acyclicity to the adult pattern of regular estrous cycles. Stimulating or ablating the hypothalamus or amygdala affects the age at which puberty occurs. Spectral analysis was applied to the EEGs of the cortex, medial amygdala, and arcuate nucleus of the hypothalamus in prepubertal and pubertal animals to describe those spontaneously occurring events which may be associated with puberty. Fifty to one hundred 10-s samples of EEG recorded between 1100 and 1800 were analyzed for each of 2 to 3 days from 10 animals at or near puberty. Average daily values for 80 variables were compared and the directions of significant (P < 0.01) changes were correlated with the occurrence of the first proestrus. Intensitities of the 1- to 5-Hz arcuate and cortical EEGs decreased in prepubertal animals, and that of 6- to 25-Hz activity in all three regions increased. Coherence between 1- and 5-Hz amygdaloid and arcuate activity increased as puberty approached, suggesting that communication between these regions participates in preparations for puberty. In animals exhibiting proestrus, this day was characterized by maximal intensity of 1- to 5-Hz amygdaloid EEG and by 1- to 5-Hz arcuate activity higher than that at estrus. Minimal coherences in the 6- to 25-Hz range at proestrus indicated the occurrence in both amygdaloid and arcuate activity of signals specific to the day of proestrus but which are unshared temporally, spatially, or both.     

Stimulation of Luteinizing Hormone-Beta Messenger Ribonucleic Acid and Post-Translational Modification of Luteinizing Hormone Isoforms by Second Messengers Mediating the Action of Gonadotrophin-Releasing Hormone

Several second messenger systems have been implicated in mediating the action of gonadotrophin-releasing hormone on the pituitary gonadotrophs and numerous studies have shown that activation of these systems induces luteinizing hormone (LH) secretion. However, it is not known how gonadotrophin-releasing hormone or the second messenger systems induce de novo LH biosynthesis and post-translational modification of the hormone. In these experiments hemipituitary glands have been perifused with drugs which activate second messengers or stimulate protein kinase C directly. The LH secretory responses have been correlated with measurements of common a and LHβ mRNA and the molecular species of LH which were present in the pituitary perifusate after exposure to the drugs. Gonadotrophin-releasing hormone (50 ng/ml, 42 nM), with and without the presence of extracellular Ca²⁺, the Ca²⁺ ionophore, A23187 (10 μM), and phorbol 12-myristate (1 μM) all stimulated an increase in LHβ mRNA compared with controls and the appearance of a different isoform of LH to that found stored in and released from the unstimulated pituitary gland. Phospholipase C was without effect on LHβ mRNA levels and showed minimal efficacy in inducing the appearance of the different LH isoform.     

Repeated immobilization stress alters tyrosine hydroxylase, corticotropin-releasing hormone and corticosteroid receptor messenger ribonucleic Acid levels in rat brain

