Hypothalamo-pituitary Disconnection Research Articles

The Regulation of Prolactin Receptor Messenger Ribonucleic Acid Levels in the Sheep Liver before Birth: Relative Roles of the Fetal Hypothalamus, Cortisol, and the External Photoperiod

We have investigated the separate actions of hypothalamo-pituitary disconnection (HPD), with or without cortisol administration, and changes in the external photoperiod on the regulation of the levels of messenger RNA (mRNA) encoding long (PRLR1) and short (PRLR2) forms of PRL receptor in the liver of the fetal lamb. In pregnant Merino ewes (n = 20), the hypothalamus and pituitary were surgically disconnected in 13 fetuses (HPD group), and fetal vascular catheters were implanted in the HPD group and in an additional 7 fetuses (intact+ saline group) between 104–120 days gestation (d). Fetal sheep in the HPD group were infused with either cortisol (3.5 mg/4.8 ml saline/24 h; HPD + F; n = 5) or saline for 5 days between 134–141 d, and saline was also infused in the intact group within the same gestational age range. A second group of pregnant ewes (n = 12) was kept in a 12-h light, 12-h dark cycle from 70 d until implantation of fetal vascular catheters between 106–120 d, after which ewes were allocated to either a long photoperiod (16 h of light, 8 h of darkness; LL group; n = 6) or a short photoperiod (8 h of light, 16 h of darkness; SL group; n = 6) regimen. Circulating cortisol concentrations were higher (P < 0.05) in the intact fetal sheep (18.7 ± 3.8 nmol/liter) than in the HPD + saline group (1.5 ± 0.6 nmol/liter), and were further increased (P < 0.05) in the HPD + cortisol group (97.4± 23.7 nmol/liter). Fetal PRL concentrations were lower (P < 0.05) in the HPD + saline (10.6 ± 4.3 ng/ml) and HPD ± cortisol (5.6 ± 2 ng/ml) groups compared with those in the intact group (38.9 ± 6.8 ng/ml). The levels of hepatic PRLR mRNA were higher (P < 0.05) in the intact (PRLR1, 27.4 ± 6.1; PRLR2, 17.7 ± 2.5) and HPD + cortisol (PRLR1, 23.4 ± 0.4; PRLR2, 15.3 ± 3.0) groups than in the HPD + saline group (PRLR1, 10.6 ± 1.8; PRLR2, 8.9 ± 1.8) at 140/141 d. The mean plasma PRL concentration in the LL group (70 ± 9 ng/ml) was higher (P < 0.05) than that in the SL group (34 ± 15 ng/ml), whereas the levels of hepatic PRLR1 mRNA (LL group, 4.6 ± 0.9; SL group, 4.3 ± 0.8) and PRLR2 mRNA (LL group, 3.4 ± 0.4; SL group, 3.0 ± 0.5) at 140–141 d were not different. These data indicate that cortisol acts directly or indirectly to maintain hepatic PRLR mRNA levels in the sheep fetus during late pregnancy. In contrast, changes in the external photoperiod and circulating PRL concentrations in the sheep fetus do not directly alter PRLR expression in the fetal liver. These studies provide further insight into the role that the PRL axis may play in the transduction of signals about the external environment to the fetus as it prepares for the transition to extrauterine life.

The regulation of prolactin receptor messenger ribonucleic acid levels in the sheep liver before birth: relative roles of the fetal hypothalamus, cortisol, and the external photoperiod.

