京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18929号 / 薬科博第43号 / 新制||薬||5(附属図書館) / 31880 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

Le RFRP est une neuropeptide impliqué dans la régulation de l’axe reproducteur, mais ses effets varient en fonction du sexe et des espèces. Le but de cette étude était de décrire en détails l’organisation du système RFRP et de caractériser son rôle dans le contrôle circadien et saisonnier de l’axe reproducteur de hamsters femelles. Les résultats montrent que le système RFRP est régulé par la photopériode et que son niveau d’expression est plus élevé chez les femelles que chez les mâles. Cela se traduit par des actions spécifiques sur l’axe gonadotrope femelle. En effet, L’activité des neurones à RFRP est diminuée au moment du pic pré-ovulatoire de LH et des injections centrales de RFRP-3 dans l’heure qui précède le pic de LH induisent une diminution de l’amplitude de la sécrétion de LH, démontrant une implication du RFRP dans la régulation circadienne du pic pré-ovulatoire de LH. Par ailleurs, des infusions chroniques de RFRP-3 chez des hamsters femelles sexuellement inactifs sont capables de réactiver le fonctionnement de l‘axe reproducteur, ce qui montre que le RFRP a un également un rôle régulateur essentiel dans le contrôle saisonnier de la reproduction. / RFRP neurons regulate the reproductive axis, however, their effects depend on species and sex. Here, we aimed at providing a neuroanatomical description of the RFRP system in the Syrian hamster and at investigating the role of RFRP in the daily and seasonal control of female reproduction. We show that besides being regulated by annual changes in photoperiod, the RFRP system is more strongly expressed in females than in males. In line with this, we unveil that RFRP has multiple roles in regulating female reproduction. RFRP neuronal activity is specifically reduced at the time of the pre-ovulatory LH surge and central RFRP-3 administration prior to the surge decreases LH peak levels, altogether pointing towards a daily down-regulation of the inhibitory RFRP signal necessary for proper generation of the LH surge. Moreover, chronic RFRP-3 infusion in sexually inactive females, with endogenous low RFRP expression, completely reactivates the reproductive axis. Taken together, we demonstrate that RFRP is a key component in the seasonal control of reproduction while at the same time specifically regulating cyclic events controlling reproductive activity in females.

