Ovine Pituitary Research Articles

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion of Follicle-Stimulating Hormone in Ovine Pituitary Cells1

In several physiological paradigms, secretion of FSH and LH are not coordinately regulated. Because these hormones appear to be produced by a single cell type in the anterior pituitary gland, their discordant regulation must be related to differential intracellular responses to various stimuli. Estradiol-17beta (estradiol) has been shown to influence secretion of both FSH and LH and some of its effects are mediated directly on the gonadotrope. Changes in expression of intrapituitary factors such as activin and follistatin may mediate effects of estradiol and account for discordant patterns of FSH and LH. The aims of this study were 1) to determine if estradiol alters expression of genes encoding activin, follistatin, or both in ovine pituitary cells; and 2) to observe the effects of immunoneutralizing activin B in vitro on gonadotropin secretion. Pituitary cells from five ewes in the anestrous season were cultured for 24 h with estradiol (0.01 or 1.0 nM). Estradiol reduced basal secretion of FSH in a dose-dependent manner (P: < 0.001) and simultaneously increased basal secretion of LH (P: < 0.001). Decreased secretion of FSH in estradiol-treated cultures was accompanied by suppressed levels of FSHbeta subunit mRNA (P: < 0.001). Amounts of mRNA for activin beta(B) were reduced in a dose-dependent manner by estradiol (27% +/- 4.9% at 0.01 nM, P: < 0.02; and 46% +/- 3.9% at 1.0 nM, P: < 0.002). In contrast, mRNA for follistatin was not affected by treatment with estradiol. Treatment of pituitary cells with an antibody to activin B reduced secretion of FSH by 50% (P: < 0.01) without influencing secretion of LH. These data lead us to conclude that discordant secretion of gonadotropins can be induced by estradiol acting directly at the pituitary level. The inhibitory effect of estradiol on FSH secretion may be mediated indirectly through decreased pituitary expression of the activin gene.

Stimulating effect of glycoprotein hormone free alpha-subunit and daily gonadotropin releasing hormone treatment on prolactin release from 50-day ovine foetal pituitary explants.

The aim of our study was to determine whether free alpha of glycoprotein hormones (free alpha) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free alpha, luteinzing hormone beta-subunit (LHbeta) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free alpha- and LHbeta-containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin-containing cells were first identified on day 42. Analysis of serial sections suggested that free alpha immunoreactive cells were also LHbeta-positive, indicating that free alpha was mainly synthesized by gonadotrophs. In early foetal stages, free alpha occurred in the antero-medio ventral region of the pituitary gland, whereas prolactin-containing cells were more dorsally and more caudally localized. The free alpha-, LHbeta- and prolactin-immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free alpha or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42-day-old ovine foetuses for 8 days with 10-9 or 10-7 M ovine free alpha did not affect prolactin release. By contrast, free alpha administration on pituitary explants from male and female 50-day-old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10-8 M GnRH had similar stimulatory effect to free alpha whereas a 'first day' treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free alpha at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free alpha on prolactin release.

Cell type-specific adenoviral transgene expression in the intact ovine pituitary gland after stereotaxic delivery: an in vivo system for long-term multiple parameter evaluation of human pituitary gene therapy.

Ablative therapies for pituitary tumors commonly cause irreversible damage to normal pituitary cells. Toxin gene therapy should therefore ideally be targeted to specific cell types to avoid collateral cell damage. To evaluate cell-type-specific adenoviral gene transfer in the intact pituitary gland we have used stereotaxic transcranial delivery of recombinant adenoviruses in the sheep with continuous assessment of endocrine function. Adenoviral ss-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL gene promoter. The hCMV promoter directed adenoviral ss-galactosidase expression in all pituitary cell types, but the PRL promoter restricted this exclusively to lactotropic cells, indicating that this promoter conferred appropriate cell type specificity in the context of adenoviral transduction in vivo. Serial measurements of plasma hormones showed no disruption of endocrine function over 7 days after intrapituitary injection. In summary, this work shows cell type-specific expression of an adenoviral transgene in the mixed cell population of the intact pituitary gland in vivo in a large animal model and indicates that stereotaxic intrapituitary delivery does not disrupt normal endocrine function.