We have investigated the separate actions of hypothalamo-pituitary disconnection (HPD), with or without cortisol administration, and changes in the external photoperiod on the regulation of the levels of messenger RNA (mRNA) encoding long (PRLR1) and short (PRLR2) forms of PRL receptor in the liver of the fetal lamb. In pregnant Merino ewes (n = 20), the hypothalamus and pituitary were surgically disconnected in 13 fetuses (HPD group), and fetal vascular catheters were implanted in the HPD group and in an additional 7 fetuses (intact + saline group) between 104-120 days gestation (d). Fetal sheep in the HPD group were infused with either cortisol (3.5 mg/4.8 ml saline/24 h; HPD + F; n = 5) or saline for 5 days between 134-141 d, and saline was also infused in the intact group within the same gestational age range. A second group of pregnant ewes (n = 12) was kept in a 12-h light, 12-h dark cycle from 70 d until implantation of fetal vascular catheters between 106-120 d, after which ewes were allocated to either a long photoperiod (16 h of light, 8 h of darkness; LL group; n = 6) or a short photoperiod (8 h of light, 16 h of darkness; SL group; n = 6) regimen. Circulating cortisol concentrations were higher (P < 0.05) in the intact fetal sheep (18.7 +/- 3.8 nmol/liter) than in the HPD + saline group (1.5 +/- 0.6 nmol/liter), and were further increased (P < 0.05) in the HPD + cortisol group (97.4 +/- 23.7 nmol/liter). Fetal PRL concentrations were lower (P < 0.05) in the HPD + saline (10.6 +/- 4.3 ng/ml) and HPD + cortisol (5.6 +/- 2 ng/ml) groups compared with those in the intact group (38.9 +/- 6.8 ng/ml). The levels of hepatic PRLR mRNA were higher (P < 0.05) in the intact (PRLR1, 27.4 +/- 6.1; PRLR2, 17.7 +/- 2.5) and HPD + cortisol (PRLR1, 23.4 +/- 0.4; PRLR2, 15.3 +/- 3.0) groups than in the HPD + saline group (PRLR1, 10.6 +/- 1.8; PRLR2, 8.9 +/- 1.8) at 140/141 d. The mean plasma PRL concentration in the LL group (70 +/- 9 ng/ml) was higher (P < 0.05) than that in the SL group (34 +/- 15 ng/ml), whereas the levels of hepatic PRLR1 mRNA (LL group, 4.6 +/- 0.9; SL group, 4.3 +/- 0.8) and PRLR2 mRNA (LL group, 3.4 +/- 0.4; SL group, 3.0 +/- 0.5) at 140-141 d were not different. These data indicate that cortisol acts directly or indirectly to maintain hepatic PRLR mRNA levels in the sheep fetus during late pregnancy. In contrast, changes in the external photoperiod and circulating PRL concentrations in the sheep fetus do not directly alter PRLR expression in the fetal liver. These studies provide further insight into the role that the PRL axis may play in the transduction of signals about the external environment to the fetus as it prepares for the transition to extrauterine life.

Adrenal responsiveness and the timing of parturition in hypothalamo-pituitary disconnected ovine foetuses with and without constant adrenocorticotrophin infusion.

Ovine parturition results from an increase in foetal cortisol secretion in late gestation which is dependent on an intact hypothalamo-pituitary connection. The cortisol surge and parturition fails in hypothalamo-pituitary disconnected (HPD) foetuses but, paradoxically, immunoreactive (ir)-ACTH concentrations and secretory dynamics appear normal. This study compares the occurrence and timing of labour, basal ir-ACTH and cortisol concentrations and adrenal responsiveness in HPD foetuses (HPD/ACTH) receiving constant ACTH(1-24) infusion (43 ng/h/kg) from surgery (114+/-1 days gestational age (GA)) with those of saline-infused HPD or intact foetuses (HPD/SAL and INT/SAL). HPD/ACTH foetuses initiated labour at 147+/-2 days GA, which was not significantly different from INT/SAL foetuses (149+/-1 day GA). HPD/SAL foetuses were killed electively at 146+/-3 days GA with no signs of labour. Foetal ir-ACTH concentrations in all groups were indistinguishable, but only HPD/ACTH and INT/SAL foetuses had a significant cortisol surge. Adrenal responsiveness to ACTH(1-24)(1 microg/kg) was greater in HPD/ACTH foetuses than in HPD/SAL or INT/SAL foetuses at all GAs studied. Adrenal responsiveness in HPD/SAL foetuses exceeded that in INT/SAL foetuses at 120 and 130 days GA but did not change with GA. In summary, the basal cortisol and parturition defect in HPD foetuses was reversed by low-dose ACTH(1-24) infusion. Basal cortisol concentrations were unrelated to adrenal responsiveness. HPD/SAL foetuses had hyper-responsive adrenals compared to those of INT/SAL foetuses until 130 days GA, suggesting that the foetal hypothalamus exerts a negative influence on adrenal cortisol responses before 130 days GA, after which time stimulatory influences predominate.

Review: Central non-glucocorticoid inhibitors of the hypothalamo-pituitary-adrenal axis.