In situ hybridization histochemistry was used to localize and quantify the effects of acute and repeated immobilization stress on mRNA levels of tyrosine hydroxylase (TH) in catecholaminergic neurons in the locus ceruleus and substantia nigra and on mRNA levels of relevant markers of the hypothalamic-pituitary-adrenal axis, namely corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN), proopiomelanocortin in the pituitary, and mineralocorticoid receptors (MR, type I) and glucocorticoid receptors (GR, type II) in the hippocampus, PVN and pituitary. Control, acutely stressed (1 × lMO, sacrificed immediately after 2 h of immobilization), and repeatedly stressed (6 × IMO plus delay, sacrificed 24 h after 6 daily 2-h immobilizations and 6 × lMO plus challenge, sacrificed immediately after the seventh daily 2-h immobilization) male Sprague-Dawley rats were examined. TH mRNA expression was increased in the locus ceruleus in the acutely stressed and repeatedly stressed animals. The increase in TH mRNA levels was greatest in the repeatedly stressed (6 × IMO plus challenge) group. TH mRNA levels were not altered in the substantia nigra. CRH mRNA levels in the PVN were significantly increased in the three stressed groups and the increase was greatest in the 6 × IMO plus challenge group. CRH mRNA levels were increased in the central nucleus of the amygdala only after acute stress. Proopiomelanocortin mRNA levels were elevated in the anterior pituitary during acute and repeated stress, but the magnitude of the effect was largest after acute stress. The changes in the hypothalamic-pituitary-adrenal axis were accompanied by an acute stress-induced increase in MR mRNA levels in the hippocampus, MR and GR mRNA levels in the PVN and GR mRNA levels in the pituitary. MR mRNA levels continued to be elevated in the PVN in the 6 × IMO plus challenge animals. Plasma corticosterone levels were elevated in the acute and repeated stress conditions. The results show that repeated immobilization stress produces a rapid and persistent increase in mRNA expression of TH in the locus ceruleus, CRH in the PVN, and proopiomelanocortin in the anterior pituitary. The TH-containing neurons in the locus ceruleus and the CRH-containing neurons in the PVN appear to preserve the capability to respond to repeated stimulation (6 × IMO plus challenge) indicating altered feedback mechanisms under repeated stress conditions. GR and MR mRNA levels are differentially regulated in the hippocampus, PVN and pituitary by acute and repeated stress. It is of interest that the central nervous system systems which are activated during repeated stress, namely the locus ceruleus-norepinephrine system and hypothalamic-pituitary-adrenal axis, are dysregulated in melancholic depression. Further studies of the central nervous system effects of prolonged exposure to stress may help elucidate the mechanisms underlying dysregulation of the locus ceruleus-norepinephrine system and hypothalamic-pituitary-adrenal axis in depression and other stress-related psychiatric diseases.     

Acute Action of Oestrogen on Medial Preoptic Gamma-Aminobutyric Acid Neurons: Correlation with Oestrogen Negative Feedback on Luteinizing Hormone Secretion

The acute effects of oestrogen on the medial preoptic area (MPOA) γ-aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17β-oestradiol (5 μg/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen-min blood samples were taken to determine the time-course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17β-oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle-treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17β- oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17β-oestradiol compared with controls (P<0.05). To determine the effects of the 17β-oestradiol treatment on pituitary responsiveness to LH-releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17β-oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17β-oestradiol-treated rats was approximately 50% that of control-treated values. This study demonstrates the acute and potent action of 17β-oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.     

Effect of Hypophysectomy and Growth Hormone Replacement on Hypothalamic Growth Hormone-Releasing Factor Messenger Ribonucleic Acid Levels

The mechanisms by which the pituitary gland, and growth hormone (GH) in particular, affect growth hormone-releasing factor (GRF) gene expression have been addressed using the technique of in situ hybridization. Anatomically matched sections through the mediobasal hypothalamus of control and hypophysectomized male rats, with or without GH hormone replacement, were analysed to obtain information on GRF mRNA levels within the arcuate nucleus and around the ventromedial hypothalamus. Hypophysectomy resulted in a 70% increase in the amount of GRF mRNA per cell (P<0.001), within neurons in the arcuate nucleus. GH replacement and T4 replacement separately partially attenuated this increase (GH replacement P< 0.001 versus hypophysectomy, T4 replacement P<0.05 versus hypophysectomy). Additionally, after hypophysectomy there was an 80% increase in the number of cells expressing the GRF gene in neurons around the ventromedial hypothalamus, when compared to shamoperated controls (P<0.01). Both GH and T4 replacement separately partially attenuated this phenomenon (P<0.01 versus hypophysectomized animals). Hypothyroidism alone did not affect GRF mRNA levels in either the arcuate nucleus or in the area surrounding the ventromedial hypothalamus. These results show that hypophysectomy increases GRF mRNA levels in two separate ways: by increasing the amount of mRNA produced per cell within the arcuate nucleus, and by increasing the number of cells expressing the gene in the area surrounding the ventromedial hypothalamus. This increase in the number of GRF mRNA-containing cells after hypophysectomy could result from the recruitment of neurons which previously did not express the GRF gene, and may reflect the plasticity of the adult central nervous system in response to a changing endocrine environment. This could represent part of a sensor mechanism to drive the production of GRF in the arcuate nucleus in response to extreme disruption of the GRF/ GH feedback loop.     