Cell Type-Specific Adenoviral Transgene Expression in the Intact Ovine Pituitary Gland after Stereotaxic Delivery: An in VivoSystem for Long-Term Multiple Parameter Evaluation of Human Pituitary Gene Therapy

Ablative therapies for pituitary tumors commonly cause irreversible damage to normal pituitary cells. Toxin gene therapy should therefore ideally be targeted to specific cell types to avoid collateral cell damage. To evaluate cell-type-specific adenoviral gene transfer in the intact pituitary gland we have used stereotaxic transcranial delivery of recombinant adenoviruses in the sheep with continuous assessment of endocrine function. Adenoviral ss-galactosidase expression was driven either by the human cytomegalovirus (hCMV) promoter or the human PRL gene promoter. The hCMV promoter directed adenoviral ss-galactosidase expression in all pituitary cell types, but the PRL promoter restricted this exclusively to lactotropic cells, indicating that this promoter conferred appropriate cell type specificity in the context of adenoviral transduction in vivo. Serial measurements of plasma hormones showed no disruption of endocrine function over 7 days after intrapituitary injection. In summary, this work shows cell type-specific expression of an adenoviral transgene in the mixed cell population of the intact pituitary gland in vivo in a large animal model and indicates that stereotaxic intrapituitary delivery does not disrupt normal endocrine function.

Evidence for regulation of basic fibroblast growth factor gene expression by photoperiod and melatonin in the ovine pars tuberalis

In the ovine pituitary the in vivo expression of the bFGF gene was studied using in situ hybridisation and Northern analysis. Expression of bFGF mRNA was highest in the pars tuberalis (PT) and zona tuberalis (ZT) and this expression was higher in the pituitaries of animals acclimited to long days than in those from short day housed animals. Expression in the pars intermedia (PI) was also detectable but the pars distalis (PD) showed negligible expression compared with the PT. Regulation of bFGF gene was investigated in primary cultured PT cells. Both forskolin and PMA increased bFGF mRNA expression significantly and melatonin was able to inhibit these effects partially, however there was no independent effect of melatonin on bFGF mRNA levels. Despite the inducibility of the bFGF gene, bFGF protein levels in PT culture media were insensitive to the same stimuli and detectable bFGF protein declined with time. Exogenous bFGF had no effect on c-fos mRNA levels and did not increase prolactin secretion from ovine lactotrophs. In contrast c-fos mRNA was induced in GH3 cells by bFGF. These data suggest that although basic fibroblast growth factor (bFGF) mRNA expression in the ovine PT is photoperiodically-sensitive, it is not directly involved in the seasonal regulation of lactotrophic activity.

Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture

Identification of High Affinity Binding Sites for Inhibin on Ovine Pituitary Cells in Culture

Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture.

The aim of this study was to identify and characterize binding sites for inhibin in primary cultures of ovine anterior pituitary cells. Recombinant human 31-kDa inhibin A was iodinated by an optimized lactoperoxidase procedure. Fractionation of the labeled protein by gel filtration chromatography on Sephadex G-100 in 0.1 M HCl yielded two immunoactive peak regions, the second of which was bioactive as assessed by in vitro bioassay, with a ratio of bioactivity/immunoactivity of 0.62-0.77 and an iodine incorporation ratio of 1.7-2.0 mol 125I/mol inhibin. The specific binding of purified [125I]inhibin to cultured ovine pituitary cells varied with time, temperature, and cell number. Displacement of the tracer by unlabeled inhibin, as assessed by Scatchard analysis, revealed two binding sites with average Kd values of 0.28 and 3.9 nM and with approximately 250 and 3100 binding sites/anterior pituitary cell, respectively. There was little cross-reaction between inhibin and activin A (<2%), transforming growth factor-beta (<0.2%), or follistatin (<<0.1%). Examination of cell lines that were not expected to have inhibin receptors showed that there was no specific binding of inhibin to human leukemia (Jurkat) cells, whereas the binding to human embryonic kidney (293) cells was displaced by both inhibin and activin with a similar degree of cross-reaction, which suggests binding to an activin receptor. It is concluded that inhibin-binding sites with high affinity and specificity have been identified on ovine pituitary cells, consistent with both inhibin action on the pituitary and the presence of the putative inhibin receptor.

