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Recent studies showed that GPR30, a seven-transmembrane G protein-coupled receptor, is a novel estrogen receptor (ER) that mediates some biological events elicited by estrogen in several types of cancer cells. However, its physiological or pathological role in vivo is unclear. For the first project of my dissertation, I investigated the physiological role(s) of GPR30 in energy metabolism by using transgenic mouse model as well as immortalized cell lines and primary stromal cells. We discovered for the first time that GPR30 knockout (GPRKO) female mice were protected from high-fat diet (HFD)-induced obesity, glucose intolerance, and insulin resistance. The decreased body weight gain in GPRKO female mice is due to the reduction in body fat mass. These effects occurred in the absence of significant changes in food intake, intestinal fat absorption, or triglyceride metabolism. However, GPR30 had no significant metabolic effects in male mice fed the HFD and both sexes of mice fed a chow diet. Further, GPR30 expression levels in fat tissues of WT obese female mice greatly increased, whereas ERα/β expression was not altered. Deletion of GPR30 reduced adipogenic differentiation of adipose tissue-derived stromal cells. Conversely, activation of GPR30 enhanced adipogenic differentiation of 3T3-L1 preadipocytes. For the second project, I explored whether estrogen acts through GPR30 to affect adiposity in female mice. For this study, I generated and examined three independent transgenic mouse models, aromatase (Ar) knockout (ArKO) mice, GPRKO, and GPR30 and Ar double knockout (DKO) mice. We discovered that GPR30 deficiency had limited effects on energy metabolism in mice fed a standard chow diet (STD). However, deletion of GPR30 promoted metabolic flexibility in both genders fed a HFD regardless of the presence of estrogen, suggesting that GPR30 may not solely act as an ER. Consistent with our previous findings, GPRKO mice had higher body temperature, indicating that GPR30 deficiency may promote thermogenesis and energy metabolism, resulting in the reduced fat depots and enhanced metabolic flexibility. For the third project, I further explored whether GPR30 is involved in regulating browning of adipose tissue and thermogenesis in mice. The results show that the expression of UCP-1, the key regulator of thermogenic browning, was higher in the adipose tissue of HFD-fed GPRKO female mice as compared with that of WT mice. Consistently, deletion of GPR30 enhanced mitochondrial respiration in brown adipose tissue (BAT), suggesting that GPR30 deficiency at least partially suppressed the fat accumulation by promoting thermogenesis and dissipating energy. Ex vivo, the expression of thermogenic genes and UCP-1 protein level were upregulated in beige adipocytes differentiated from GPR30-deficient stromal vascular fraction (SVF) cells. These findings provide evidence for the first time that deletion of GPR30 reduces adiposity, promotes white adipose beigeing and thermogenesis, therefore preventing the development of obesity in female mice exposed to excess energy. Further investigations elucidating the underlying mechanism by which GPR30 promotes obesity in females could provide a novel therapeutic target to fight obesity in females. / Doctor of Philosophy / Estrogen can elicit pleiotropic genomic and rapid nongenomic cellular responses via a diversity of estrogen receptors (ERs). Unlike the genomic responses, where the classical nuclear ERα and ERβ initiate gene transcription in estrogen target tissues, the nongenomic cellular responses to estrogen are believed to start at the plasma membrane, leading to rapid activation of second messengers-triggered cytoplasmic signal transduction cascades. The recently acknowledged ER, GPR30, was discovered in human breast cancer cells two decades ago and subsequently in many other cells. Since its discovery, it has been claimed that estrogen, ER antagonist fulvestrant, as well as some estrogenic compounds can directly bind to GPR30, and therefore initiate the rapid nongenomic cellular responses. We are interested to investigate the physiological role(s) of GPR30 in energy metabolism by using transgenic mouse model as well as immortalized cell lines and primary stromal cells. We discovered for the first time that deletion of GPR30 protects female mice from high fat-diet (HFD)-induced obesity and the expression of GPR30 increased in fat tissues of wild type (WT) obese mice, while no alterations of classical ERα/β observed. Consistently, activation of GPR30 by the selective agonist G-1 promotes adipogenic differentiation of 3T3-L1 preadipocytes. ERα is known to exert a protective effect against excess fat accumulation whereas GPR30 may acts as an “obesity gene” and counteracts the classical ERα’s action in regulating fat metabolism. We speculated that there might be a “Yin-Yang” relationship between GPR30 and ERα regarding their actions in the development of obesity. Therefore, we generated three