A parte ventral da área pré-óptica medial (MPOA) apresenta o RNAm do prépró-hormônio concentrador de melanina (ppMCH) e o MCH apenas durante a lactação, especialmente entre os dias 19º e 21º. Fibras imunorreativas ao MCH (MCH-ir) e ocitocinérgicas (OT-ir) de origem desconhecida transitam na lâmina interna da eminência mediana (MEi) e na hipófise posterior (PPit). Utilizamos ratas lactantes da linhagem Long-Evans para identificar a origem dessas fibras, caracterizar sua relação na MEi e PPit e quantificar o ppMCH e OT na PPit. A imuno-histoquímica mostrou que não há colocalização entre as fibras MCH-ir e OT-i e há uma proximidade entre suas fibras na MEi e PPit. A dupla hibridização in situ e imuno-histoquímica mostrou que os neurônios retrogradamente marcados com FG após injeção intravascular expressam o RNAm do ppMCH na MPOA e no núcleo paraventricular do hipotálamo. O Western Blotting mostrou uma maior presença do ppMCH na PPit no 19º dia de lactação. Os dados sugerem que o MCH atua no controle neuroendócrino para o término do período de lactação. / The ventral part of the medial preoptic area (MPOA) has been described as the novel harbor of the expression of prepro-melanin-concentrating hormone (ppMCH) mRNA and MCH peptide, only during the end of lactation, especially between the 19th and 21st days. MCH-ir and OT-ir fibers are described as passing in the internal layer of the median eminence (MEi). However, the origin of MCH-ir fibers and its possible relationship with the OT-ir fibers are not known. Long-Evans rats were used to identify the origin of these fibers and characterize their relationship into MEi and PPit and quantify ppMCH and OT in PPit. Immunohistochemistry showed no colocalization between MCH-ir and OT-ir fibers, but a closeness between these fibers into MEi and PPit. Using a combination of in situ hybridization and immunohistochemistry we have showed that neurons retrogradely labeled with FG after intravascular injection express the ppMCH mRNA into MPOA and paraventricular nucleus of the hypothalamus. The Western blotting showed an increase of ppMCH in the PPit parallel with a decrease of OT-ir on the 19th day of lactation. The data suggest that MCH acts on the neuroendocrine control towards the end of the lactation period.

The purpose of this research is, first, to determine by laboratory methods, that species specificity does not exist in closely allied taxonomic animals; second, to determine a unit of activity for the gonadotropic hormone. For this purpose a quantitative method for determining potency is necessary to ascertain the seasonal production of the gonadotropic factor in fish. A further aim in this investigation is to demonstrate that the diversity of the gonadotropic factor, in relation to phylogenetic variations, is not as ineffectual as previously reported.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22743号 / 医博第4661号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 花川 隆, 教授 渡邉 大, 教授 黒田 知宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Selected prognostic and predictive factors of pituitary tumours in patients with acromegaly (abstract) Pituitary tumours in patients with acromegaly (somatotroph adenomas, pituitary neuroendocrine tumours - PitNETs) are a rare disease with significant morbidity and high impact on economic resources of the health care system. The surgery is the first line of treatment, and while it cures a subset of the patients, another therapeutical approach combining radiotherapy and pharmacotherapy is needed in the rest. This usually includes treatment with costly somatostatin analogues (SSA). On the opposite, the therapeutic response to SSA is not a rule. This work aimed to perform precise clinicopathological characterisation of available tissue samples of PitNETs in acromegalic patients (n=110) from REgistr SEllárních Tumorů (RESET registry) and further explore potential factors related to clinically aggressive disease phenotype (tumour size, invasion, biochemical parameters) and factors that might predict therapeutic response to the first generation SSA. An extensive immunohistochemical evaluation was performed: tumours were histologically subclassified (expression of cytokeratins, prolactin, TSH) and proliferative activity was assessed (Ki67, p53, mitotic count) as well as expression of possible pharmacological...

