O transplante de células germinativas tem sido uma importante abordagem experimental para o estudo da preservação genética de espécies ameaçadas de extinção ou economicamente importantes. A técnica consiste na remoção das células germinativas indiferenciadas das gônadas do animal doador e na transferência das mesmas para a gônada de um indivíduo receptor. A fim de aumentar a eficiência da técnica, a identificação prévia das células germinativas a serem transplantadas torna-se preferível, visto que a cavidade que as receberá apresenta tamanho limitado. Sendo assim, é importante o desenvolvimento de marcadores moleculares que identifiquem precisamente as células a serem transplantadas na cavidade do individuo receptor, e os genes mais utilizados para esta finalidade são o dead end e o gene vasa, os quais são expressos apenas nas células da linhagem germinativa. Devido à importância do Colossoma macropomum (tambaqui) para a economia brasileira, esta espécie foi escolhida como uma espécie modelo de preservação para este estudo. Através do isolamento e sequenciamento dos genes dead end e vasa, desenvolvemos sondas de hibridização capazes de reconhecer as células onde estes genes são expressos e estudar o seu padrão de expressão nas gônadas. Ambos os genes apresentaram intensa expressão nos oócitos pré-vitelogênicos e fraca expressão em algumas espermatogônias. Pela primeira vez na literatura, diferentes isoformas causadas por splicing alternativo foram identificadas no gene dead end. A quantificação da expressão temporal dos diferentes transcritos mostrou que o padrão de expressão da sequência completa do gene teve uma tendência distintiva comparada ao padrão dos transcritos curtos, sugerindo que as diferentes isoformas desempenham papéis específicos e importantes para o desenvolvimento da linha germinativa nesta espécie. / Germ cell transplantation has been an important experimental approach to the study of the genetic preservation of endangered or economically important species. The technique consists in removing undifferentiated germ cells from the donor animal's gonads and transferring them to the gonad of a recipient individual. In order to increase the efficiency of the technique, the prior identification of the germ cells to be transplanted becomes preferable, since the receiving cavity presents limited size. Therefore, it is important to develop molecular markers to precisely identify the cells to be implanted in the recipient cavity, and the genes most used for this purpose are the dead end and the vasa genes, which are expressed only in germline cells. Due to the importance of Colossoma macropomum (tambaqui) for the Brazilian economy, this species was chosen as a model species for preservation in this study. By isolating and sequencing the dead end and vasa genes, we developed hybridization probes capable of recognizing the cells where these genes are expressed and better studying their expression pattern in the gonads. Both genes presented intense expression in pre-vitellogenic oocytes and poor expression in some spermatogonia. For the first time in the literature, different isoforms caused by alternative splicing were identified in the dead end gene. Quantification of the temporal expression of the different transcripts showed that the expression pattern of the full-length sequence had a distinctive tendency compared to the short transcripts pattern, suggesting that the different isoforms play specific roles for the germline development in this species.