by Lau Hoi Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-96). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Endocrine control of normal and abnormal growth of prostate --- p.1 / Chapter 1.1.1 --- Androgen regulation of prostate gland --- p.1 / Chapter 1.1.2 --- Estrogen regulation of prostate gland --- p.4 / Chapter 1.2 --- Gonadotropin-releasing hormone plays a central role in reproduction --- p.6 / Chapter 1.2.1 --- GnRH gene --- p.7 / Chapter 1.2.2 --- GnRH receptor --- p.9 / Chapter 1.3 --- Therapeutic strategies using GnRH analogs to treat prostate cancer --- p.12 / Chapter 1.4 --- Expression of GnRH or its receptor in reproductive tissues --- p.12 / Chapter 1.4.1 --- Expression of GnRH in reproductive --- p.13 / Chapter 1.4.2 --- Expression of GnRH and its receptor in pituitary and reproductive tissues --- p.13 / Chapter 1.5 --- Animal models for the study of prostate cancer --- p.15 / Chapter 1.5.1 --- Nobel rat inducible model --- p.15 / Chapter 1.5.2 --- Androgen dependent rat Dunning prostatic tumor --- p.16 / Chapter 1.5.3 --- Androgen-independent prostatic carcinoma line of Noble rat --- p.18 / Chapter 1.6 --- Aim of study --- p.18 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and supply of Nobel rat --- p.20 / Chapter 2.2 --- Induction of dysplasia in Nobel rat prostate gland by long-term treatment with steroids --- p.20 / Chapter 2.2.1 --- Chemicals --- p.20 / Chapter 2.2.2 --- Preparation of steroid hormone-filled Silastic tubings --- p.20 / Chapter 2.2.3 --- Surgical implantation of Silastic® tubings --- p.21 / Chapter 2.2.4 --- Protocols of hormonal treatments --- p.21 / Chapter 2.3 --- Androgen- dependent Dunning rat prostatic adenocarcinoma --- p.22 / Chapter 2.4 --- Androgen- independent prostatic carcinoma line (ALT) of Noble rat --- p.22 / Chapter 2.5 --- Detection of mRNA expression of gonadotropin- releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.23 / Chapter 2.5.1 --- Preparation of tissue for total RNA extraction --- p.23 / Chapter 2.5.2 --- Total RNA extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription Polymerase Chain Reaction (RT-PCR) --- p.25 / Chapter 2.5.4 --- Purification of DNA fragments from agarose gels --- p.27 / Chapter 2.5.5 --- Subcloning of DNA into vector --- p.27 / Chapter 2.5.6 --- Nucleotide sequencing --- p.30 / Chapter 2.5.7 --- Southern blot analysis --- p.32 / Chapter 2.5.7.1 --- Southern blotting --- p.32 / Chapter 2.5.7.2 --- Preparation of α-32P-dCTP labelled GnRH probe --- p.32 / Chapter 2.5.7.3 --- Hybridization --- p.33 / Chapter 2.6 --- Detection of mRNA expression of gonadotropin-releasing hormone receptor (GnRH-R) in normal and neoplastic rat prostates --- p.34 / Chapter 2.6.1 --- Cloning of GnRH-R cDNA and synthesis of its probe --- p.34 / Chapter 2.6.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates by Southern blot --- p.36 / Chapter 2.6.3 --- Detection of GnRH receptor mRNA expression in Dunning tumor --- p.37 / Chapter 2.6.4 --- Detection of the GnRH receptor mRNA expression in AIT tumor by RT-PCR --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Detection of mRNA expression of gonadotropin-releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.38 / Chapter 3.1.1 --- Reverse -transcription Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 3.1.2 --- Purification of DNA fragments amplified by PCR from the agarose gel --- p.38 / Chapter 3.1.3 --- Subcloning of DNA into vector --- p.39 / Chapter 3.1.4 --- Nucleotide sequencing --- p.39 / Chapter 3.1.5 --- Southern-blot analysis --- p.39 / Chapter 3.2 --- Detection of gonadotropin-releasing hormone receptor mRNA expression in normal and neoplastic rat prostates --- p.40 / Chapter 3.2.1 --- Cloning of gonadotropin-releasing hormone receptor (GnRH) cDNA and synthesis of probe from the normal Noble rat pituitary gland --- p.40 / Chapter 3.2.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates --- p.42 / Chapter 3.2.3 --- Detection of GnRH receptor mRNA expression in rat Dunning tumor by PCR --- p.43 / Chapter 3.2.4 --- Detection of GnRH receptor mRNA expression in AIT tumor --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Detection of mRNA expression of gonadotropin-releasing releasing hormone(GnRH) in normal and neoplastic rat prostates --- p.69 / Chapter 4.1.1 --- Expression of GnRH mRNA in normal Nobel rat prostate gland --- p.69 / Chapter 4.1.2 --- Expression of GnRH mRNA in dysplastic Nobel rat prostate --- p.71 / Chapter 4.1.3 --- Expression of GnRH mRNA in androgen-dependent rat Dunning prostatic tumor --- p.72 / Chapter 4.1.4 --- Expression of GnRH mRNA in AIT tumor --- p.74 / Chapter 4.2 --- Detection of GnRH receptor in normal and dysplastic rat prostates --- p.75 / Chapter 4.2.1 --- Negative expression of GnRH receptor in normal and dysplastic Nobel in rat prostates --- p.75 / Chapter 4.2.2 --- Positive expression of GnRH receptor mRNA in rat Dunning tumor --- p.77 / Chapter 4.2.3 --- Negative expression of GnRH receptor mRNA in ALT tumor --- p.78 / Chapter Chapter 5 --- Summary and Conclusions --- p.80 / References --- p.83

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 6, 2010). Thesis advisor: M. Cathleen Kuehl-Kovarik. Includes bibliographical references.