Oestrogen receptor beta-immunoreactivity in gonadotropin releasing hormone-expressing neurones: regulation by oestrogen

Double-label immunohistochemistry was employed to establish whether immunoreactivity for the beta subtype of the oestrogen receptor (ER beta-IR) is present in gonadotropin releasing hormone (GnRH)-containing cells. In the immortalized GnRH cell line, GT1-7, almost all nuclei were immunoreactive for ER beta. In the preoptic area of ovariectomized rats, more than one-half of the GnRH neurones (52.0-63.5%) contained ER beta-IR within the nucleus; a smaller proportion of these neurones (5-10%) displayed a particularly intense nuclear signal for ER beta. The presence of ER beta-IR in the nuclei of GT1-7 cells and GnRH neurones is consistent with recent reports of ER beta mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ER beta-IR. The range of signal intensity for ER beta and the incidence of the ER beta signal in GnRH neurones were comparable following double-label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ER beta receptors mediate direct effects of oestrogen on GnRH neurones.     

Evidence for Oestrogen Receptor α-Immunoreactivity in Gonadotrophin-Releasing Hormone-Expressing Neurones

Having used the cingulate cortex to demonstrate the validity of our methods for detecting hitherto unrecognized oestrogen receptor alpha (ERalpha)-immunoreactive neurones, we have now employed immunoprecipitation and double-label immunohistochemistry to investigate whether the ERalpha protein is present in gonadotrophin-releasing hormone (GnRH)-containing cells. The immortalized GnRH cell line GT1-7 and GnRH neurones within the rat preoptic area were found to possess ERalpha-immunoreactivity (ERalpha-IR). These observations indicate that oestrogen may regulate the synthesis and release of GnRH by direct actions on GnRH neurones.     

Dysregulation of the Hypothalamus-Pituitary-Adrenal Axis in Male and Female, Genetically Obese (ob/ob) Mice

The autosomal, recessive obesity of ob/ob mice is associated with hypercorticosteronemia and amelioration of most symptoms of obesity following adrenalectomy. Increased adrenocorticotropic hormone (ACTH) secretion has been hypothesized on the basis of several reports of higher pituitary ACTH content in ob/ob mice compared to lean littermates. However, the only measurement of ACTH blood concentration found lower levels in ob/ob mice than in leans suggesting that hypercorticosteronemia might result solely from an enhanced adrenal response to ACTH and also suggesting that the ob/ob's elevated pituitary ACTH content might be due to decreased ACTH secretion rather than increased ACTH synthesis. In our study, basal serum ACTH levels were higher in ob/ob males and females compared to sex-matched lean littermates. Anterior pituitary ACTH synthesis was also elevated as indicated by increased content of ACTH and proopiomelanocortin mRNA in obese mice of both sexes; however hypothalamic corticotropin-releasing factor content was not different in lean and obese mice. Basal serum ACTH and corticosterone (CS) levels showed normal circadian rhythm in both phenotypes and sexes, but the circadian increase in CS level was much greater in obese mice than in leans despite equal serum ACTH increases in the two phenotypes. Ether stress at both peak and trough of the circadian rhythm also stimulated much larger serum CS increases in obese mice even though ACTH increases were again equal in the two phenotypes. Taken together, these results strongly indicate that ob/ob mice have increased synthesis and secretion of pituitary ACTH despite the presence of chronically elevated serum CS. This hyperactivity of the hypothalamo-pituitary-adrenal axis appears to be most pronounced in ob/ob females since pituitary ACTH content was equal in obese males and females despite much higher circulating CS levels in the females. Furthermore, the results also indicate an enhanced response to ACTH by the adrenal cortex of the obese mouse. Thus, ob/ob mice exhibit abnormal hypothalamo-pituitary-adrenal axis function with hyperactivity occurring at the level of pituitary ACTH synthesis/secretion as well as at the level of adrenocortical response to ACTH.     