O estudo visou elucidar o desenvolvimento embrionário e fetal de pacas (Agouti paca) e demarcação dos sítios germinativos nos embriões em diferentes estágios. Foram utilizados sete espécimes de Agouti paca, sendo dois embriões e três fetos doados do pacário mantido pela UNESP - Jaboticabal e, dois doados do acervo da FMVZ-USP. Os fetos 1 e 2 apresentaram imaturidade facial acentuada, olhos recobertos por uma lente proeminente e lóbulos das orelhas; os fetos 3 e 4, mostravam-se com orelhas bem desenvolvidas, membros torácicos e pélvicos em grau equalitário de desenvolvimento, como vibrissas ao redor das bordas nasais e olhos também protegidos; no feto 5, haviam pêlos distribuídos por todo o corpo, membros torácicos e pélvicos com garras, vibrissas na face, olhos proeminentes e orelhas bem desenvolvidas. O embrião 1, apresentou a vesícula óptica com pigmentação da retina, o quarto ventrículo encefálico e curvatura cervical acentuada e broto dos membros em desenvolvimento; o embrião 2, possuiu divisão dos arcos branquiais e neuróporo cranial aberto; presença da área cardíaca e fígado; vesícula óptica sem pigmentação da retina, abertura do tubo neural na região do quarto ventrículo encefálico, rombencéfalo e mesencéfalo em desenvolvimento. Na microscopia de luz, visualizamos a medula espinhal, abertura do 4º ventrículo encefálico, presença das vesículas encefálicas (prosencéfalo, mesencéfalo e rombencéfalo), coluna vertebral, hipófise, cavidades oral e nasal, olho, átrio e ventrículo cardíacos, pulmão e diafragma, além das cristas metanéfrica e mesonéfrica, fígado, intestino e pedículo umbilical. Nas reações de imunohistoquímicas para OCT-4 não houve marcação expressiva em órgãos tais como pulmão, intestino e somitos, o coração apresentou uma leve positividade à reação, enquanto que nas cristas meso e metanéfrica e fígado obtiveram uma marcação expressiva, sendo mais acentuada no último. Nos testes com vimentina todos os órgãos mostraram-se imunopositivos em diferentes áreas; e em se tratando da reação a testes com actina apenas a região de somitos não obteve marcação positiva. Concluímos que o período embrionário/fetal da paca não pode ser comparado com o modelo clássico de roedor; sua embriogênese pode ser comparada à de ratos, Guinea pig, coelhos e humanos. A imunolocalização positiva de OCT-4 apresenta diferenças de acordo com a idade gestacional, devido às mudanças embriológicas dos tecidos. A imunolocalização positiva de OCT-4 apresenta diferenças de acordo com a idade gestacional, devido às mudanças embriológicas dos tecidos. A vimentina como marcador de mesênquima se expressou positivamente em todos os órgãos do embrião de paca. A actina como imunomarcador de músculo liso foi expressiva nas áreas contendo musculatura. / The study aimed elucidates the development of embryonic and fetal of pacas (Agouti paca) and demarcation of the germ sites in embryos at different stages. Seven specimens were used; two embryos and three fetuses from UNESP- Jaboticabal and other two fetuses were from FMVZ-USP collection. The fetuses 1 and 2 showed immaturity facial sharp, eyes covered by a lens and prominent lobes of the ears, the fetuses 3 and 4, showed up with well-developed ears, members thoracic and pelvic in equal level of development, vibrisses around the nasal edges and eye also protected;. The fetus 5 had hairs distributed throughout the body, members thoracic and pelvic with claws, vibrisses on the face, prominent eyes and ears well developed. The embryo 1, presented the optic vesicle with pigmentation of the retina, the fourth encephalic ventricle and cervical curvature and button members in development. The embryo 2, had split the branchial arches and open neuropore cranial; heart and liver were identified, optical vesicle without pigmentation of the retina, the neural tube was opening in the region of the fourth encephalic ventricle, rombencephalon and mesencephalon were in development. Light microscopy, shows the spinal cord, 4 th encephalic ventricle, resence of encephalic vesicles (prosencephalon, mesencephalon and rombencephalon), vertebral column, pituitary gland, oral and nasal cavities, eye, atrium and ventricle heart, lung and diaphragm, as well the metanephron and mesonephron, liver, intestine and mbilical pedicle. The immunohistochemical reactions for OCT-4 were non expressive in organs such as lung, intestine and somites; heart presented a discrete positive reaction, while kidneys and liver obtained an expressive expression, more pronounced in the last one. Vimentina\'s tests showed that all organs stained in different areas, and the reaction whit the actin was negative just in the region of somites. We conclude that the period embryonic/fetal of paca can not be compared with the classical model of rodents; its embryogenesis can be compared with the rats, Guinea pig, rabbits and humans ones. The positive immunolocalization of OCT-4 presents differences according to the gestational age, due to changes embryological tissue. The vimentine as a mesenchyme marker is expressed positively in all organs of the embryo of paca. The actin as immunomarker of smooth muscle was expressive in areas containing muscles.