The popular perception of stress is that of social or work tensions and pressures that have an overall negative influence often accompanied by increased frequency of illness. However, stress is not all bad. A healthy response to acute stress is crucial for our interaction with the environment and for our ability to deal with everyday challenges, either physical or psychological. The key is to be able to terminate a response to stress when the stressor is no longer present. Failure to terminate a response to acute stress can result in the syndrome of chronic stress which is associated with inappropriately elevated secretion of glucocorticoids, with consequent immunosuppression and predisposition to infection and illness. Responses of the hypothalamo–pituitary–adrenal (HPA) axis to stress are mediated principally through the neuropeptides corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) which are synthesised in the parvocellular subdivision of the hypothalamic paraventricular nucleus (PVN) (for review see Buckingham et al. 1997). CRH and AVP are transported down axonal projections to the median eminence (ME) where they are secreted into the hypophysial portal blood system to synergistically stimulate the release of adrenorticotrophin (ACTH) from corticotroph cells in the anterior pituitary, leading to increased secretion of glucocorticoids from the adrenal cortex (Fig. 1). Glucocorticoid feedback at the hippocampal and hypothalamic levels is a well-recognised mechanism for inhibiting basal and stress-induced HPA axis activity (for a review see De Kloet et al. 1998), but there is also considerable evidence for the existence of non-glucocortocoid mechanisms of inhibition. In adrenalectomised animal models, basal plasma ACTH concentrations are elevated but not maximally, since stressors such as ether (Rees et al. 1971), restraint (Chowdrey et al. 1991) and haemorrhage (Darlington et al. 1990) are capable of further stimulating ACTH release. In addition, a chronic osmotic stimulus inhibits ACTH release through a mechanism which, because it occurs in adrenalectomised rats, cannot be regulated by glucocorticoids ( Jessop et al. 1990). Thus the HPA axis is subject to mechanisms of tonic inhibition which operate independently of glucocorticoids. Inhibitory agents may act at any or all levels of the HPA axis. Lesioning of selective hypothalamic nuclei can enhance the HPA axis response to some stressors (Herman & Cullinan 1997), providing evidence for endogenous hypothalamic inhibitors acting on CRH and/or AVP expression within the PVN. Observations of pulsatile ACTH secretion after complete lesioning of the PVN (and elimination of CRH from the ME) have revealed a surprising degree of residual pituitary–adrenocortical activity (Boyle et al. 1997). Total PVN lesioning was still accompanied by an ACTH response to some cytokines (Kovacs & Elenkov 1995), and electrical lesions that destroyed the entire hypothalamus did not prevent a response to the stress of laparotomy (Witorsch & Brodish 1972). One explanation for these results is that the lesions eliminate not only hypothalamic corticotrophin-releasing factors but also inhibitory factors which may be acting to block the secretion of ACTH from the anterior pituitary. Further evidence for the release of ACTH inhibitors into hypophysial portal blood was supplied by the report that hypothalamo–pituitary disconnection in sheep was accompanied by increased ACTH and cortisol secretion (Engler et al. 1988). Although some of these results could be explained by the existence of novel extrahypothalamic ACTH-releasing factors, cumulative evidence is compelling for the existence of compounds acting on the hypothalamus and/or anterior pituitary that can inhibit the stress response. The purpose of this review is to distil the literature on non-glucocorticoid inhibitors of the HPA axis (briefly reviewed by Grossman & Tsagarakis 1989 and Engler et al. 1994) and to assess the extent to which this information has contributed to our understanding of the biochemical mechanisms of stress control. The emphasis throughout will be on inhibitory agents that have the potential to operate at the hypothalamus or the anterior pituitary, rather than at the adrenal cortex. Compounds directly affecting glucocorticogenesis have been the 169

Prolactin receptor expression in the testis of the ram: localisation, functional activation and the influence of gonadotrophins

The present study investigated the pattern and site of expression of the prolactin receptor gene in the testis of the seasonally breeding Soay sheep. In experiment 1, Northern blot analysis confirmed expression of the prolactin receptor gene in the testis which was encoded by RNA transcripts of ≈3.6, 11.2, 12.6, and 14.1 kb. In situ hybridisation localised expression of the receptor within the interstitial and seminiferous tubule compartments of the testis and immunohistochemistry localised expression of the receptor to Leydig cells and to pachytene spermatocytes, round and elongating spermatids. In experiment 2, phosphorylation of Jak2, Stat1 and Stat5 proteins in response to prolactin was investigated by Western blotting following incubation of testicular samples with 100 ng/ml ovine prolactin. Jak2 and Stat1 phosphorylation were induced by prolactin within 10 min and Stat5 within 30 min. In experiment 3, intact and hypothalamo-pituitary disconnected (HPD) rams were transferred from a short to a long day photoperiod regimen for a period of 8 weeks. By week 8, testicular diameter had declined in intact rams (52.71±1.06 cm vs. 48.00±0.49 cm for weeks 1 and 8 after transfer respectively, P<0.01, n=3) and increased in HPD rams (27.00±0.45 cm and 29.66±0.99 cm for weeks 1 and 8 after transfer respectively, P<0.05, n=3). RT-PCR using RNA extracted from intact and HPD rams confirmed expression of the prolactin receptor in the testis of both groups. Immunohistochemistry localised prolactin receptor expression in Leydig cells and in pachytene spermatocytes, round and elongating spermatids of intact sheep testis. In HPD rams, prolactin receptor expression was localised in Leydig cells and germ cells which were arrested predominantly at the pachytene spermatocyte stage. These data demonstrate expression of functional prolactin receptors in the testis of Soay rams. The site and pattern of expression of the receptor gene suggest a role for prolactin in the regulation of steroidogenesis and spermatogenesis.