independent transgenic mouse models, GPR30 and aromatase (Ar) double knockout (DKO), GPR30 knockout (GPRKO), and Ar knockout (ArKO) to test our hypothesis that the excess fat accumulation in HFD-fed WT mice could be, or at least partially, caused by the enhanced estrogen-GPR30 signaling. Ar is the key enzyme that catalyzes the biosynthesis of C18 estrogens from C19 androgens in men and postmenopausal women, thereby the ArKO and DKO mouse models allowed us to investigate the role of GPR30 in the absence of endogenous estrogen. We discovered that GPR30 deficiency had limited effects on energy metabolism in young mice fed a standard chow diet (STD). However, deletion of GPR30 promoted metabolic flexibility in both genders fed a HFD regardless of the presence of estrogen, suggesting GPR30 may not solely acts via the ligation of estrogen. Interestingly, consistent with our previous findings, GPRKO mice had higher body temperature, indicating that GPR30 deficiency may promote thermogenesis and energy metabolism, resulting in the reduced fat depots and enhanced metabolic flexibility. Hence, we explored that deletion of GPR30 exerted thermo-promoting effect via upregulation of the mitochondrial uncoupling protein-1 (UCP-1) and enhanced mitochondrial respiration in brown adipose tissue (BAT). Further, the expression of thermogenic genes were significantly higher in the stromal cells-differentiated beige adipocytes, suggesting that GPR30 deficiency suppressed fat accumulation by promoting thremogenic browning of white adipose tissue (WAT) and dissipating excess energy as heat. In summary, my dissertation work provide valuable insight regarding the role of GPR30 in energy metabolism. Further investigations testing whether GPR30 acts as a pro-obesity gene would facilitate our understanding of obesity development and provide a novel therapeutic target to fight obesity.

Afin de mettre en évidence les principaux éléments de la voie androgénique et œstrogénique dans l’épididyme du rat des sables adulte, capturé dans la région de Beni Abbès, en Algérie, l’aromatase, l’œstradiol, les récepteurs des androgènes (RA) et des œstrogènes (REα, REβ, GPR30) ont été recherchés chez des animaux en saison d’activité, en saison de repos sexuel, chez des animaux castrés, castrés puis traités par la testostérone et chez des animaux ayant subi la ligature des canaux efférents. En saison d’activité, les RA sont ubiquitaires, l’aromatase est cytoplasmique par contre l’œstradiol est nucléaire et cytoplasmique. Les REα et le GPR30 sont principalement dans le cytoplasme apical par contre les REβ sont nucléaires. En saison de repos sexuel, les RA, l’aromatase, l’œstradiol, les REα et le GPR30 persistent, cependant, les REβ subissent une translocation cytoplasmique. Chez les animaux castrés, les RA, l’aromatase et l’œstradiol sont réduits par contre les REα persistent avec une faible intensité. Le GPR30 est cytoplasmique et nucléaire. Chez les animaux castrés puis traités, les RA, l’aromatase, l’œstradiol, les REα, les REβ et le GPR30 sont restaurés. Chez les animaux ligaturés, le RA est faiblement conservé uniquement dans l’épididyme proximal. L’aromatase et l’œstradiol sont conservés. Le signal des REα, des REβ et du GPR30 est fortement exprimé dans le noyau et le cytoplasme dans l’épididyme proximal par contre il est fortement exprimé uniquement pour les REα dans l’épididyme distal. Par western blot, les RA, REα, REβ et GPR30 sont de 122, 64, 55 et 55 kDa respectivement. / In order to highlight the main elements of androgen and estrogen pathway in the epididymis of sand rat, captured in Beni Abbès area, in Algeria, androgen receptor (AR), aromatase, estradiol, estrogen receptors (ERα, ERβ and GPR30) were explored in breeding season, in resting season and in animals underwent castration, castration then testosterone treatment and ligation of efferent ducts. In breeding season, AR has a ubiquitous distribution, aromatase is exclusively cytoplasmic and estradiol is nuclear and cytoplasmic. The ERα and GPR30 were distributed with a high intensity in the apical cytoplasm contrarily to ERβ which were nuclear. In resting season, AR, aromatase, estradiol, ERα persist with lower staining. However, ERβ undergo cytoplasmic translocation and GPR30 persist in cytoplasm. In castrated animals, AR, aromatase and estradiol are reduced. ERα persist with low intensity in the apical cytoplasm. GPR30 is distributed in the cytoplasm and the nucleus. In castrated then treated animals, AR is restored; aromatase and estradiol reappear with a cytoplasmic localization for aromatase, nuclear and apical for ERα. ERβ and GPR30 are restored and have a cytoplasmic localization. In ligatured, RA is preserved in the caput, aromatase and estradiol persist caput and cauda. The signal of ERα, ERβ and GPR30 is highly expressed in the nucleus and cytoplasm of caput epididymis and highly expressed of ERα exclusively in cauda. By Western blot, RA, ERα, ERβ and GPR30 are found with molecular weights of 122, 64, 55 and 55 kDa respectively.