Immunohistochemical distribution of oestrogen receptor and luteinizing hormone B subunit in the ovine pituitary gland during foetal development.

The presence of oestrogen receptor in the developing hypothalamo-hypophyseal system is an essential prerequisite for the development of sex-steroid feedback on gonadotrophin secretion. We have used dual immunocytochemistry to examine the ontogeny and regional distribution of oestrogen receptor and LHbeta subunit in the ovine pituitary gland during foetal development. At day 65 gestation (term= 145 days) oestrogen receptor and LH/ immunopositive cells are found in a small region at the base of the anterior pituitary gland, and also in a band immediately adjacent to the neurointermediate lobe. By day 100 gestation there was a significant increase in the number of immunopositive LHbeta cells accounting for around 12% of the total cell population, and these were widely distributed throughout the anterior pituitary gland. There was also a significant increase in the proportion of gonadotrophs which contain oestrogen receptor compared with day 65. By day 130 gestation the percentage of LH containing cells had declined to around 7% of the total population, but the proportion which also contained oestrogen receptor remained the same. There were no differences in the numbers or distribution of cells containing LH or oestrogen receptors between male and female foetuses, at any age. These data describing a parallel change in the number of oestrogen receptors and LHbeta containing cells in the pituitary gland throughout gestation suggest that the development of pituitary sex-steroid feedback is not solely dependent on changes in the numbers of oestrogen receptor containing cells alone.

Ovine luteinizing hormone heterogeneity: Androgens increase the percentage of less basic isohormones

The heterogeneity of luteinizing (LH) is hormonally regulated with androgens having been reported to increase the percentage of extremely basic LH isohormones in the ovine pituitary. This study re-examined the effects of androgens on the heterogeneity of LH in the ovine pituitary because the extremely basic isoforms of LH reported previously were subsequently demonstrated to be artifacts resulting from the conditions used for chromatofocusing. Pituitary extracts from wethers, rams, and dihydrotestosterone (DHT)-implanted wethers were desalted by flow dialysis and chromatofocused on pH 10.5 to 7.0 gradients. LH was quantitated by radioimmunoassays. When compared with rams, wethers had increased percentages of LH isoforms eluting between pH 9.6 and 10. Patterns of intrapituitary LH isohormones were comparable in rams and DHT-implanted wethers except that rams had a higher percentage of LH eluting at pH 9.2 (20.9 ± 2.1% vs. 13.3 ± 1.3%). In contrast to what was previously reported, rams and DHT-implanted wethers had higher percentages of ovine LH isoforms eluting at pH's 9.4 or lower. Thus, androgens alter LH heterogeneity yielding increased percentages of isohormones eluting at pH's of 9.4 or lower.

Components of the Neuronal Exocytotic Machinery in the Anterior Pituitary of the Ovariectomised Ewe and the Effects of Oestrogen in Gonadotropes as Studied with Confocal Microscopy

We have investigated exocytotic proteins in ovine pituitary cells and sought to identify changes in expression of these proteins related to the effects of estrogen on luteinising hormone (LH) secretion in the ovariectomised ewe. Sheep were treated with either oestradiol benzoate, or oil (i.m.) and blood samples collected for LH assay. Pituitaries were perfusion-fixed and dual-label immunohistochemistry was performed to identify hormone-secreting cells, and colocalise synaptic proteins within different cell types. Synaptophysin, SNAP-25, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in gonadotropes and somatotropes. Lactotropes were positive for SNAP-25 and synaptophysin (other synaptic proteins not investigated). Synaptotagmin I was detected in gonadotropes and lactotropes, but not somatotropes. Synaptophysin, SNAP-25, synaptotagmins I, II and III, VAMP-2, rab3A, Munc-18-1, α/β-SNAP, csp, and secretogranin II were detected in nerve fibres of the posterior lobe. Membrane staining for SNAP-25 and weak cytoplasmic labelling for both synaptotagmin I and secretogranin II were detected in the intermediate lobe. Syntaxin and complexin II antibodies did not label any region of the ovine pituitary. Oestrogen treatment, to induce a pre-ovulatory-like LH surge, caused migration of LH-containing secretory granules toward the plasma membrane of gonadotropes, but did not alter the percentage of gonadotropes expressing each exocytotic protein. Oestrogen treatment caused a similar redistribution of csp and secretogranin II staining in gonadotropes. We conclude that synaptic protein expression is not altered in the anterior pituitary at the time when LH secretion is maximal. The ubiquitous distribution of many exocytotic proteins suggests that all hormone-secreting cells of the pituitary gland contain the same, or similar exocytotic machinery, but distinct ‘activating factors’ are required to selectively trigger the secretion of individual hormones.