Orientadores: Rejane Maira Góes, Maria Etelvina Pinto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T07:52:43Z (GMT). No. of bitstreams: 1 Christante_CarolineMaria_M.pdf: 8386652 bytes, checksum: 6c501e195d790722221976fe7452ee67 (MD5) Previous issue date: 2013 / Resumo: Os gonócitos são os precursores das células germinativas masculinas, encontrados durante o período fetal e neonatal. Sua diferenciação neonatal é fundamental para a função reprodutora e envolve retomada da atividade proliferativa, movimentação para a base do epitélio seminífero e diferenciação em espermatogônia. Sabe-se que, no período fetal, a atividade proliferativa dessas células é andrógeno dependente, e que a obesidade altera o cenário dos hormônios sexuais, levando a um aumento dos níveis de andrógenos em mulheres. Contudo, pouco é conhecido sobre os efeitos da obesidade materna sobre o desenvolvimento do aparelho genital masculino, em particular no período neonatal, e as implicações com o cenário hormonal. Sendo assim, o objetivo desse estudo foi avaliar se a obesidade materna interfere no desenvolvimento neonatal do testículo de ratos Wistar, tanto no que se refere às suas características morfológicas gerais, quanto à diferenciação das células germinativas. A obesidade materna foi induzida pelo tratamento, por 15 semanas, com dieta contendo 20% de lipídeos saturados. Foram utilizados filhotes machos de mães normais e obesas nas idades de 0,5, 4,5, 7,5 e 14,5 dias pós-parto (dpp). Os testículos foram processados para microscopias de luz e eletrônica de transmissão. Os cortes histológicos submetidos à reação imunocitoquímica para hormônio anti-Mülleriano (AMH) foram utilizados para a determinação da densidade numérica (Nv) de gonócitos e estimativa da densidade de gonócitos reposicionados para a periferia do túbulo. Também foi avaliado os níveis de apoptose, após imunocitoquímicas para Caspase 3 ativada, e a localização dos receptores de andrógeno (AR) e de estrógeno (ER? e ER?). Nossos resultados revelaram uma diminuição no peso dos filhotes de mães obesas, ao nascimento, mas nenhuma variação foi observada para a distância anogenital. O peso das gônadas foi significantemente menor para os animais obesos de 4,5 dpp, resultando em uma variação do índice gonadossomático, nessa idade. A Nv de gonócitos e os níveis de apoptose não variaram nos neonatos de mães obesas em comparação com o grupo controle, em nenhuma das idades consideradas. No entanto, o número de gonócitos reposicionados para a base do epitélio seminífero, aos 4,5 dpp, foi aproximadamente o dobro nos testículos de animais controle em comparação com os submetidos à obesidade materna. Também foi observado que esse tipo celular não possui imunoreatividade para AR. Entretanto, as células de origem mesenquimal e o citoplasma das células de Leydig fetais, de maneira geral, são imunomarcados. Com relação aos níveis de andrógenos, verificou-se que as médias obtidas para os animais sujeitos à obesidade materna, de 4,5 até 14,5 dpp, são muito semelhantes às encontradas para as idades anteriores no grupo controle, sendo possível inferir que a obesidade materna afete a esteredoigênese e promova alterações na concentração de testosterona, no período peri-natal. De maneira geral, nossos dados indicam que a obesidade materna afeta os níveis de esteroides sexuais e, consequentemente, o padrão de diferenciação dos gonócitos. Contudo, tais alterações parecem ocorrer nos primeiros dias de vida, principalmente na idade de 4,5 dpp, com uma recuperação do desenvolvimento testicular ainda no período pré-púbere / Abstract: The gonocytes are the precursors of male germ cells, found during fetal and neonatal period. Their differentiation is critical for neonatal reproductive function and involves resumption of proliferative activity, drive to the base of the seminiferous epithelium and differentiation into spermatogonia. It is also known that in the neonatal period the proliferative activity of these cells is androgen-dependent, and that the obesity changes the sex hormones scenario, leading to increased androgens levels in women. However, little is known about the effects of maternal obesity on the male genital tract development, particularly in the neonatal period, and the implications with hormonal scenario. Therefore, the objective