The G protein-coupled estrogen receptor GPER/GPER1, also known as GPR30, was originally cloned as an orphan receptor and later shown to be specifically activated by 17-ß-estradiol. This has led to its classification as an estrogen receptor and expanded the perspective on the mechanisms underlying the rapid estrogenic effects reported over the years. GPER is strongly expressed in the central nervous system and peripheral tissues and appears to be involved in a wide variety of physiological and pathological processes. Estrogens are known to alter the processing of nociceptive sensory information and analgesic responses in the central nervous system. Both analgesic and pro-nociceptive effects of estrogens have been reported. Some pro-algesic estrogenic responses have a short latency, suggesting a non-genomic mechanism of action. Immunohistochemical studies in rodents prove the existence of GPER in pain-relevant areas of the nervous system such as dorsal root ganglia, superficial dorsal horn of the spinal cord, periaqueductal gray (PAG), amygdala, trigeminal sensory nucleus and thalamus. In the periphery, activation of GPER results in pro-nociceptive effects. However, GPER involvement in pain processing at central levels is largely unexplored. Thus, the work presented in this thesis was aimed at investigating whether GPER modulates nociception at spinal and supraspinal sites. The behavioral response to GPER activation in the spinal cord and PAG was evaluated in an acute grooming test (scratching, biting and licking behavior) and in the hot plate test, respectively. Intrathecal challenge of mice with the GPER agonist G-1 (0.1-1 nmol) induced a dose-dependent increase in pain-related behaviors, that was abolished by pre-treatment with the GPER antagonist G15 (1-10 nmol), confirming GPER specificity of the response. Likewise, intra-PAG microinjection of G-1 (10-100 pmol) to rats reduced the nociceptive threshold in the hot plate test, an effect that was G15 sensitive. To obtain further insight on the mechanisms involved in the behavioral effects observed in whole animals, we tested the effect of GPER ligands on neuronal membrane potential, intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) accumulation. The membrane depolarization and the increases in [Ca2+]i and ROS levels are markers of neuronal activation, underlying pain sensitization in the spinal cord and pain facilitation in the PAG. Electrophysiological recordings from superficial dorsal horn and lateral PAG neurons indicate neuronal depolarization upon G-1 application, an effect that was fully prevented by G15 pre-treatment. Both cultured spinal neurons and cultured PAG neurons responded to G-1 administration by elevating [Ca2+]i and mitochondrial and cytosolic ROS levels. In the presence of G15, G-1 did not elicit the calcium and ROS responses. Collectively, these results demonstrate that GPER modulates both the ascending and descending pain pathways to increase nociception via cytosolic calcium elevation and ROS accumulation in spinal and PAG neurons, respectively. These findings broaden the current knowledge on GPER involvement in physiology and pathophysiology, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the mechanisms involved. Moreover, we show for the first time ROS accumulation downstream of GPER activation, extending the current understanding of GPER signaling. / Pharmacology