Effect of adrenocorticotrophic hormone on gonadotrophin releasing hormone-induced luteinizing hormone secretion in vitro.

An in vitro perifusion study investigated the effect of different forms of adrenocorticotrophic hormone (ACTH) on gonadotrophin releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, particularly GnRH self-priming, and oestradiol sensitisation of the ovine pituitary. Fragments of pituitaries were obtained from mixed-breed adult nonpregnant female sheep (without corpora lutea, unless otherwise stated). The amount of LH released by different doses of GnRH (2.5 x 10(-10) M (n = 9 chambers), 1 x 10(-10) M (n = 9), or 5 x 10(-11) M (n = 6)) was evaluated by giving two GnRH pulses (5 min each) 2 h apart. In a duplicate set of chambers, ACTH1-24 (5 x 10(-7) M) was included in the perifusate 0.5 h before the first GnRH challenge. Potassium chloride (KCl; 100 mM) was administered 2 h after the second GnRH challenge to assess the viability of the tissue and the size of the releasable LH pool. Results were expressed as percentage of LH secretion. The influence of ACTH1-24 on oestradiol sensitisation was also examined using pituitaries obtained during the luteal phase. Pituitary tissues were perifused throughout with 1 x 10(-9) M or 6 x 10(-11) M oestradiol in the medium. The LH response to the second GnRH challenge (GnRH 2) was significantly greater (p < 0.01) than after the first (GnRH 1) at the highest dose of GnRH (2.5 x 10(-10) M; 2547 +/- 804 vs. 4547 +/- 1013%), but at the lower doses (1 x 10(-10) M or 5 x 10(-11) M), the self-priming effect of GnRH was not evident (3016 +/- 550 vs. 2932 +/- 490% and 841 +/- 205 vs. 711 +/- 87%). Treatment with ACTH1-24 (5 x 10(-7) M) did not affect tonic LH secretion nor the LH response to the first or second GnRH challenge at any of the GnRH doses tested. The LH released in response to KCl was also similar from control and ACTH1-24-treated tissue at all GnRH doses. Both lower doses of GnRH (1 x 10(-10) M or 5 x 10(-11) M) produced the self-priming effect when the pituitary tissue was sensitised with the higher dose of oestradiol (1 x 10(-9) M; 1711 +/- 239 vs. 5085 +/- 1307%, and 1502 +/- 376 vs. 2619 +/- 629%). In the presence of lower concentrations of oestradiol (6 x 10(-11) M), self-priming was observed only after the higher dose of GnRH (1 x 10(-10) M; 1293 +/- 214 vs. 2865 +/- 436%), not the lower dose (5 x 10(-11) M; 985 +/- 203 vs. 1271 +/- 436%). In spite of these differences, ACTH1-24 treatment did not affect LH secretion (neither basal nor potassium-induced). The effect of ACTH1-39 (1 x 10(-8) M or 5 x 10(-7) M; n = 6 chambers per combination) on GnRH-induced LH secretion was examined using the higher (2.5 x 10(-10) M) or lower dose of GnRH (1 x 10(-10) M), with or without oestradiol sensitisation (1 x 10(-9) M). At the lower dose (1 x 10(-8) M), ACTH1-39 influenced neither tonic nor GnRH-induced LH secretion. The LH released by KCl was also similar to the control and ACTH-treated tissue. In contrast, the higher dose of ACTH1-39 (5 x 10(-7) M) increased tonic LH secretion immediately after inclusion in the medium (104 +/- 3 vs. 161 +/- 20%), but suppressed the GnRH self-priming effect after 2.5 x 10(-10) M, i.e., the LH responses to GnRH 1 and 2 were similar (1786 +/- 294 vs. 1553 +/- 373%). However, the LH response to KCl was not significantly different (p > 0.05) between the control and ACTH-treated tissues (2333 +/- 286 vs. 2638 +/- 431%). When the effect of this higher dose of ACTH1-39 on oestradiol-priming was investigated, ACTH increased tonic LH secretion but suppressed the self-priming effect of GnRH (1 x 10(-10) M GnRH; 945 +/- 274 vs. 922 +/- 323%; p > 0.05), and decreased (p < 0.05) the LH released in response to KCl compared to the controls (1803 +/- 409 vs. 4302 +/- 1017%). In summary, in vitro, ACTH1-24 did not affect either tonic LH secretion, the GnRH self-priming effect, or oestradiol sensitisation. The entire ACTH1-39 increased tonic LH secretion, but reduced GnRH self-priming and oestradiol sensitisation. (ABSTRACT TRUNCATED)

