Growth Hormone (GH), also known as somatotrophin, is of cardinal importance in the regulation of somatic growth. Decreased GH expression results in short stature, whereas increased levels of GH lead to disorders such as gigantism or acromegaly. Mutations in the transcription factor Pit-1 have been shown to decrease GH, as well as prolactin (PRL; Parks et al., JCEM, 1999). However, understanding the regulation by selective GH transcription regulators, such as Zn16, a protein encoding 16 zinc fingers that binds to the GH promotor DNA (Wojtkiewicz et al., Endocrine, 2002), will require comparison of currently available rodent cell lines that express GH. Two current rat somatotroph cell lines are the somatotroph MtT/S and lactosomatotroph GH3 cell lines, both of which have been implemented in studies on the regulation of GH expression (Schaaf et al., Endocr Relat Cancer, 2009). MtT/S cells almost exclusively express GH, whereas GH3 cells are less differentiated and co-express PRL as well as GH. GH3 cells have been available longer, and thus are more frequently used for in vitro GH experiments, but there may be some increased utility in utilizing MtT/S cells. MtT/S cells were procured from Riken Cell Bank in Japan, and GH3 cells were acquired from the ATCC. These lines were cultured, then secretion of GH and PRL was examined after Insulin-like Growth Factor (IGF-1) treatment, which is inhibitory to GH and PRL secretion. Further, both cell lines were treated with stimulatory factors GH releasing hormone (GHRH); retinoic acid; cortisone; GH Releasing Peptide-6; and both cortisone and GHRH. GH secretion was on average higher in MtT/S cells than in GH3 cells. On the other hand, PRL secretion was extremely lower in MtT/S cells than in GH3 cells. This result confirms that the MtT/S cells are further differentiated as somatotrophs than GH3 cells. Although hormone release in response to treatment did not appear different, the overall difference in GH vs. PRL secretion may be useful for evaluating the role of Zn16 in selective control of the GH promoter. Therefore, mRNA levels of GH, PRL, Pit-1 and Zn16 in these cell lines are currently being measured using quantitative real-time PCR with ribosomal protein L19 (RPL19) expression as a standard. With the investigation of the characteristics of these differentiated pituitary cell types, we hope to advance the knowledge of GH transcription and regulation, particularly the study of Zn16 effects during pituitary development. Recently it was observed that Zn16 may have a role in a tumor suppressor gene that undergoes mutation leading to a form of colorectal cancer and childhood leukemia. This augments the utility of determining the proper cell lines to examine Zn16, and its role in tumor suppression and cell division as well as gene expression during hypophyseal development.

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A detailed study was made in this doctoral thesis on the development and differentiation of the vertebrate pituitary gland, with the aim to establish a fate map in Rathke's pouch of the origin of different hormone producing cells present in the adult pituitary gland, that explain if the differences observed in the distribution pattern of different hormone producing cells in the adult is the consecuence of differences in their development. For this reason, the study was made in two vertebrate groups, Mammals and Avian, that present notable differences in their hormone producing cell distribution patterns. The results allowed us to conclude that the origin of different hormone producing cells in Rathke’s pouch determine their definitive distribution in the adult gland. At the same time, the relationship between proliferation and differentiation was studied, showing us that after differentiation, hormone producing cells continue proliferating with a low rate, contributing to the establishment of differentiated populations. Using immunochemicals and in situ hidridization techniques, the expression of different molecules such as hypothalamic releasing factors; different peptides, whose role as modulators in different pituitary axis have been proposed in the adult animal; different calcium binding proteins and transcription factors in relation to the differentiation of different hormone producing cells, was also studied in this work, allowing us to establish different relationships between some of these factors and specific aspects of the development and differentiatin of the pituitary gland.