Im 1997 wurden der Orphan GPR30 aus HUVECs kloniert, die FSS ausgesetzt waren. In dieser Studie konnte gezeigt werden dass die Expression von GPR30 durch die FSS-Behandlung im Vergleich zu unbehandelten HUVEC-Zellen deutlich induziert wurde. Daraufhin wurde in einer Studie von Isensse et al. die zelluläre und gewebsspezifische Expression von GPR30 in GPR30-LacZ Reportergen-Mäusen untersucht. Es konnte eine Expression von GPR30 vorwiegend in den Endothelzellen der kleinen Arterien verschiedenster Gewebetypen nachgewiesen werden. GPR30 war postuliert dass E2 direkt binden kann und dadurch rasche nicht-genomische Signale vermittelt. Im Gegensatz dazu haben verschiedene andere Veröffentlichungen gezeigt dass E2 nicht spezifisch an GPR30 bindet. Trotz der Kontroverse ob es sich bei GPR30 um einen Östrogenrezeptor oder nicht ist bislang nichts über seine Interaktion zu anderen Proteinen und deren Wechselwirkung bekannt. Deswegen war ein Ziel dieser Arbeit, Interaktionspartner von menschlichen GPR30 zu identifizieren und folglich ein humanes vaskuläres in vitro Modell zu etabliren, um die potentiellen Interaktionen von GPR30 sowie die downstream-Effekte der Wechselwirkung zwischen GPR30 und den neuen Interaktionspartner des vaskulären Modells auf Transkriptebene zu evaluieren. Ein Screening einer humanen kardiovaskulären cDNA-Bibliothek mit Hilfe des Y2H-Systems führte zur Identifizierung mehrerer Interaktionspartner für GPR30 darunter PATJ und FUNDC2. Durch anschließende CoIP konnte die Interaktion von GPR30 mit PATJ validiert werden. Des Weiteren konnte in dieser Arbeit die Wirkung von FSS auf die Expression von GPR30 in HUVECs bestätigt und ebenfalls in weiteren anderen Endothelzellen gezeigt werden. Abschließend wurde die Rolle von GPR30 und PATJ bei der Reaktion auf FSS auf transkriptioneller Ebene in HMEC-1-Zellen genomweit untersucht. Interessanterweise war eine Gruppe von Genen aufgrund von FSS in Zellen die GPR30 überexprimierten dereguliert als alleine durch FSS. / In 1997, the orphan G protein-coupled receptor 30, GPR30, was cloned using HUVECs exposed to FSS. It was shown that the level of GPR30 expression was up-regulated in response to FSS. Subsequently, in a study performed in the laboratory where the work for this thesis was carried out, the cellular and tissue distribution of GPR30 were investigated in GPR30-LacZ reporter mice and the expression was found predominantly in the endothelial cells of small arteries in several tissue types. GPR30, was also claimed to bind 17-β-estradiol (E2) directly and to mediate rapid non-genomic signalling. In contrast, various reports have indicated that E2 fails to bind GPR30 in a specific manner. Despite the controversy on whether GPR30 is an estrogen receptor or not, nothing is known at present about its relation and interaction with other proteins. Therefore, the aim of the work described in this thesis was to identify human GPR30 protein interaction partners and to establish a human vascular in vitro model in order to evaluate the potential role of GPR30 and the downstream effects of the interaction between GPR30 and new interaction partners in a vascular model at transcript level. The screening of a human heart cDNA library using the yeast two-hybrid assay led to the identification of several interaction partners for GPR30, among them PATJ and FUNDC2. These interactions were verified by CoIP experiments and the interaction of GPR30 with PATJ could be confirmed. The effect of FSS on the expression of GPR30 was confirmed in HUVECs and was detected in other endothelial cell types. In HUAECs, HAoECs and HMEC-1 cells GPR30 was also found up-regulated upon FSS, suggesting that GPR30 may indeed play a key role in vascular physiology. Finally, the role of GPR30 and PATJ in the FSS response was investigated at the genome-wide transcript level in HMEC-1 cells. Interestingly, a different panel of genes was deregulated owing to FSS in cells over-expressing GPR30 compared to FSS alone.