Dopaminergic regulation of pituitary function in the late-gestation fetal sheep.

Development of the fetal ovine pituitary is essential for normal maturation and initiation of the parturition process, as well as for orchestrating endocrine responses to stress in utero. Increases in the biosynthesis of ACTH and prolactin (PRL) occur in the late-gestation fetal sheep pituitary. In the anterior lobe (AL) of the pituitary, pro-opiomelanocortin (POMC) biosynthesis and processing are primarily regulated by corticotrophin-releasing hormone and vasopressin. However, POMC in the intermediate lobe (IL) and PRL in the AL are known to be primarily regulated by dopamine, via the D2 receptor, in adult sheep. Because of the importance of ACTH and PRL during gestation we have investigated a potential role of dopamine in the control of both IL melanotrophs and AL lactotrophs and corticotrophs, in late gestation. Catheters were implanted into a maternal femoral artery and vein, fetal carotid artery and jugular vein as well as into the amniotic cavity. At day 130 of gestation, fetuses were infused intravenously with either the specific D2 receptor agonist bromocriptine (n = 5) or vehicle (n = 5), for 5 days. Blood samples were taken throughout the experiment and pituitaries were removed at the end of the treatment period. Bromocriptine caused a significant decrease (> 50%) in POMC mRNA levels in the IL. In contrast, bromocriptine had no significant effect on POMC mRNA levels or distribution in the AL. Fetal arterial ACTH and cortisol concentrations were unaffected by the bromocriptine infusion, compared with vehicle-infused controls. There was a dramatic decrease (> 80%) in plasma PRL concentrations, compared with the control fetuses. However, PRL mRNA levels in the AL were not significantly affected by bromocriptine. In conclusion, we have found that bromocriptine inhibits aspects of both melanotroph and lactotroph function in late-gestation fetal sheep. The data indicate that the fetal pituitary possesses functional D2 receptors in late gestation.

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.

Regulation of the Mel 1a melatonin receptor mRNA and protein levels in the ovine pars tuberalis: evidence for a cyclic adenosine 3',5'-monophosphate-independent Mel 1a receptor coupling and an autoregulatory mechanism of expression.

The Mel 1a receptor gene has recently been cloned and sequenced, and its product has been found to represent a new class of the G protein-coupled receptor superfamily. A unique aspect of its endogenous hormonal ligand, melatonin, is the prolonged duration (16 h) required to initiate appropriate photoperiodic responses in the animal. This study provides the first data on the transcriptional and translational regulation of this receptor in primary cultures of its native tissue, the pars tuberalis of the ovine pituitary. Using RNAse protection assays we show Mel 1a mRNA expression is rapidly induced through changes in intracellular cAMP levels. Forskolin enhances Mel 1a mRNA expression, whereas melatonin reverses this effect. RNA stability is also a factor, as Mel 1a mRNA expression decreases rapidly in the presence of actinomycin D. Although the expression of receptor protein is sensitive to forskolin stimulation, the lag between induction of mRNA and protein suggests that other factors (e.g. RNA splicing) contribute to functional receptor expression. Furthermore we observed that both mRNA and receptor protein levels increase spontaneously during primary culture, and these rises could be blocked by melatonin. A spontaneous rise in Mel 1a mRNA can also occur in serum-depleted cultures. In the absence of corresponding changes in cAMP, these results suggest not only that the Mel 1a receptor can autoregulate its own expression by an undefined pathway, but also they provide the first evidence that melatonin can act via a cAMP-independent signal transduction pathway to repress transcription in this tissue.

Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin

Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin

Regulation of mitogen-activated protein kinase in the pars tuberalis of the ovine pituitary: interactions between melatonin, insulin-like growth factor-1, and forskolin.

The pars tuberalis (PT) of the anterior pituitary is notable for the expression of levels of melatonin receptors that consistently exceed those in all other tissues in mammals. For this reason and because of its anatomical position, it has been suggested that the PT may play a role in seasonal reproductive responsiveness. However, no data have been forthcoming on the nature of the melatonin-responsive cells in this tissue or on the interaction of melatonin with other hormonal signals in the control of PT cells. A number of recent studies have reported that the tubero-infundibular region of the pituitary in several species contains binding sites for insulin-like growth factor-1 (IGF-1). The present study, therefore, sought to address the question of whether functional receptors for IGF-1 exist in the ovine PT (oPT). Primary cultures of cells from the oPT contained a widespread distribution of cells staining positively with a monoclonal antibody to the human IGF-1 receptor, with the strongest staining occurring over the small phase-bright cells that predominate in this culture system and are thought to constitute the melatonin-responsive cell type. As a functional assay for responsiveness to IGF-1, primary cultures of oPT cells were assayed for activation of mitogen-activated protein kinase (MAPK) using a previously validated phosphotransferase assay. Cytosolic extracts from PT cells treated with IGF-1 (100 pM-10 nM) caused a dose-dependent increase in the rate of phosphorylation of myelin basic protein; in contrast, treatment with melatonin had no significant effect on myelin basic protein phosphorylation. Immunostaining of Western blots of PT cell extracts with a pan-extracellular regulated kinase antibody demonstrated that both p42 and p44 MAPK are strongly expressed in this tissue. To confirm that the effects observed in the cytosol assay were indeed attributable to increased activation of p42/p44, gel renaturation assays of protein kinase activity were performed. These experiments revealed that IGF-1 (10 nM) and forskolin (1 microM) were both potent activators of 42- and 44-kDa moeities; however, neither of these agents had any significant effect on the phosphotransferase activity associated with several other higher molecular weight kinases also detected by the gel-renaturation assay procedure. Melatonin (10 nm) was consistently found to be a highly potent inhibitor of the activation of MAPK induced by forskolin; in contrast, melatonin did not inhibit the activation of MAPK induced by IGF-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Pituitary-adrenal function in the immature ovine foetus

Pituitary-adrenal responses to intravenous infusion of ovine corticotrophin-releasing hormone (oCRH) or arginine vasopressin (AVP) and to haemorrhage were examined in the ovine foetus prior to 90 days of gestation (term 145-150 days). In chronically cannulated foetuses (n = 8), between 74 and 84 days of gestation, basal ACTH levels were less than 20 pg/ml while cortisol levels were 6.5 +/- 1.5 nmol/l (mean +/- S.E.M.). Intravenous infusion of oCRH (1 microgram/h for 60 min) or AVP (1 microgram/h for 60 min) significantly increased ACTH (P < 0.05 for both treatments) and cortisol (P < 0.01 for both treatments) levels, although the response to both hormones was modest. In acutely studied foetuses of a similar age (70-90 days of gestation, mean 82.0 +/- 1.4 days, n = 7), exteriorization and progressive haemorrhage significantly (P < 0.05) elevated ACTH levels from 117.4 +/- 32.1 pg/ml to a maximal value of 329.2 +/- 112.8 pg/ml, the maximal ACTH response corresponding to the removal of a volume of blood equivalent to 6.6 +/- 1.2% of the pre-haemorrhage body weight. The present study has demonstrated that the ovine foetal pituitary, in vivo, is responsive to exogenous and endogenous stimuli by mid-gestation and, at this age, although basal cortisol levels are low, the foetal adrenal is capable of responding to elevated ACTH levels in the short term.