No / Gestational stress (GS) produces profound behavioural impairments in the offspring and may permanently programme hypothalamic¿pituitary¿adrenal (HPA) axis function. We investigated whether or not GS produced changes in the maternal behaviour of rat dams, and measured depression-like behaviour in the dam, which might contribute to effects in the progeny. We used the Porsolt test, which measures immobility in a forced-swim task, and models depression in rodents, while monitoring maternal care (arched-back nursing, licking/grooming, nesting/grouping pups). Pregnant rats underwent daily restraint stress (1 h/day, days 10¿20 of gestation), or were left undisturbed (control). On post-parturition days 3 and 4, dams were placed into a swim tank, and time spent immobile was measured. GS significantly elevated immobility scores by approximately 25% above control values on the second test day. Maternal behaviours, in particular arched-back nursing and nesting/grouping pups, were reduced in GS dams over post-natal days 1¿10. Adult offspring showed increased immobility in the Porsolt test, and also hypersecreted ACTH and CORT in response to an acute stress challenge. These data show that GS can alter maternal behaviour in mothers, and this might contribute to alterations in the offspring. GS may be an important factor in maternal post-natal depression, which may in turn detrimentally effect the offspring because depressed mothers do not sufficiently care for their offspring.

The purpose of this experiment is to determine if immature female white mice will react to the gonadotropic hormone from the anterior pituitary of several species of fish. If they can be shown to do so, a second purpose is to establish an assay unit for this fish gonadotropin in order that it may be used with predictable results in inducing extra-seasonal spawning for fresh-water conservation practices.

Adenomas que se desenvolvem a partir da linhagem corticotrófica (corticotropinomas) secretam ACTH (hormônio adrenocorticotrófico) de modo autônomo. Esta secreção induz a produção crônica e excessiva de cortisol, pelo córtex das glândulas suprarrenais, caracterizando a doença de Cushing (DC). A grande maioria dos adenomas visível à ressonância magnética é microadenoma ( < 10 mm) e apenas 10-30 % dos indivíduos com DC possuem macroadenomas ( > 10 mm), enquanto macroadenomas invasivos são considerados raros. Para investigar os diferentes fenótipos destes tumores, estudamos o padrão de expressão gênica entre microadenomas e macroadenomas, incluindo como critério de classificação sua capacidade de invasão. Utilizando a metodologia de microarray, estudamos 12 amostras de corticotropinomas de indivíduos com diagnóstico clínico, laboratorial e histopatológico de DC (microadenomas não-invasivos n = 4, macroadenomas não-invasivos n = 5 e macroadenomas invasivos n = 3). Além disso, foi investigada a presença de mutações do gene USP8. Observamos que micro e macrocorticotropinomas não-invasivos possuem uma assinatura gênica semelhante, com apenas 48 genes diferencialmente expressos entre si. Por outro lado, macroadenomas invasivos apresentaram um perfil de expressão diferencial mais acentuado, com 168 genes diferencialmente expressos em relação aos não-invasivos (ANOVA p-valor < 0,05; fold change cut off = 2; FDR = 0,05). Nenhum dos pacientes apresentou variantes do USP8. Baseado em sua significância de expressão e funcionalidade, destacamos os genes CCND2, ZNF67 (hiper-expressos, DAPK1 e TIMP2 9 (hipo-expressos). A expressão desses transcritos foi validada por QPCR em 15 corticotropinomas não-invasivos e 3 invasivos, onde 28% destes tumores apresentou mutações somáticas para o gene da USP8. Dentre as vias biológicas comprometidas com pelo menos dois genes hipo ou hiperexpressos estão: via do receptor de Vitamina D, TGF-beta, sinalização por proteína G, resposta ao dano no DNA e controle do ciclo celular. Nossos resultados podem ser úteis para identificar novos marcadores envolvidos no fenótipo invasivo dos corticotropinomas clinicamente ativos. Apesar das funções específicas destes potenciais marcadores ainda precisarem ser elucidadas nos corticotropinomas, nossos resultados podem apresentar um impacto positivo na escolha e eficácia terapêutica, no prognóstico e na previsão de recorrência destes tumores / Adenomas that develop from the