Der G Protein-gekoppelte Rezeptor 30 (GPR30) wurde vornehmlich im Kontext von schnellen Östrogeneffekten auf zelluläre Signaltransduktionskaskaden untersucht und stellt möglicherweise einen neuen Östrogenrezeptor dar. Die physiologische Funktion von GPR30 in vivo konnte jedoch bisher nicht ermittelt werden. Daher wurde in dieser Arbeit ein Gpr30-defizientes Mausmodell charakterisiert, bei dem ein Teil der kodierenden Sequenz durch einen LacZ-Reporter ersetzt wurde (Gpr30-lacZ). Die Integration des Konstruktes in den Gpr30-Locus wurde mittels Southern blotting und Real-time PCR verifiziert. Gpr30-positive Zelltypen wurden durch Kolokalisation von LacZ mit zelltyp-spezifischen Markerproteinen identifiziert. Weitere Versuche dienten der Aufklärung des Phänotyps von Gpr30-lacZ Mäusen. Zur Identifizierung von Proteinen des GPR30-Signalkomplexes wurden Yeast-Two-Hybrid Analysen mit der N- bzw. C-terminalen Domäne des Rezeptors durchgeführt. Die wesentlichen LacZ-positiven Zellpopulationen waren (i) Endothelzellen in kleinen arteriellen Gefäßen, (ii) glatte Muskelzellen, Perizyten und neuronale Subpopulationen im Gehirn, (iii) Hauptzellen in der Magenschleimhaut, (iv) Zellpopulationen in der Adenohypophyse und dem Hypophysenzwischenlappen sowie (vi) chromaffine Zellen im Nebennierenmark. Während der Phenotypisierung des Mausmodells wurde eine Reduktion der CD62L+ T-Zellen von ca. 50% im peripheren Blut festgestellt. Mittels Yeast Two-Hybrid Analyse wurden Pals1-associated tight junction protein (PATJ) und FUN14 domain-containing 2 (FUNDC2) als mögliche Interaktionspartner identifiziert. Zusammenfassend wurde in dieser Arbeit eine zelluläre Basis für die Funktion von Gpr30 in vivo ermittelt. Der Phänotyp in Gpr30-lacZ Mäusen ist wahrscheinlich durch eine verringerte Produktion von naiven T-Zellen im Thymus bedingt. PATJ bindet die C-terminalen Aminosäuren von GPR30 mit einer PDZ-Domäne und könnte ein Gerüst-protein des GPR30-Signal¬komplexes darstellen. / The orphan G protein coupled receptor 30 (GPR30) was predominantly analyzed in the context of membrane-initiated estrogen signaling suggesting that GPR30 represents a novel estrogen receptor. However, the physiological function of GPR30 in vivo remained unknown. To unravel the physiological role of murine Gpr30 in vivo, a Gpr30-deficient mouse model was analyzed that harbors a LacZ reporter (Gpr30-lacZ) within the Gpr30 locus. The targeting of Gpr30 was verified by Southern blotting and real-time PCR. Gpr30-expressing cell types were identified by colocalization of LacZ along with cell type-specific markers. Further experiments aimed to decipher the phenotype of Gpr30-lacZ mice. To gain information about the signaling complex of human GPR30, yeast two-hybrid screenings were performed with the N- and C-terminal domains as bait. The main LacZ-positive cell populations were (i) endothelial cells in small arterial vessels of various tissues, (ii) smooth muscle cells, pericytes, and neuronal subpopulations in the brain, (iii) gastric chief cells in the stomach, (iv) cells in the intermediate and anterior pituitary, and (v) chromaffin cells in the adrenal glands. Extensive phenotype screening at the German Mouse Clinic revealed reduced numbers of T cells in the peripheral blood of Gpr30-lacZ mice. Especially the proportion of CD62L+ cells was decreased by approx. 50%. Yeast two-hybrid screening led to the identification of Pals1-associated tight junction protein (PATJ) and FUN14 domain-containing 2 (FUNDC2). In conclusion, this study provides a cellular basis for the function of Gpr30 in vivo. Since CD62L+ cells represent the naive T cell compartment, the phenotype of Gpr30-lacZ mice suggests an impaired production of T cells in the thymus. PATJ likely binds the C-terminus of GPR30 with one of its PDZ domains and may represent a scaffolding protein of the GPR30 signaling complex.