Hypothalamo-pituitary disconnection of the late-gestation ovine fetus results in profound changes in cortisol secretion that are not reflected in commensurate changes in adrenocorticotropin secretion.

A prepartum increase in fetal glucocorticoid concentrations is essential for the perinatal transition to extrauterine life for many mammalian species. In the case of the sheep, this increase in cortisol is also the trigger for parturition, and depends upon an intact hypothalamo-pituitary unit. Fetal sheep that have undergone hypothalamo-pituitary disconnection (HPD) fail to have a prepartum cortisol surge or initiate labor, despite apparently normal fetal ACTH concentrations in late gestation. We have investigated whether a defect exists in the regulation of pulsatile neurohormone secretion in the pituitaryadrenal axis of the HPD sheep fetus, by comparing immunoreactive (ir) ACTH and cortisol secretory dynamics in intact and HPD fetuses at 126 and 145 days of gestation (normal gestation length, 147 days). The fetal surgery was conducted at 115 days of gestation. Blood samples were collected at 5-min intervals for 2 h on each experimental day, and the resulting irACTH and cortisol concentrations were analyzed by multiple-parameter deconvolution and cross-correlation analysis. Basal irACTH secretion was less (P < 0.01) in HPD fetuses than intact fetuses at 126 days, but it had recovered by 145 days. There were no differences in irACTH half-life or the number or duration of irACTH secretory bursts between the two groups of fetuses or the two gestational ages (GAs). The size of the irACTH secretory bursts was not affected by the operation, but it increased with GA to a similar extent in both groups of fetuses (P < 0.01). In keeping with the observations for irACTH secretion, there was no effect of age or the operation on cortisol half-life or on the number or duration of cortisol secretory bursts. In contrast, there were dramatic age-related increases (P < 0.01) in the basal cortisol secretion rate and the size of the cortisol secretory bursts in the intact, but not the HPD, fetuses. Cross-correlation analysis revealed a significant (P < 0.01) concordance between irACTH and cortisol secretion in only the intact fetuses at 126 days; this was not apparent in the intact fetuses at 145 days, or in the young or old HPD fetuses. These findings confirm a major defect in cortisol secretion in the late-gestation HPD fetus but suggest that this is not caused by defects in irACTH secretion. Together with other observations, these data suggest that ACTH may not be the sole, or primary, regulator of adrenal cortisol secretion in the late-gestation ovine fetus.

Hypothalamo-Pituitary Disconnection of the Late-Gestation Ovine Fetus Results in Profound Changes in Cortisol Secretion that Are Not Reflected in Commensurate Changes in Adrenocorticotropin Secretion

Hypothalamo-Pituitary Disconnection of the Late-Gestation Ovine Fetus Results in Profound Changes in Cortisol Secretion that Are Not Reflected in Commensurate Changes in Adrenocorticotropin Secretion

Melatonin receptors in the brain and pituitary gland of hypothalamo-pituitary disconnected Soay rams.

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(125I)-iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel 1a(b)) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.

Refractoriness to a static melatonin signal develops in the pituitary gland for the control of prolactin secretion in the ram.

Hypothalamo-pituitary disconnected (HPD) and control Soay rams were treated chronically for 48 wk with s.c., continuous-release implants of melatonin while under long days (16L:8D). The implants produced continuously elevated blood concentrations of melatonin 2-3 times higher than the normal nocturnal maximum. The long-term treatment induced a biphasic effect on prolactin secretion in both the HPD and control rams, with a marked decrease in the blood prolactin concentrations for 10 wk followed by a gradual increase. The introduction of a second melatonin implant after 20 wk failed to affect prolactin secretion. The treatment with melatonin also caused a dynamic effect on FSH secretion, but this occurred in the control rams only. Blood concentrations of FSH in the HPD rams were very low throughout, but there were minor changes in testicular diameter that were correlated with variations in prolactin. Overall, the results support the conclusion that 1) melatonin acts primarily in the pituitary gland to affect prolactin secretion, and partial refractoriness develops at this level for control of prolactin; and 2) melatonin acts most probably in the hypothalamus to affect gonadotropin secretion, and refractoriness develops at the level of the neural tissue regulating GnRH release for control of gonadotropins.