corticotrophic lineage (corticotrophinomas) secrete ACTH (adrenocorticotropic hormone) autonomously. This secretion leads to chronic and excessive production of cortisol, by the cortex of the adrenal glands, featuring Cushing\'s disease (CD). Most of the adenomas visible to the MRI are microadenomas (< 10 mm) and macroadenomas (> 10 mm) occur in only 10-30 % of individuals with CD, while invasive macroadenomas, although rare, have great clinical relevance. To investigate the different phenotypes of these tumors, we studied the pattern of differential gene expression between microadenomas and macroadenomas, including their invasiveness as classification a criterion. Using DNA microarray methodology, we studied 12 samples of corticotrophinomas of patients with clinical, laboratory and histopathologic diagnosis of CD (non-invasive microadenomas n = 4, non-invasive macroadenomas n = 5 and invasive macroadenomas n=3). In addition, we investigated the presence of USP8 mutations. We observed that non-invasive corticotrophinomas have a similar genic signature with each other, with only 48 genes differentially expressed between them. Moreover, invasive macroadenomas showed a more pronounced differential expression profile, with 168 differentially expressed genes compared to sellar corticotrophinomas (ANOVA p value < 0.05; fold change cut-off = 2; FDR = 0.05). None of them exhibited USP8 variants. Based on expression significance and functionality, we highlighted CCND2, ZNF676 (overexpressed), DAPK1 and TIMP2 (underexpressed). These results were validated through alfaRT-PCR in another cohort of 15 sellar and 3 invasive corticotrophinomas, in which 28% of these tumors harbored USP8 somatic mutations. Among the biological pathways committed with at least two under or overexpressed genes are: Vitamin D receptor pathway, TGF-beta, G-protein signaling, response to DNA damage and control of the cell cycle. Our results can be useful to identify new markers involved in the invasive phenotype of clinically active corticotrophinomas. Although the specific functions of these potential markers still need to be elucidated in corticotropinomas, our results may have a positive impact on choice and therapeutic efficacy, prognosis and prediction of recurrence of these tumors

Cortisol, the principal glucocorticoid secreted from the adrenal glands, is essential for life. Healthy cortisol levels are maintained through negative feedback on the central nervous system (CNS) – pituitary stimulatory apparatus which regulates production of adrenocorticotropin (ACTH) and contains a light–entrained intrinsic CNS driven diurnal rhythm. Cortisol participates in a regulatory mechanism where inflammatory cytokines stimulate cortisol release and cortisol in turn suppresses cytokine release. The effects of cortisol in inflammatory states include elevating blood pressure and metabolic regulation. This thesis contains three exploratory studies examining circulating cortisolaemia using the best available methodologies (total and free cortisol and corticosteroid-binding globulin (CBG)) in clinical states characterized by immune activation/ inflammation and altered blood pressure. These clinical states include: (1) septic shock, (2) hypertensive disorders of pregnancy and (3) obesity-induced hypertension. Prior to the studies described here, little was know about cortisolaemia in these common pathological states. Septic shock is a life threatening condition that complicates severe infection and is characterized by systemic inflammation and refractory hypotension. High plasma total cortisol levels and attenuated responses to synthetic ACTH stimulation are associated with increased mortality. The use of corticosteroids in septic shock has been highly controversial for decades, however recent trials have reported haemodynamic and survival benefits associated with the use of physiologic steroid replacement in patients with relative adrenal insufficiency (RAI) – currently defined as a total cortisol increment of 248 nmol/L or less following ACTH (250 μg) stimulation. However, CBG and albumin levels fall by around 50% with an increase in plasma free cortisol in critical illness. Hence, total cortisol may not reflect the biologically active free (unbound) cortisol, suggesting that standard assays for plasma cortisol (which measure total plasma