Publikationen die in letzter Zeit veröffentlicht wurden zeigten den G Protein-gekoppelte Rezeptor 30 (Gpr30) als neuer potenzieller Östrogen Rezeptor. Dieser Befund wird kontrovers diskutiert, zudem wurde die physiologische Funktion von Gpr30 bisher noch nicht vollständig geklärt. Ziel der vorliegenden Arbeit war die Erforschung der Rolle von Gpr30 in vivo. In einer primären und sekundären Untersuchung wurde eine phänotypische Charakterisierung einer Gpr30-defizienten Mauslinie vorgenommen. Diese Mauslinie wurde generiert, indem eine beta-Galactosidase-Neomycin Vektorkassette in den open reading frame des Gpr30 Gens eingesetzt wurde. Im Rahmen der primären Untersuchung zeigte die immunologische Analyse eine Reduzierung der T-Zellen sowohl bei den männlichen als auch bei den weiblichen mutanten Mäusen. In einer Thymus-Genexpressionanalyse konnten einige Gene identifiziert werden, die möglicherweise in der Regulation der Anzahl an T-Zellen involviert waren. Auf der Grundlage dieser Ergebnisse wurde eine Erhöhung der Kalzium-vermittelten T-Zellen Apoptose hypothetisiert. Gegenstand der sekundären Untersuchung war die Bestimmung eines möglichen metabolischen und kardiovaskulären Phänotyps, da Gpr30 überwiegend in den Blutgefäßen verschiedener Organe, sowie in der Pankreas und im Magen exprimiert ist. Zu diesem Zweck wurden die Mäuse einer Hochfettdiät unterzogen und es wurden metabolische sowie hemodynamische Tests durchgeführt. Um den Phänotyp dieser ersten Mauslinie zu bestätigen, wurde eine zweite Mauslinie ohne Selektionsmarker generiert. Insgesamt tragen die Ergebnisse der vorliegenden Studie zu einem besseren Verständnis der Funktion von Gpr30 in vivo bei. Eine Rolle des Rezeptors bezüglich der Regulation des Körpergewichts konnte widerlegt werden, während ein Einfluss auf den Lipid- und Muskelstoffwechsel angenommen werden kann. Zudem wurde gefunden, dass Gpr30 für einige Östrogen-regulierende, physiologische Prozesse nicht erforderlich ist. / Recent studies identified the G protein-coupled receptor 30 (Gpr30) as a potential new estrogen receptor. However, these findings remain still controversial and the physiological role of Gpr30 has not been clarified yet. In order to decipher the role of Gpr30 in vivo, we investigated the phenotype of a Gpr30 mutant mouse line, generated by the insertion of a beta-galactosidase-neomycin cassette into the Gpr30 open reading frame, in a primary and a secondary screen. The primary screen revealed a decrease of T cell levels in both male and female mutants. Thymus gene expression analysis allowed to detect some of the genes potentially involved in regulating T cell levels in these mice. On this basis a hypothesis of an increase in T cell calcium-mediated apoptosis was formulated. The secondary screen aimed at unraveling a potential metabolic and cardiovascular phenotype, being Gpr30 mainly expressed in the vasculature of several organs, as well as in the pancreas and in the chief gastric cells of the stomach. Therefore, mice were challenged with a defined high fat diet, and metabolic and hemodynamic tests were performed. To confirm the phenotype achieved in this first mouse line, a second one, devoid of any selection marker, was analyzed. Altogether the results achieved may contribute to a better understanding of Gpr30 function in vivo, disproving a role of Gpr30 in body weight regulation, suggesting a role in lipid and muscular metabolism, and providing evidence that Gpr30 may not be required for several estrogen-regulated physiological processes.

Orientador: Wellerson Rodrigo Scarano / Resumo: Citocinas e proteínas quinases são fundamentais para o controle do processo espermatogênico, estando diretamente envolvidas na dinâmica da barreira hematotesticular. Diferentes mecanismos de controle são modulados por receptores como o GPR30, que ativa rapidamente diferentes vias de sinalização, responsáveis pelos processos de proliferação, sobrevivência e morte celular. Os desreguladores endócrinos (DEs) possuem grande afinidade pelo GPR30, além de potencial para ativar vias de estresse oxidativo e a abertura da barreira. Antagonistas funcionais dos DEs, como o Panax ginseng, podem ser protetores contra seus efeitos. Considerando a importância das vias de sinalização que regulam a espermatogênese e a constante exposição ambiental aos DEs a que estamos submetidos, este trabalho objetiva estudar a possível modulação da via não genômica ativada por GPR30 e do estresse oxidativo em células de Sertoli expostas a baixas doses do DE Monobutil Ftalato (MBP) bem como o potencial papel citoprotetor do GIM-1 (metabólito do P. ginseng) sobre essas células. Para tal, as células de Sertoli humanas (HSec) foram mantidas sobre matriz artificial, simulando o ambiente in vivo. A exposição ao MBP e ao GIM-1 foi realizada nos tempos de 30min, 1, 12, e 48 horas, em doses pré-estabelecidas pelo ensaio do MTT (teste de toxicidade) em 4 grupos: controle, MBP, GIM-1 e MBP + GIM-1. A morfologia celular foi avaliada pela coloração com Hematoxilina e Eosina, evidenciando efeitos deletérios do MBP sobre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cytokines and kinases protein are essential to control the spermatogenic process, being directly involved in the blood-testis barrier control. Activation of these mechanisms is modulated by receptors such as GPR30, which rapidly activates different signaling pathways responsible for proliferation and cell death processes. Endocrine Disruptors (EDs) have high affinity for GPR30, causing oxidative stress and possible barrier rupture. Functional antagonists of EDs, such as Panax ginseng, may be protective against their effects. Considering the importance of the signaling pathways that regulate spermatogenesis and the constant environmental exposure to the EDs to which we are subject, this work aims to study the possible modulation of the non-genomic pathway activated by GPR30 and oxidative stress in Sertoli cells exposed to low doses of the ED Monobutyl Phthalate (MBP) and the possible cytoprotective role of GIM-1 (P. ginseng metabolite) on these pathways. To this end, HSec human lineage cells were maintained on artificial matrix, simulating in vivo environment. Exposure to MBP and GIM-1 was performed at 30 min, 1, 12 and 48 hours at pre-set MTT (toxicity assay) levels in 4 groups: control, MBP, GIM-1 and MBP + GIM-1. Morphology and cell adhesion were evaluated by staining with Hematoxylin and Eosin, evidencing deleterious effects of MBP above cell distribution and adhesion in basement membrane; GIM-1 group was similar to Control and MBP+GIM-1 showed an intermediate aspect. In o... (Complete abstract click electronic access below) / Doutor

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.