of this study was to evaluate whether maternal obesity interferes with rat testis development, both with regard to their overall morphology, as the differentiation of germ cells. Obesity was induced by treating, for 15 weeks, with a diet containing 20% of saturated lipids. We used male offspring from normal and obese mothers at 0.5, 4.5, 7.5 and 14.5 days postpartum (dpp). The testes were processed for light microscopy and transmission electron microscopy. The sections subjected to immunocytochemistry for anti- Müllerian Hormone (AMH) were used to determine the numerical density (Nv) of gonocytes and density estimation of repositioned gonocytes to the periphery of the tubule. We also assessed the apoptosis levels after immunocytochemistry for activated Caspase 3, and location of androgen receptors (AR) and estrogen (ER? and ER?). Our results showed a decrease in the weight of pups born from obese mothers, at birth, but no change was observed for the anogenital distance. The gonad weight was significantly lower in obese animals at 4.5 dpp, resulting in a variation of the gonadosomatic index in this age. The Nv of gonocytes and apoptosis levels did not differ in newborns subjected to maternal obesity compared with the control group, for any age considered. However, the number of repositioned gonocytes to the base of the seminiferous epithelium at 4.5 dpp was approximately twice in control group compared with the maternal obesity group. It was also observed that this cell type has no AR immunoreactivity. However, mesenchymal cells and the cytoplasm of the fetal Leydig cells generally are immunostained. With respect to androgens levels, it was found that the mean for the animals subjected to maternal obesity from 4.5 to 14.5 dpp are very similar to those found in earlier ages in the control group, it is possible to infer that maternal obesity affects steroidogenesis and promotes changes in testosterone concentration, in the perinatal period. Overall, our data indicate that maternal obesity affects the sex steroids levels and, consequently, the pattern of differentiation of gonocytes. However, these changes seem to occur in the first days of life, especially at the age of 4.5 dpp, with a recovery of testicular development in the pre-pubertal age / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientador: Irani Quagio Grassiotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T07:25:27Z (GMT). No. of bitstreams: 1 Wildner_DanielDantas_M.pdf: 4539782 bytes, checksum: 657949aff7cd6fcd4b59c61e584bebac (MD5) Previous issue date: 2012 / Resumo: Na maioria dos Teleostei o desenvolvimento gonadal é periódico e acompanha as estações reprodutivas anuais. Ao longo da vida reprodutiva feminina a renovação das células germinativas é constante e responde pela produção ilimitada dos folículos ovarianos. Aqui a dinâmica de renovação das células germinativas femininas, que resulta na formação dos folículos ovarianos, foi estudada em duas diferentes espécies de Ostariophysi com distintos comportamentos reprodutivos: o Serrasalmus maculatus (Characiformes) e o Pimelodus maculatus (Siluriformes). O estudo teve por base dados biométricos, morfométricos e a análise histológica das gônadas, interpretado segundo novas propostas de classificação dos estágios do desenvolvimento oocitário e das fases do ciclo reprodutivo. Adicionalmente, a formação dos folículos ovarianos a partir das oogônias isoladas presentes no epitélio das lamelas, sua proliferação formando ninhos e entrada em meiose dando origem aos oócitos, é descrita tendo como modelo S. maculatus. Os estágios do desenvolvimento oocitário são caracterizados para ambas as espécies. S. maculatus com período reprodutivo longo compreendido principalmente entre os meses de inverno e primavera (hemisfério sul), tem fecundidade indeterminada. Em S. maculatus a foliculogênese é mais intensa nos ovários em Regeneração, reconhecidos por conterem apenas oócitos pré-vitelogênicos, alguns já com alvéolos corticais. No epitélio das lamelas os ninhos contendo oogônias em proliferação são proporcionalmente mais numerosos do que as oogônias isoladas. Nas gônadas em Desenvolvimento, os ninhos contendo oogônias e aqueles contendo oócitos profásicos iniciais, predominam em relação às oogônias isoladas. Nelas, parte dos oócitos