cortisol) underestimate HPA axis activity. In this study, we have showed that plasma free cortisol is a better guide to circulating glucocorticoid activity in systemic infection than total cortisol. We have also validated the use of Coolens’ method in estimating free cortisol in systemic infection, using plasma total cortisol and CBG measurements as plasma free cortisol is not performed in clinical laboratories. Free cortisol measurement allows better categorization of RAI and non-RAI groups with a free cortisol increment of 110 nmol/L as cut-off. Moreover, we have shown that survivors of RAI have normal adrenocortical function on follow-up testing suggesting a lack of functional adrenal reserve rather than adrenal damage during critical illness. Larger randomized controlled trials will be required to redefine RAI using free cortisol measurements and relate that to clinical outcomes and responses to corticosteroid therapy. Nitric oxide (NO) is normally produced in the endothelium by the constitutive form of the NO synthase and this physiologic production is important for blood pressure regulation and blood flow distribution. Studies have shown that an overproduction of NO by the inducible form of NO synthase (iNOS) may contribute to the hypotension, cardiodepression and vascular hyporeactivity in septic shock. Clinical studies of non-selective inhibitors of the L-arginine nitric oxide pathway showed increased mortality from cardiovascular complications. However, glucocorticoids, which improve vasopressor sensitivity, may act by partially suppressing NO synthesis through selective direct inhibition of iNOS, and suppression of inflammatory cytokine synthesis. Hence, plasma nitrate/ nitrite (NOx) levels may provide a titratable end point to individualize glucocorticoid therapy in sepsis. The NOx study in this thesis showed that cortisol (total and free), CBG and NOx correlated to illness severity. Free cortisol, and to a lesser extent total cortisol, but not NOx levels, predicted septic shock. NOx levels were characteristically stable within individuals but inter-individual differences were only partly accounted for by illness severity or renal dysfunction. NOx levels correlated weakly with cortisol, did not relate to the need for vasopressors and were not suppressed by hydrocortisone treatment. Thus, NOx is not a suitable target for glucocorticoid therapy in septic shock. Pregnancy is the only sustained physiologic state of hypercortisolism in humans. A large body of data suggests that excessive foetal and prenatal glucocorticoid exposure leads to reduced birth weight and adverse health in offspring such as elevated blood pressure and insulin resistance. Pre-eclampsia and gamete donor pregnancies are associated with immune activation, elevated inflammatory cytokines as well as elevated blood pressure. Prior to the study described in this thesis however, there was no prospective data on maternal cortisolaemia in these complicated pregnancies. My study has demonstrated for the first time that there was a substantial fall in plasma CBG levels in the last few weeks of gestation with a corresponding rise in free cortisol in normal pregnancy, a finding obscured for methodological reasons in past studies. This free cortisol elevation in late pregnancy may facilitate organ maturation in the foetus and perhaps prepare the mother for the metabolic demands of labour. In pre-eclampsia and gestational hypertension, plasma CBG, total and free cortisol levels were lower in late third trimester; and in IUGR, plasma CBG levels were suppressed from 28 weeks gestation until delivery but with no significant difference in plasma total and free cortisol. Women with assisted reproduction using donor gametes/ embryos had significantly lower plasma CBG, total and free cortisol levels even in those with normal pregnancy outcomes. Low CBG may be due to reduced synthesis or enhanced inflammation-driven degradation. Low maternal cortisol may be due to a lack of placental corticotropin-releasing hormone, or reduced maternal ACTH, driving cortisol production. This unanticipated maternal hypocortisolism in complicated pregnancies may trigger precocious activation of the foetal HPA axis and could have implications for postnatal and adult health. Speculatively, since excess prenatal GCs increase HPA axis activity, we proposed that