The hypothalamic hormones, thyrotrophin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH), play pivotal roles in the growth and sexual maturation of chickens. In chickens, TRH regulates the release and synthesis of thyrotrophin (TSH) and also acts as a growth hormone-releasing factor. GnRH stimulates the release and synthesis of gonadotrophins (LH and FSH). TRH and GnRH are released and stored in the median eminence, and both hormones are transported into the pituitary gland via the hypophysial portal circulation. TRH and GnRH exert their physiological functions by binding to their specific receptors (TRH receptor and GnRH receptor, respectively) on the surface of cells in the pituitary gland. The activated receptors couple to guanine nucleotide-binding regulatory proteins (G proteins), Gq and/or G11, which in turn triggers the secondary messenger [1,2- diacylglycerol (DAG) and inositoltrisphosphate (IP3)] signalling cascade. The signalling generates the physiological effects of the hormones. The TRH-R and GnRH-R are members of G-protein coupled receptor (GPCR) family. The objective of this thesis was to clone and characterise the chicken TRH and GnRH receptors as useful tools for investigating the regulatory roles of TRH and GnRH receptors in the growth and sexual maturation of chickens. In addition, sequence information of the receptors would potentially assist in elucidating the binding sites and the molecular nature of the processes involved in receptor activation.

The use of static in vitro pituitary cultures has been indispensible for examining the regulation of anterior pituitary hormone secretion. While the static cultures have shown the effects of various chemical stimulators of hormone secretion, the dynamics of such secretion has not been assessed before in vitro. The in vitro superfusion method, which partially stimulates in vivo physiologic conditions, allows for the observation of the dynamics of pituitary hormone secretion.

There are two distinct communication systems in the brain, term wiring and volume transmission (Agnati et al., 2010). Volume transmission refers to a way of communication lacking any wire-like channel connecting the source of signal and its target. This way of signaling is the focus of the current thesis. Portal systems are one aspect of volume transmission in which they provide a pathway for diffusible signaling between bodily fluids (blood and cerebrospinal fluid) and the nervous system. A portal system entails two capillary beds linked by connecting veins. This connection allows signals from one capillary bed to be transported to a target in high concentrations without being diluted in the systemic circulation (Dorland, 2020). For the past decades, the only identified portal system in the brain is pituitary portal system (Popa, 1930; Popa & Fielding, 1933). Here, hypothalamic neurosecretions are released into the fenestrated capillaries of median eminence, a circumventricular organ, and transported to the capillaries of anterior pituitary via portal veins. The median eminence, due to its location on the surface of the ventricle and its contact to the cerebrospinal fluid, is categorized as a circumventricular organ. According to the classification (Oldfield & McKinley, 2015), there are three sensory circumventricular organs in the brain, all are characterized by fenestrated capillaries allowing contact between brain parenchyma and blood. For this reason, the circumventricular organs are known as “windows to the brain” (Gross et al., 1987). Whether other circumventricular organs also form portal systems is unknown. This thesis examines whether sensory circumventricular organs, specifically the organum vascular organ of the lamina terminals, the subfornical organ and the area postrema, bear portal systems. Although there have been prior studies of the vascularity of these CVOs in many species (reviewed in Duvernoy and Risold (2007)), the tissue preparation methods available limited the possibility of tracking small vessels over relatively large volumes in these structures. In the present work, to preserve the blood vessel structure, brain clearing and light sheet microscopy were combined to acquire volumetric images of the regions containing the circumventricular organs. In vivo two-photon microscopy was used to study the blood flow of the sensory circumventricular organs and the adjacent neuropil. The results indicate that organum vasculosum of the lamina terminalis is connected to the brain’s clock located in the suprachiasmatic nucleus by portal vessels. The direction of blood flow is from the suprachiasmatic nucleus to the organum vasculosum of the lamina terminalis, and speed of blood flow is faster during the night compared to the day. Volumetric imaging of the suprachiasmatic nucleus also shows portal veins emerging from the rostral shell region of this nucleus. Also, the subfornical organ connects to the septofimbrial nucleus and the triangular nucleus of the septum via portal veins. The arrangement of the vasculature of the area postrema differs from the other sensory CVOs: the AP and the nucleus of the solitary tract share a common capillary bed directly joining the vasculature of these morphologically distinct nuclei. In summary, there are multiple portal systems connecting the circumventricular organs. These newly discovered portal systems represent new pathways for diffusible signaling, bridging the systemic circulation, cerebrospinal fluid, circumventricular organs and the portal veins connected regions.