Melatonin receptors couple through a cholera toxin-sensitive mechanism to inhibit cyclic AMP in the ovine pituitary.

The nature of melatonin receptor-G-protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins. ADP-ribosylation of ovine PT membrane proteins using 32P-NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins. Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX. Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for 125I-MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%). A CTX-sensitive component also mediates the inhibition of forskolin-stimulated cyclic AMP accumulation as pre-treatment of PT cells with CTX (5 micrograms/ml) for 16 h blocked this response. Gs alpha is a major substrate for ADP-ribosylation by CTX, and 16 h pre-treatment of PT cells with CTX (5 micrograms/ml) caused a down-regulation of Gs alpha. Northern analysis showed only one major transcript of Gs alpha of about 2 kb, which would encompass all of the known splice variants of the Gs gene. Screening of a cDNA library from ovine PT for Gs-related genes and sequencing of clones, combined with RT-PCR of PT mRNA, revealed no novel products. On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the Gs class of G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in glucocorticoid receptor mRNA in the developing ovine pituitary and the effects of exogenous cortisol

Developmental changes in pituitary glucocorticoid receptor (GR) mRNA were examined during gestation and early neonatal life using in situ hybridization. Pituitaries were harvested from sheep fetuses at days 60-80, 100-120, 130-135, 140-142 and term, and from lambs of days 0-7 and 30-60, and adults. GR mRNA was present in the pars distalis by day 60, levels increased through gestation, and there was a redistribution of GR mRNA, resulting in a relatively greater abundance at the base of the pars distalis. At term, there was a significant (P < 0.05 compared with the day 140-142 fetuses) elevation of GR mRNA, which was maintained in the newborn lamb, reaching highest levels at days 30-60 of neonatal life. GR mRNA was undetectable in the pars intermedia until day 120, but subsequently increased to high levels at term. Interestingly, the expression of GR mRNA in the pars intermedia dropped precipitously in the newborn (P < 0.05 compared with term), though levels recovered in the older lambs and adults. The regional and cellular distribution of GR mRNA correlated closely with the presence of immunoreactive GR (irGR) in the pituitary; the majority of irGR was present in the nuclei. Intrafetal infusion of cortisol (12 h; 5 micrograms/min) in late gestation (day 135) had no effect on Gr mRNA expression in either the pars distalis or pars intermedia.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental and glucocorticoid regulation of pituitary 11 beta-hydroxysteroid dehydrogenase 1 gene expression in the ovine fetus and lamb.

To examine the role of 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1) in the control of glucocorticoid actions in the ovine pituitary during development, we have sought developmental changes in the distribution and the level of 11 beta-HSD1 mRNA by in situ hybridization. In the pars distalis, 11 beta-HSD1 mRNA was present by day 60; its amount did not change significantly until term (days 145-147) when it increased dramatically. The level of 11 beta-HSD1 mRNA increased further during the postnatal period. In contrast, 11 beta-HSD1 mRNA in the pars intermedia was not detectable until day 135; it increased in amount at days 140-143, but did not change significantly thereafter through to adulthood. We have also measured levels of both dehydrogenase and reductase activities of 11 beta-HSD1 in the pars distalis of fetal sheep at day 140 and term, and of postnatal sheep at 1-2 months of age, to determine whether changes in 11 beta-HSD1 mRNA are reflected in the levels of enzyme activities. There were progressive increases in both dehydrogenase and reductase activities from day 140 to 1-2 months postnatally, although dehydrogenase activity was consistently higher than reductase activity. Finally, we have determined the effect of short-term intrafetal cortisol infusion (5 micrograms/min for 12 h) on levels of pituitary 11 beta-HSD1 mRNA by in situ hybridization. There was no effect of cortisol infusion on 11 beta-HSD1 mRNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)