pré-vitelogênicos entram e avançam na vitelogênese. Nas gônadas consideradas Aptas à Desova, a quantidade de ninhos de oogônias e oócitos profásicos iniciais é menor relativamente às demais fases e as oogônias isoladas aparecem com maior frequência. Os oócitos vitelogênicos tardios e completamente desenvolvidos convivem com os oócitos pré-vitelogênicos. Resultantes da liberação dos oócitos maduros aparecem os complexos foliculares vazios. Nos ovários em Regressão, a retomada da proliferação das oogônias é detectada pelo aumento do número de ninhos, alguns deles já contendo oócitos profásicos iniciais. A atresia dos folículos contendo oócitos não liberados convive com os oócitos pré-vitelogênicos. Pimelodus maculatus com período reprodutivo nos meses de verão (hemisfério sul), tem fecundidade determinada. Também nessa espécie, os eventos iniciais da foliculogênese são mais intensos nos ovários em Regeneração, nos quais os únicos oócitos presentes são os pré-vitelogênicos. No epitélio das lamelas, os ninhos contendo oogônias em proliferação são proporcionalmente mais frequentes que as oogônias isoladas e ninhos contendo oócitos profásicos iniciais. Nas gônadas em Desenvolvimento, a frequência do numero de ninhos contendo oogônia difere estatisticamente da frequência de ninhos de oócitos e as oogônias isoladas no epitélio. Nessas gônadas parte dos oócitos pré-vitelogênicos entram em vitelogênese. Nos ovários Aptos à Desova, os ninhos de oogônias e oócitos profásicos iniciais aparecem em menor quantidade relativamente às oogônias isoladas. Neles, os oócitos completamente desenvolvidos predominam sobre aqueles vitelogênicos e pré-vitelogênicos. Nos ovários em Regressão, a retomada da proliferação das oogônias é detectada pelo aumento do número de ninhos, alguns deles contendo oócitos profásicos iniciais. A atresia dos folículos contendo oócitos não liberados convive com os oócitos pré-vitelogênicos / Abstract: In most Teleostei the gonadal development is cyclical and follows the annual reproductive seasons. Throughout the female reproductive life the germ cell renewal is constant, and accounts for the unlimited production of ovarian follicles. Here the dynamics of female germ cell renewal, resulting in the formation of ovarian follicles was studied in two different species of Ostariophysi with different reproductive strategies: Serrasalmus maculatus (Characiformes) and Pimelodus maculatus (Siluriformes). The study was based on biometrics, morphometric and histological analysis of gonads, interpreted according to new proposals for the classification of the phases of the reproductive cycle and for the stages of the oocytes development. Additionally, using S. maculatus as a model, we describe the formation of ovarian follicles from the isolated oogonia present in the lamellar epithelium, their proliferation forming cell nests and entrance into meiosis giving rise to the oocytes. The stages of the oocyte development are characterized for both species. S. maculatus with long reproductive period comprising the months of winter and spring (southern hemisphere) has an indeterminate fecundity. In S. maculatus the folliculogenesis is more intense in the Regenerating ovaries, recognized by containing only pre-vitellogenic oocytes, some of them with cortical alveoli. At this time, in the lamellar epithelium the nests with proliferating oogonia are proportionally more numerous than the isolated oogonia. In the Developing ovaries, the nests containing oogonia and those with the early prophase oocytes, are predominate over the isolated oogonia. In them, the part of the pre-vitellogenic oocytes enters and advances in vitellogenesis. In the Spawning Capable ovaries, the number of nests of oogonia and early prophase oocytes is lower compared with other reproductive phases as the isolated oogonia appear more frequently. In these, the late vitellogenic and full-grown oocytes co-occur with pre-vitellogenic ones. The postovulatory follicle complex appear resulting from the release of mature oocytes. In the Regressing ovaries, the resumption of oogonia proliferation is detected by an increase in the number of nests, some of them already containing early prophase oocytes. Atresia of follicles, containing oocytes that were not released, co-occurs with the pre-vitellogenic