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

Neste estudo foi verificado que a hipófise de S. brasiliensis era composta por dois tecidos, a neuro hipófise (NH) e a adeno-hipófise (AH). Nesta última, foram distinguidos sete tipos celulares. Na RPD foram detectadas as células adrenocorticotrópicas e prolactínicas, na PPD as células gonadotrópicas, somatotrópicas e tireotrópicas, e na PI, as células melanotrópicas e somatolactínicas. Foi evidenciada única célula gonadotrópica, produtora de LH e FSH. O primórdio da hipófise foi detectado 12 horas após a eclosão (hpe), a NH com 72 hpe e o início da formação do pedúnculo com 300 hpe. Nos juvenis (600 hpe), a hipófise apresentou uma morfologia semelhante àquela observada nos adultos. As células prolactínicas foram detectadas com 12 hpe, juntamente com as células adrenocorticotrópicas e melanotrópicas, seguidas das somatotrópicas e somatolactínicas, com 36 hpe. Por outro lado, nas larvas e juvenis foram detectadas duas populações distintas de células gonadotrópicas, as células produtoras de FSH foram detectadas com 600 hpe, enquanto as produtoras de LH com 120 hpe. / This study showed that the pituitary gland of S. brasiliensis was formed by two tissues, neurohypophysis and adenohypophysis. In the latter, seven cell types were distinguished. In RPD, prolactin and adrenocorticotropic cells were present. In PPD, gonadotropic, somatotrope and thyrotropic cells were detected, and in PI, somatolactin and melanotropic cells were found. Interestingly, was detected a single gonadotropic cell responsible for producing both gonadotropins. The primordium of the pituitary gland was detected 12 hours after hatching (hah), the neurohypophysis was detected 72 hah and formation of the stalk 300 hah. In juveniles, 600 hah, the pituitary showed a similar morphology to that observed in adults of this species. Prolactin cells were detected 12 hah together with adrenocorticotropic and melanotropic cells, followed by somatotropic and somatolactin cells 36 hah. Unlike adults, larvae and juveniles have shown two distinct populations of gonadotropic cells. FSH-producing cells were detected 600 hah, while LH-producing cells were detected 120 hah.

Após a reprodução de Astyanax altiparanae, foram obtidas larvas e juvenis da espécie, os quais foram coletados desde a eclosão até 120 dias após a eclosão (dpe). Por meio de métodos histoquímicos e imuno-histoquímica foram detectadas a neuro (NH) e adeno-hipófise (AH). Nas 3 regiões da AH, rostral (RPD) e proximal (PPD) pars distalis, e pars intermedia (PI), foram encontrados 8 tipos celulares produtores de hormônios, sendo, na RPD, as células produtoras de ACTH e PRL; na PPD, GTH (FSH e LH), GH e TSH; e na PI, MSH e SL. As prolactínicas foram as primeiras células identificadas com 1 dpe no início da formação da glândula. A NH foi diferenciada com 3 dpe junto com as células produtoras de ACTH, MSH, TSH e FSH. A identificação das células produtoras de LH, SL e GH ocorreu com 5 dpe. Com 20 dpe houve um grande aumento da hipófise e o aparecimento do pedúnculo. Com 60 dpe a hipófise apresentou-se semelhante à observada nos adultos. Os resultados desta pesquisa colaboram para o conhecimento do eixo hipotálamo-hipófise-gônada em teleósteos sul-americanos. / After reproduction of A. altiparanae, was made larvae and juveniles of this specie, were collected from zero to 120 days post hatching (dph). By histochemical and immunohistochemical techniques were detected neuro (NH) and adenohypophysis (AH). In 3 regions of AH, rostral (RPD), proximal (PPD) pars distalis and in pars intermedia (PI), eight producing hormones cell types are distinguished, being, in the RPD, ACTH and PRL producing cells; in PPD, GTH (FSH and LH), GH and TSH; and in the PI, MSH and SL. The first cells identified were prolactins at 1dph, in the beginning of the formation of the gland. NH differentiated at 3dph along with identification of ACTH, MSH, TSH and FSH producing cells. The identification of LH, SL and GH producing cells was possible at 5dph. At 20dph, there was a large increase in the pituitary, and it was observed the presence of the pituitary stalk. At 60 dph the pituitary showed a similar morphology to that observed in adults. The results of this research collaborate to knowledge of the hypothalamic-pituitary-gonadal axis in South American teleosts.