The characterization, localization and regulation of endothelin in ovine pars intermedia

The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP-HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with α-melanocyte stimulating hormone (α-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol-treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels ( t=9.4 P<0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

Photoperiodic history and hypothalamic control of prolactin secretion before birth.

We investigated whether the fetal lamb can construct a photoperiodic history in utero. We measured the fetal PRL response to a 12-h photoperiod in intact fetal sheep and in fetal sheep after hypothalamo-pituitary disconnection (HPD), following exposure of the ewe to either a long (16 h L) or short (8 h L) photoperiod for 50 days in early pregnancy. Ewes were maintained on either a long light (LL, n = 20) or a short light (SL, n = 19) regimen from 57 days gestation until fetal HPD (pre-LL, n = 7; pre-SL, n = 7) or sham surgery (pre-LL, n = 13; pre-SL, n = 12) was performed at 99-113 days gestation. All ewes were housed in a 12-h photoperiod from surgery until 140 days gestation. In HPD fetal sheep previously exposed to SL, fetal PRL concentrations were significantly higher (P < 0.05) after 20 days in the 12-h L regimen than previously (0-5 days, 3.2 +/- 0.6 ng/ml; 21-25 days, 5.6 +/- 1.4 ng/ml). In the HPD fetal sheep previously exposed to LL, however, fetal PRL concentrations significantly decreased (P < 0.05) after 5 days exposure to the 12-h L regimen (6.7 +/- 2.9 ng/ml) and remained low throughout the remaining study period (31-35 days, 1.7 +/- 0.5 ng/ml). In contrast, in the sham group there was no effect of photoperiodic history on the gestational age profile of fetal PRL, and PRL concentrations increased significantly (F = 22.4, P < 0.001) in fetal sheep previously exposed to either SL or LL. Fetal PRL concentrations were significantly higher (P < 0.05) after 121 days gestation in the 12-h L regimen in all sham fetal sheep (<110 days, pre-SL 6.4 +/- 0.3 ng/ml, pre-LL 12.0 +/- 3.3 ng/ml; 121-125 days, pre-SL 20.0 +/- 3.9 ng/ml, pre-LL 25.9 +/- 4.4 ng/ml). TRH (50 microg) was administered i.v. to all fetal sheep at 130-134 days gestation. There was a significant fetal PRL response to TRH in both the HPD (F = 20.9, P < 0.001) and sham (F = 31.3, P < 0.001) groups. There was no difference, however, in the PRL response to TRH in fetal sheep previously exposed to SL or LL in either the HPD or sham groups. The maximum percentage changes in PRL occurred at +10 min after TRH administration in the HPD (pre-SL, 421 +/- 75%; pre-LL, 555 +/- 76%) and sham groups (pre-SL, 394 +/- 68%; pre-LL, 369 +/- 59%). In summary, therefore, we have demonstrated that there is an effect of photoperiodic history on the PRL response to an intermediate photoperiod in utero in HPD fetal sheep. It appears, however, that the effect of photoperiodic history on PRL secretion in intact fetal sheep is either masked or suppressed by the stimulatory effect of factors associated with an increase in gestational age acting at the fetal hypothalamus.

Role of the new growth hormone-releasing secretagogues in the diagnosis of some hypothalamopituitary pathologies

Growth hormone (GH)—releasing hormone (GHRH) and somatostatin have a dominant role in regulating GH secretion. However, results of studies using the new class of GH secretogogues, particularly GHRP-6, indicate that there may also be other, as yet undefined, hypothalamic mechanisms involved. Studies in adults with hypothalamopituitary disconnection (functional pituitary stalk transection), show GHRP-6—mediated GH release to be completely blocked, indicating a main action at the hypothalamic rather than the pituitary level. The synergistic effect of GHRH plus GHRP-6 administration on GH release seen in normal adults (and virtually unaffected by age, obesity, or sex) is also absent in these patients, providing further support for this conclusion. Studies of the effects of GHRP-6 in children with GH deficiency due to perinatal pituitary stalk transection have produced similar findings. It is suggested that the combined GHRH plus GHRP-6 test should be a promising tool for diagnosing GH deficiency states in both children and adults, and may identify a subgroup of patients with GH deficiency caused by interruption of the hypothalamopituitary connection.