Hermaphroditism, associated with territoriality and dominance behavior, is common in the marine environment. Male sex-specific coloration patterns and behavior are particularly evident in species where males are territorial and guard harems of females such as wrasses and groupers. Protogynous hermaphrodites that change sex from female to male are good models to study sexual behavior and related changes in the brain due to their abilities to reorganize their sexual phenotype as adults. Two hormones produced in the brain and implicated in the process of sex-specific behavior and reproductive development are arginine vasotocin (AVT) and gonadotropin releasing hormone (GnRH). While a wealth of data exists regarding these hormone systems separately, little is known about linkage between these two systems. Especially there is no data tracking these two systems together in any protogynous fish. This study was conducted to test the hypothesis that coordinated interactions between AVT and GnRH facilitate the process of behavioral and gonadal sex change in the rock hind Epinephelus adscensionis. Four topics were addressed to investigate the relationship between behavior and reproduction: i) rock hind sex change, sexual characteristics and conditions causing sex change to occur in captivity were detailed as a basis for examining the AVT system and GnRH during this process, ii) the distribution of a vasotocin V1a type receptor identified in rock hind brain was examined for the first time in a fish species using a custom designed antibody then the receptor protein was co-localized with GnRH producing cells within the brain to confirm that a pathway exists for AVT action on GnRH, iii) levels of AVT, AVT receptors, and GnRH messenger RNA (mRNA) were compared between male and female rock hind phenotypes, and iv) female rock hind at early stages of sex change were compared for brain mRNA expression of AVT, AVT receptors, and GnRH to determine the order of hormonal change during the process of sexual inversion in this species. This study provides a better understanding of the relationship between sex-specific behavior and reproductive development via AVT and GnRH systems that are conserved in all vertebrates. / text

Fertility is dependent on the precisely orchestrated communication of an array of effectors within the reproductive axis, all of which impinge on gonadotropin-releasing hormone (GnRH) neurons. A novel reproductive inhibitor was identified in avian species and growing evidence suggests that the functional mammalian homologue, RFamide-related peptide-3 (RFRP-3 or GnIH) can inhibit GnRH neuronal activity and gonadotropin release. To date, the regulation and effects of RFRP-3 at the hypothalamic level are poorly understood. We established an Rfrp-expressing neuronal cell model to investigate the mechanisms of transcriptional regulation of the genes for RFRP and the RFRP receptor, GPR147 by dexamethasone and estradiol. We show that the RFRP system is a direct target for stress-associated transcriptional regulation. Further, employing a novel GnRH-secreting cell line, we report that GnRH neurons express Gpr147 and RFRP-3 represses the transcription of GnRH. These data further our understanding of the level and regulatory effects at which RFRP-3 modulates reproduction.

Fertility is dependent on the precisely orchestrated communication of an array of effectors within the reproductive axis, all of which impinge on gonadotropin-releasing hormone (GnRH) neurons. A novel reproductive inhibitor was identified in avian species and growing evidence suggests that the functional mammalian homologue, RFamide-related peptide-3 (RFRP-3 or GnIH) can inhibit GnRH neuronal activity and gonadotropin release. To date, the regulation and effects of RFRP-3 at the hypothalamic level are poorly understood. We established an Rfrp-expressing neuronal cell model to investigate the mechanisms of transcriptional regulation of the genes for RFRP and the RFRP receptor, GPR147 by dexamethasone and estradiol. We show that the RFRP system is a direct target for stress-associated transcriptional regulation. Further, employing a novel GnRH-secreting cell line, we report that GnRH neurons express Gpr147 and RFRP-3 represses the transcription of GnRH. These data further our understanding of the level and regulatory effects at which RFRP-3 modulates reproduction.