Cellular levels of messenger ribonucleic acids encoding vasoactive intestinal Peptide and gastrin-releasing Peptide in neurons of the suprachiasmatic nucleus exhibit distinct 24-hour rhythms

There is strong evidence supporting the view that the Suprachiasmatic nucleus (SCN) functions as a circadian clock; however, the neural and molecular events underlying SCN function remain unclear. A specific subpopulation of neurons within the ventrolateral aspect of the SCN that contains three peptides, vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP), play an important role in SCN function. VIP-containing neurons of the SCN receive synapses from photic projections, and co-injection of all three peptides mimics the phase-delaying effects of light on circadian activity rhythms. In principle, the signaling potential of a neuron containing several transmitters may be affected by the concentration ratio of co-released factors; hence, one mechanism by which VIP/PHI/GRP-containing neurons could influence SCN function is by changing the concentration ratio of these peptides throughout the light-dark cycle. The present study was performed to examine this possibility. Relative cellular levels of mRNA encoding both VIP/PHI and GRP were determined within the SCN every 4 h in rats housed in a 14 h light: 10 h dark cycle. Quantitative in situ hybridization revealed a statistically significant (P<0.005) 24-h profile of changes in VIP/PHI mRNA that peaked during the dark phase, and a significant (P<0.005) 24-h profile of changes in GRP mRNA that peaked during the light phase. These data support the interpretation that cellular levels of mRNAs encoding VIP/PHI and GRP within the SCN exhibit distinct profiles of changes throughout the light-dark cycle. Further, these findings are consistent with the hypothesis that the concentration ratio of VIP and PHI to GRP changes over the light-dark cycle, and that this may be an important mechanism by which circadian rhythms are generated or entrained.     

Involvement of central catecholamines in the feedback actions of 17beta-estradiolbenzoate on luteinizing hormone secretion in the ovariectomized female rat.

(1) Evidence has been presented, based on quantitative microfluorimetric estimations of dopamine (DA) and noradrenaline (NA) levels and turnover, and on radioimmunological measurements of serum luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and prolactin levels, that the central inhibitory feedback action of estradiol on LH secretion mainly involves a marked increase in DA turnover of the lateral palisade zone (LPZ) of the median eminence and also involves a reduction of NA turnover, mainly located in the medial preoptic area (MPOA) and in the subependymal layer (SEL). (2) The mechanism for these changes in catecholamine (CA) turnover is discussed. The possibility is favored that they involve the stimulation of central cytosol estrogen receptors which may even be located in the arcuate DA and possibly reticular NA cell bodies themselves. (3) The marked and long-lasting hypersecretion of prolactin caused by estrogen could be involved in mediating the sustained increase of DA turnover in the median eminence and the sustained reduction of NA turnover. (4) The central facilitatory feedback action of estrogen, on the other hand, may be mainly responsible for the sharp increase of NA turnover in the MPOA and SEL and also the associated reduction of DA turnover in the LPZ in the critical period of both adult cyclic rats and of immature female rats treated with PMS. (5) We take the view that these latter NA and DA turnover changes take precedence over cytosol estrogen receptors, which previous evidence indicates (see Sawyer, 1975) are located in preoptic and amygdaloid regions. (These estradiol concentrating neurons then directly and/or indirectly make connections with the NA and DA pathways). (6) FSH secretion is not controlled by DA and NA pathways. (7) The hypothesis given above is based on the assumption that different estradiol concentrating neurons are involved in the central inhibitory and facilitatory feedback action of estradiol and that the estrogen receptors of these respective neurons have differences which allow their differential activation, the inhibitory feedback leading mainly to a marked increase in the ratio DA activity/NA activity in the median eminence, whereas the facilitatory feedback causes a marked reduction of this ratio.     