Production of a prolactin releasing factor by the ovine pars tuberalis

The anterior pituitary hormone prolactin is involved in the regulation of a wide variety of processes including mammary growth, lactation, reproduction and pelage growth (1). Correspondingly, in seasonal breeders, pronounced photoperiod-driven, seasonal changes occur in the levels of prolactin secretion (2). These effects are thought to be mediated by the pineal hormone melatonin, which acts as a humoral indicator of photoperiod (3). Melatonin is thought to act through hypothalamic sites to control the gonadotrophic axis (4-6), but the sites through which melatonin modulates prolactin remain to be established. One possibility is that melatonin acts at the level of the hypothalamus to modulate the release of the hypothalamic prolactin inhibitory factor, dopamine (7). However, recent evidence from hypothalamo-pituitary disconnection experiments performed in the ram suggests that the photoperiodic modulation of prolactin secretion can occur independently of the hypothalamus, presumably due to direct effects of melatonin on the anterior pituitary (8). The only identified site of melatonin receptor expression within the ovine pituitary is the pars tuberalis (PT) (9), the cells of which are almost completely non-lactotrophic (10). Thus the possibility exists that factors released by the PT regulate the activity of lactotrophs in the pars distalis (PD). We have investigated this hypothesis using a range of co-culture and medium-conditioning experiments on primary cultures of ovine PT and PD cells, and here we report that PT cells secrete an unidentified factor that is a potent stimulus of prolactin secretion by PD cells.

The peptide ACTH(1-39), adrenal growth and steroidogenesis in the sheep fetus after disconnection of the hypothalamus and pituitary.

1. We have investigated the role of the fetal hypothalamo-pituitary axis in the control of adrenocortical growth and steroidogenesis in the sheep fetus during late gestation. Plasma concentrations of ACTH(1-39) increased between 120-125 and 136-142 days (P < 0.05), but did not change after surgical disconnection of the fetal hypothalamus and pituitary (HPD) at 106-120 days gestation. There was no effect of either gestational age or HPD on the circulating concentrations of the ACTH-containing precursors pro-opiomelanocortin (POMC) and pro-ACTH (the 22 kDa N-terminal portion of POMC). 2. In the fetal sheep adrenal, the relative abundance of the mRNAs of the steroidogenic enzymes CYPIIA1 and CYP21A1 increased between 130-135 and 136-140 days gestation (P < 0.05) and remained high after 141 days, whereas that of CYP17 mRNA increased after 141 days gestation (P < 0.05). The abundance of adrenal 3 beta-HSD mRNA did not change between 130 and 145 days. 3. Hypothalamo-pituitary disconnection significantly reduced the abundance of of CYPIIA1 mRNA, 3 beta-HSD mRNA and CYP17 mRNA by 3.4, 3.1 and 3.7 times, respectively, at 140-142 days gestation (P < 0.05). 4. In the intact group of fetal sheep, adrenal weight increased between 130-135 and 141-145 days (P < 0.05), but there was no change in the abundance of adrenal insulin-like growth factor II (IGF-II) mRNA across this gestational age range. Hypothalamo-pituitary disconnection significantly reduced fetal adrenal weight to 66% that of intact sheep (P < 0.01), but did not alter the abundance of IGF-II mRNA in the fetal adrenal at 140-142 days. 5. Our results suggest that the prepartum changes in adrenal growth and steroidogenesis are under the control of an intact hypothalamo-pituitary axis in late gestation and are dependent on an increase in circulating ACTH(1-39), rather than on ACTH precursors. We have found no evidence, however, for a direct-relationship between fetal adrenal growth or steroidogenesis and adrenal IGF-II mRNA between 130 and 145 days gestation.