maternal hypocortisolism may predispose to the hypocortisolaemic state characterized by fatigue, pain and stress sensitivity, in offspring. The third state of immune/ inflammatory activation associated with blood pressure dysregulation studied in this thesis is obesity. The epidemiologic relationship between obesity and hypertension is widely recognised. Central obesity in particular has been associated with exaggerated HPA responses to stimuli. Studies of severe dieting and starvation resulted in hypercortisolism and a significant decrease in CBG. The HPA axis and the renin-angiotensin-aldosterone system (RAAS) have been implicated in the pathophysiology of obesity-induced hypertension. However, there is little data on the effect of moderate weight loss (30% caloric restriction) on adrenocortical function, and the relation of adrenal hormones to altered blood pressure with weight loss. In this study, measures of HPA axis and RAAS and blood pressure monitoring were performed in twenty-five obese subjects before and after a 12-week diet program (6000kJ/day). Short-term, moderate weight loss (mean 8.5 kg) was associated with a small reduction in blood pressure (mean arterial pressure 6 mmHg) and significantly reduced levels of aldosterone and renin but not cortisol levels. These findings suggest that aldosterone may have an important role in the blood pressure reduction with weight loss via a renin mediated mechanism, perhaps involving renal sympathetic tone. In contrast to severe caloric restriction, HPA axis activation does not occur with moderate weight loss. This suggests a threshold effect of weight loss on the HPA axis where greater caloric restriction is required for HPA stimulation, or a counterbalancing of central and direct adrenal effects on HPA axis function. Overall, these three exploratory studies have provided novel data on HPA axis function in systemic infection, pregnancy and in diet-induced weight loss. Each study offers a basis for further studies of HPA axis function in these disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1289330 / Thesis(Ph.D.)-- School of Medicine, 2007.

Der GLI2-Transkriptionsfaktor ist eines der Haupt Effektor-Proteine des Sonic Hedgehog (SHH)-Signalweges und hat vermutlich eine Schlüsselfunktion in der Entwicklung der Hypophyse. Genomische GLI2-Veränderungen welche zu abgeschnittenen Proteinen führten, wurden beschrieben als Ursache für Holoprosenzephalie (HPE) oder HPE-ähnliche Veränderungen, teilweise in Verbindung mit einer Hypophysenunterfunktion. Ziel dieser Arbeit war die Ermittlung der Frequenz von GLI2-Mutationen in Patienten mit multiplem hypophysärem Hormonausfall (multiple pituitary hormone deficiency, MPHD) und eine funktionelle Untersuchung der gefunden Mutationen mittels Transkriptionsaktivitäts-Untersuchungen durch funktionelle Luciferase assays. Hierfür wählten wir Teilnehmer der GeNeSIS (Genetics and Neuroendocrinology of Short Stature International Study)-Studie. Patienten bei denen bereits Mutationen eines der etablierten Gene für MPHD nachgewiesen wurde, wurden ausgeschlossen. Insgesamt haben wir 168 Patienten mit MPHD untersucht. Bei allen Patienten waren mindestens ein GH- und ein TSH-Mangel dokumentiert, Auffälligkeiten in der zentralen Bildgebung mittels cMRT wurden bei 96 Patienten angegeben. In fünf Studienteilnehmern wurden vier verschiedene heterozygote missense Varianten nachgewiesen, hiervon wurden zwei bislang noch nicht in der Literatur beschrieben. Eine Variante, pR516P, führte in den in-vitro Experimenten zu einem kompletten Verlust der Proteinaktivität. Zusätzlich zu einem Wachstumshormonmangel hatte der Träger dieser Mutation einen Mangel an TSH und der Gonadotropine, sowie einen nichtdeszendierten Hypophysenhinterlappen und eine Polydaktylie, aber keine ersichtlichen Mittelliniendefekte. Anhand der funktionellen Untersuchung konnten wir erstmalig nachweisen, dass ein heterozygoter Aminosäuren-Austausch im GLI2-Protein zu einer möglichen Funktionseinschränkung der Transkriptionsaktivität führen kann und somit die Ursache für MPHD mit milden extrahypophysären Auffälligkeiten sein könnte. Der Phänotyp von GLI2-Mutationen ist variabel und die Penetranz ist unvollständig. GLI2-Mutationen sind assoziiert mit einer Hypoplasie des Hypophysenvorderlappens und treten gehäuft mit einem ektopen Hypophysenhinterlappen auf.