Effect of Ovariectomy or Oestrogen Implants upon Pituitary Function in Female Hypogonadal Mice Bearing Normal Fontal Preoptic Area Grafts

The effects of ovariectomy and oestrogen feedback for 10 days upon pituitary and serum luteinizing hormone (LH) content, pituitary glycoprotein subunit messenger ribonucleic acid (mRNA) and prolactin mRNA content in normal females, female hypogonadal mice and hypothalamic grafted female hypogonadal mice, bearing a graft of normal mouse preoptic area tissue into the third ventricle, have been investigated. In normal females ovariectomy resulted in a rise in serum LH, LHβ-subunit and common α-subunit mRNAs with no significant change in pituitary LH content or follicle-stimulating hormone (FSH) β-subunit mRNA. In the hypogonadal females, preoptic area grafting resulted in an elevation in all of the above parameters into the normal range. Ovariectomy in this group resulted in a further elevation of serum LH, LHβ-subunit and α-subunit mRNAs with no change in pituitary LH content or FSHβ-subunit mRNA, which in all cases were comparable to ovariectomized normal animals. Oestrogen treatment caused a fall in pituitary LH content and the serum LH fell below the detection of the assay. LHβ-subunit and a-subunit mRNA mirrored this fall but there was no change in FSHβ-subunit hybridization. These experiments suggest that even though normal neuronal input to the gonadotrophin-releasing hormone neurons is disrupted, oestrogen-induced negative feedback can still occur in grafted female hypogonadal animals. Gonadotrophin-releasing hormone neurons are reported to lack oestrogen receptors but feedback within this graft by co-transplanting oestrogen-sensitive neurons remains a possibility, as does feedback at the level of the host median eminence where graft axons extend to the pituitary portal vessels. The similarity of the response in normal and grafted animals indicates that these actions of oestrogen may be effected predominantly at the pituitary level.     

N-Methyl-DL-Aspartate Receptor Antagonism by D-2-Amino-5-Phosphonovaleric Acid Delays Onset of Puberty in the Female Rat

To investigate the possible role of N-methyl-DL-aspartate (NMDA) receptor activation in the initiation of puberty, we examined the effects of the selective competitive antagonist 2-amino-5-phosphonovaleric acid (AP5) on the timing of vaginal opening. Paired and weight-matched litter mates of immature female rats were implanted with osmotic minipumps for the intracerebroventricular infusion of DL- or D-AP5 or artificial cerebrospinal fluid from 27 to 30 days of age for 14 days. Each animal was weighed and examined daily for vaginal opening as the indicator of first oestrous. Infusion of 20 or 40 mM DL-AP5 beginning on Day 30 failed to delay vaginal opening. Administration 50mM of the single enantiomer D-AP5 beginning on Day 27 significantly delayed the age of vaginal opening to 40.6±1.1 (mean ± SEM) days compared to the cerebrospinal fluid-infused controls (36.5 ± 0.6 days). Blockade of NMDA receptors in the D-AP5-treated animals was confirmed on Day 32 by the suppression of luteinizing hormone response to intravenous NMDA (20 mg/kg) while the response to exogenous luteinizing hormone-releasing hormone (50 ng/kg) remained intact. AP5-treated animals had a slower rate of growth (3.1 ± 0.2 g/day) compared to controls (4.2 ± 0.2 g/day). However, a similar degree of growth retardation produced by a 75% restricted diet in untreated juvenile animals did not delay vaginal opening. This suggests that the slower growth rate in the D-AP5-treated animals could not account for the delayed onset of puberty. In conclusion, these data suggest that blockade of central NMDA receptors inhibits excitatory mechanisms which may be important in the control of pubertal onset in the female rat.