Evidence that Melatonin Acts in the Pituitary Gland through a Dopamine‐independent Mechanism to Mediate Effects of Daylength on the Secretion of Prolactin in the Ram

A previous study provided evidence that melatonin acts in the pituitary gland to mediate the effects of daylength on the secretion of prolactin in sheep. This was based on the observation that hypothalamo-pituitary disconnected (HPD) Soay rams showed normal patterns in the changes in the peripheral blood concentrations of prolactin in response to alterations in photoperiod (10-fold higher concentrations under long than short days), and in response to exogenous melatonin (rapid decline following the administration of a constant-release implant of melatonin). The purpose of this study was to establish whether dopamine (DA) might be involved in mediating the effects of melatonin on the secretion of prolactin. Groups of HPD (n = 7) and control Soay rams (n = 8) were treated with vehicle (control, 2.0 ml 0.1 M tartaric acid/saline sc), bromocriptine (DA agonist, 0.06 mg/kg sc) or sulpiride (DA antagonist, 0.6 mg/kg sc), and the acute prolactin responses were measured over the next 4 h. Treatments were carried out under short days (8L: 16D, low prolactin), long days (16L: 8D), high prolactin), and under long days in the presence of a constant-release implant of melatonin (low prolactin). The prolactin response to TRH (1.25 micrograms/kg iv) was also measured. Bromocriptine caused a decrease in the plasma concentrations of prolactin in both HPD and control rams under short and long days. Sulpiride had no effect in the HPD rams on any occasion, but caused a very marked increase in the plasma concentrations of prolactin in the control rams under short days, long days, and under long days + melatonin. TRH caused an acute increase in the plasma concentrations of prolactin in the HPD rams under both long and short days although the responses were notably reduced compared with the controls especially under long days + melatonin. Overall, the inhibitory response to the DA agonist in HPD rams indicates the presence of DA D2 receptors linked to functional lactotrophs in the isolated pituitary gland. However, the total lack of a response to the DA antagonist indicates the absence of endogenous DA mechanisms regulating the secretion of prolactin in the HPD rams. The conclusion is that melatonin acts directly on the pituitary gland to mediate effects of photoperiod through a DA-independent mechanism.

Blocked growth hormone-releasing peptide (GHRP-6)-induced GH secretion and absence of the synergic action of GHRP-6 plus GH-releasing hormone in patients with hypothalamopituitary disconnection: evidence that GHRP-6 main action is exerted at the hypothalamic level.

GH-releasing peptide (GHRP-6; His-D Trp-Ala-Trp-D Phe-Lys-NH2) is a synthetic compound that releases GH in a specific and dose-related manner through mechanisms and a point of action that are mostly unknown but different from those of GHRH. In man, GHRP-6 is more efficacious than GHRH, and a striking synergistic action on GH release is observed when GHRP-6 and GHRH are administered simultaneously. Based on such a synergistic action, it has been hypothesized that GHRP-6 acts through a double mechanism by actions exerted both at the pituitary and hypothalamic levels. The aim of the present study was 2-fold: 1) to further characterize the mechanism of action and synergistic effects of GHRP-6; and 2) to study its action in patients with hypothalamopituitary disconnection. Twelve patients with different neuroendocrine pathologies leading to a state of hypothalamopituitary disconnection (functional stalk section) and 11 age- and sex-matched normal controls were studied. Each subject underwent 3 tests on separate occasions, being challenged with GHRH (100 micrograms, i.v.), GHRP-6 (90 micrograms, i.v.), or GHRH plus GHRP-6. GH was analyzed as the area under the curve (mean +/- SE, micrograms per L/120 min). In normal subjects GH secretion was 483.7 +/- 99.2 after GHRH, 1434.8 +/- 393.0 after GHRP-6, and 3771.5 +/- 399.6 after GHRH plus GHRP-6; the level of GH secreted after GHRH plus GHRP-6 treatment was significantly (P < 0.05) higher than after the arithmetic sum of GH levels after both compounds administered separately. In the group of patients with hypothalamopituitary disconnection, the level of GH secreted after GHRH was similar to that in controls (423.4 +/- 62.8); however, a complete blockade was observed after GHRP-6 (97.3 +/- 7.9), significantly (P < 0.05) lower than after GHRH as well as lower than the GHRP-6-induced GH release in control subjects (P < 0.01). After GHRH plus GHRP-6, the patients with hypothalamopituitary disconnection showed severely reduced secretion (745.3 +/- 67.6; P < 0.01 vs. controls), a value that was not significantly different from the arithmetic addition of levels produced by both compounds administered separately.(ABSTRACT TRUNCATED AT 400 WORDS)

Blocked growth hormone-releasing peptide (GHRP-6)-induced GH secretion and absence of the synergic action of GHRP-6 plus GH-releasing hormone in patients with hypothalamopituitary disconnection: evidence that GHRP- 6 main action is exerted at the hypothalamic level

Blocked growth hormone-releasing peptide (GHRP-6)-induced GH secretion and absence of the synergic action of GHRP-6 plus GH-releasing hormone in patients with hypothalamopituitary disconnection: evidence that GHRP- 6 main action is exerted at the hypothalamic level

Effect of late hypothalamo-pituitary disconnection on the development of the HPA axis in the ovine fetus and the initiation of parturition.