Os mecanismos etiopatogênicos que levam ao desenvolvimento dos nódulos e tumores da tireoide ainda não são bem conhecidos. É fato estabelecido que a prevalência dessas lesões é maior nas mulheres. Dessa forma, o que nos motivou a realizar esta Tese foi avaliar se os hormônios sexuais femininos tem efeito direto sobre as células de tireoide humanas normais podendo contribuir para a sua etiopatogenia. Este estudo teve como objetivos padronizar um modelo de cultura primária de células foliculares de tireoide humana normal e nesse modelo validar um gene normalizador após tratamento com estradiol e progesterona, avaliar os efeitos da progesterona na expressão dos genes NIS, TG e TPO bem como avaliar a expressão gênica e proteíca e a possível localização intracelular do receptor de membrana GPR30 nestas células. Em nosso modelo de cultura primária em monocamada, as células foliculares mantiveram sua morfologia cubóide característica e permaneceram diferenciadas o que foi evidenciado pela dosagem de tireoglobulina e T4 livre, no sobrenadante do meio de cultura, e pela identificação das proteínas TG e TPO por imunocitoquímica. A estimulação com progesterona aumentou a expressão dos genes NIS, TG e TPO, respectivamente, 1.78 (p=0.003), 1.50 (p=0.034) e 1.64 (p=0.018) vezes, quando comparadas ao grupo tratado somente com TSH. Essa estimulação da progesterona foi inibida por mifepristona sugerindo que a progesterona tem efeito direto nas células foliculares da tireoide e que esse efeito é mediado por seu receptor nuclear. A normalização da expressão gênica foi realizada pelo gene β-actina, o qual demonstrou uma maior estabilidade entre os grupos analisados. Demonstramos também que as células normais da tireoide expressam tanto o gene como a proteína do receptor de membrana GPR30 com possível localização na membrana celular e no espaço perinuclear. / The mechanisms leading to the development of thyroid nodules and tumors are not well established. As these lesions are more common in women, female sex hormones could be involved in the pathogenesis of these disorders. The objectives of this study were to establish a model of primary culture of normal human thyroid follicular cells, to validate a normalizing gene for qRT-PCR after treatment with estradiol and progesterone, to evaluate the effects of progesterone on the expression of genes NIS, TG and TPO, and to evaluate the GPR30 gene and protein expression as well as its possible intracellular location, in these cells. In our model of primary monolayer culture, follicular cells maintained their characteristic cuboid differentiated morphology; and had evidence of differentiated thyroid function: the production of thyroglobulin and free T4, and identification of TG and TPO proteins by immunocytochemistry . Adding progesterone to TSH increased NIS, TG and TPO mRNA, respectively, 1.78 (p=0.003), 1.50 (p=0.034) and 1.64 (p=0.018) folds, compared to the group treated with only TSH. This stimulation was inhibited by mifepristone, suggesting that progesterone has a direct effect on the thyroid follicular cells. Normalization of gene expression was performed using β-actin as reference gene. We have also demonstrated that normal thyroid cells expressed GPR30 gene and protein, which is possibly localized in the plasma membrane and the perinuclear region.