Made available in DSpace on 2015-04-09T12:28:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-03Bitstream added on 2015-04-09T12:48:07Z : No. of bitstreams: 1 000815988.pdf: 670904 bytes, checksum: df849f5b7af31dd25a1aabd5ab80b4cd (MD5) / O Brasil é o país com a maior diversidade de psitacídeos do mundo, abrigando 72 espécies reconhecidas, com 16 espécies presentes no “Livro Vermelho da Fauna Brasileira Ameaçada de Extinção”. O papagaio-verdadeiro (Amazona aestiva) se destaca por sua popularidade como animal de estimação, sendo coletado na natureza em grande número para atender ao mercado ilegal de animais silvestres. Sua reprodução em cativeiro pode se tornar uma ferramenta para a conservação das populações na natureza, porém isso exige uma criação baseada em conhecimentos científicos e técnicas avançadas de reprodução. Esta tese teve como objetivos apresentar o perfil anual endócrino dos esteroides sexuais do Amazona aestiva e realizar um ensaio de estímulo hormonal através da aplicação de análogo de GnRH de liberação lenta na espécie. Utilizamos 10 casais e 4 machos adultos da espécie Amazona aestiva mantidos em viveiros suspensos, pertencentes ao Criadouro da Brisa, situado Jaboticabal/SP. As excretas foram coletadas ao menos uma vez por semana entre junho de 2011 e julho de 2012 para entendimento dos processos endócrinos que regem a reprodução da espécie e entre agosto de 2012 e dezembro de 2012 no ensaio de estímulo hormonal. O monitoramento da atividade gonadal foi feita de forma não invasiva por mensuração de metabólitos de andrógenos nas excretas dos machos e de progestágenos nas excretas de fêmeas. Foram coletadas amostras frescas de excretas, sempre no período entre 14h as 17h, e mantidas congeladas até o processamento. As amostras foram secas em estufa a 57oC, trituradas e os hormônios extraídos utilizando metanol a 80%. A dosagem hormonal foi realizada no Laboratório de Endocrinologia do NUPECCE (Núcleo de Pesquisa e Conservação de Cervídeos) utilizando ensaio imunoenzimático com o anticorpo para andrógenos e progestágenos. No ensaio de estimulo hormonal exógeno com analago de GnRH foi aplicado ... / Brazil is the country with the greatest diversity of parrots in the world , with to 72 recognized species , with 16 species in the Red List of Endangered Brazilian Wild Animals . The blue-fronted amazon parrot ( Amazona aestiva ) stands out for its popularity as a pet , being collected from the wild in large numbers to attend the illegal market for wildlife. His captive breeding can become a tool for the conservation of populations in nature , but this requires a creation based on scientific knowledge and advanced breeding techniques . This thesis aimed to present the annual endocrine profile of sex steroids of Amazona aestiva and a test of hormonal stimulation by applying GnRH analogue of the slow release. It was used 10 couples and 4 adult males of Amazona aestiva kept in suspended cages, properties of the commercial breeder “Criadouro da Brisa”, located in Jaboticabal / SP. The droppings were collected at least once a week between June 2011 and July 2012 for the understanding of endocrine processes of the reproduction in this specie and between August 2012 and December 2012 for testing hormonal stimulation . The monitoring of gonadal activity was noninvasively by measuring androgen metabolites in droppings of males and females of droppings progestogens. Fresh droppings samples were collected , always in between 14h to 17h , and kept frozen until processing . The samples were dried at 57oC, crushed and hormones were extracted using 80% methanol . The hormone dosage was performed at the Laboratory of Endocrinology, NUPECCE ( Center for Research and Conservation of Deer ) using enzyme immunoassay with antibody to androgens and progestins . In exogenous hormone stimulation test with GnRH was applied analago buserelin slow release in 5 couples and 5 couples were used to control the following excreta collection , processing and hormonal dosage previously described . The results of the annual listing of androgens in males ...