The studies of Liggins et al. (1) in which fetuses stalk-sectioned from day 116 onwards delivered at or near term, suggested that a connection between the fetal hypothalamus and pituitary is not essential for parturition to occur. The objective of this study was to repeat these experiments on the effects of pituitary stalk sections at different gestational ages and include information on the plasma concentrations of key fetal hormones. We have used the more sophisticated technique of hypothalamo-pituitary disconnection (HPD) at either of two gestational age ranges (123-127 days or 133-135 days). Completeness of the procedure was assessed by demonstrating an attenuated prolactin response to chlorpromazine challenge. Following HPD, gestation was prolonged for at least eight days beyond term (146.2 +/- 1.5 days) in 9 of the 10 fetuses operated. Fetal plasma ACTH1-39 concentrations were not different between the HPD and control fetuses, increasing in all groups with increasing gestation. Fetal plasma cortisol concentrations increased (P < 0.01) in control fetuses over gestation. Cortisol concentrations did not change significantly in the day 125 HPD group following HPD but increased in the day 135 HPD group (P < 0.05) with advancing gestation. These latter concentrations, however were markedly less (P < 0.001) than those for control fetuses prior to parturition. Fetal and maternal plasma PGE2 concentrations increased (P < 0.01) in the control group over gestation but did not change following HPD. Maternal plasma progesterone concentrations decreased (P < 0.05) after day 143 in the control group but did not change in the HPD group.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive growth hormone secretion in sheep after hypothalamopituitary disconnection and the direct in vivo pituitary effect of growth hormone releasing peptide 6.

The effect of hypothalamopituitary disconnection (HPD) on growth hormone (GH) secretion was studied in sheep. Plasma GH levels were measured in serial blood samples (10 min x 6-8 h) taken from 12 Romney wethers and 5 ewes which had undergone HPD 40-506 days earlier and from 4 wethers (10 min x 7 h) to serve as controls. Five wethers and 5 ewes were taken approximately 300 days after HPD and injected with vehicle or 10 micrograms/kg of the synthetic hexapeptide growth hormone-releasing peptide 6 (GHRP-6); GH responses were monitored. In a second series of sheep, 4 wethers and 6 HPD wethers were given saline, 0.5 micrograms/kg of synthetic growth hormone-releasing factor (GRF) or 5 micrograms/kg GHRP-6 and were blood-sampled to measure the plasma GH response. A further group of 4 HPD wethers and 3 control wethers were killed and the anterior pituitary glands collected for the quantification of GH and LH beta mRNA by Northern analysis. Three HPD wethers and 1 HPD ewe and 1 control ewe were killed and their brains were perfused; the hypothalami were sectioned and immunostained for the presence of GRF-containing fibres in the median eminence. Normal episodic GH secretion was abolished by HPD in both wethers and ewes but plasma values did not fall below the assay detection limit, indicating constitutive secretion. Northern blot analyses showed that the GH mRNA was detectable in HPD wethers at a lower (p < 0.05) level than in control animals but mRNA for LH beta was undetectable in the HPD wethers. Immunohistochemistry revealed GRF-positive staining in the median eminence of the controls but GRF-positive staining was not seen below the operation site in the median eminence of the HPD animals. In the first series, 3/5 wethers and 3/5 of the ewes responded to GHRP-6 challenge; the magnitude of the response was similar in both sexes. In the second series, responses to GRF were lower (48%) (p < 0.03) in HPD wethers than in control wethers, and responses to GHRP-6 were much lower (p < 0.01) than those to GRF in both controls and HPD wethers. These studies show that HPD removes GRF inputs to the pituitary gland and abolishes pulsatile GH secretion in most cases but constitutive non-pulsatile secretion continues. The HPD wether pituitary glands had lower GH mRNA levels than controls. Accordingly, HPD sheep were able to respond to a single injection of GRF although the response was half that seen in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Short‐Term Regulation of Gonadotropin Subunit mRNA Levels by Estrogen: Studies in the Hypothalamo‐Pituitary Intact and Hypothalamo‐Pituitary Disconnected Ewe

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone beta (LHbeta), follicle stimulating hormone beta (FSHbeta) and the common alpha-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 micrograms of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary alpha-subunit and FSHbeta mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHbeta mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injections and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHbeta subunit mRNA seen after estrogen injection of OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHbeta mRNA are reflected in changes in FSH secretion; the same is not true for LH.