oocytes. Pimelodus maculatus with a reproductive period concentrated in the summer (southern hemisphere), has a determinate fecundity. Also in this species, the initial events of folliculogenesis in the ovaries are more intense in Regenerating phase, in which only oocytes present are the pre-vitellogenic ones. In the lamellar epithelium the nests containing proliferating oogonia are proportionally more frequent than the isolated oogonia and than the nests containing early prophase oocytes. In the Developing ovaries, the frequency of the number of nests containing oogonia differs statistically from the frequency of nests with oocytes and from the isolated oogonia and in the epithelium. In these gonads of the pre-vitellogenic oocytes enter in vitellogenesis. In the Spawning Capable ovaries, the nests of oogonia and early prophase oocytes appear in a lower number than the isolated oogonia. In them, the full-grown oocytes predominate over those pre-vitellogenic and vitellogenic. In the Regressing ovaries, the resumption of the oogonia proliferation is detected by an increase in the number of nests, some containing early prophase oocytes. Atresia of follicles, containing oocytes that were not released, co-occurs with the pre-vitellogenic / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

La détermination du sexe et la différenciation des cellules germinales est un processus très organisé qui commence au stade embryonnaire et se termine à la vie adulte. Dans les gonades embryonnaires l'expression de Sry suivie par l'expression de Sox9 initie le développement testiculaire tandis que, en l'absence de l'expression de Sry, les gènes associés au sexe féminin initient le développement des ovaires. Les cellules germinales qui ont migré vers les gonades nouvellement formées continuent leur prolifération intense jusqu'à ce qu'ils s'engagent dans la voie le mâle ou femelle. La décision de devenir des cellules germinales mâle ou femelle ne dépend pas seulement du chromosome sexuel des cellules germinales, mais aussi du microenvironnement des gonades. Si les cellules germinales entrent dans la gonade femelle, ils doivent arrêter la prolifération, dépasser l'arrêt mitotique et entrer en méiose et alors s'arrêter en prophase I. Alors que si les cellules germinales entrent dans la gonade mâle, ils doivent arrêter la prolifération et entrer en arrêt de la mitose. Ici, nous montrons que, lors de la détermination du sexe embryonnaire, la prostaglandine D2 (PGD2) produite par chacune des deux enzymes: la L-PGDS et H-PGDS dans les cellules somatiques et les cellules germinales du testicule mâle participe au programme de différenciation des cellules germinales. La voie de signalisation PGD2 entraine l'arrêt de la mitose par l'activation de l'expression et de la localisation nucléaire de l'inhibiteur du cycle cellulaire p21Cip1 et en réprimant les marqueurs de pluripotence et aussi Stra8. En outre, PGD2 est responsable de l'activation du gène spécifique au mâle Nanos2. Par conséquent, ces données suggèrent que la signalisation par le récepteur de PGD2 DP2 est requise pour la différenciation correcte de la cellule germinale mâle. / The sex determination and subsequent germ cell differentiation is highly ordered process that starts at embryonic stage and completes at adult life. In the embryonic gonads Sry expression followed by Sox9 expression initiates testis development while in the absence of Sry expression, genes associated to female fate initiate ovary development. The germ cells that migrated towards newly formed gonads continue extensive proliferation until they commit to the male or female pathway. The fate decision of germ cells as male or female does not depend only on germ cell chromosomal sex but also on gonadal micro-environment. If germ cells enter into female gonad, they have to stop proliferation, pass through mitotic arrest and enter into meiosis; then arrest into prophase I. While if germ cells enter into male gonad, they have to stop proliferation and enter into mitotic arrest. Here we show that during embryonic sex determination, Prostaglandin D2 (PGD2) produced by each of the two enzymes: L-Pgds and H-Pgds in somatic cells and germ cells of testis participates in male germ cell differentiation program. PGD2 signalling supports mitotic arrest by activating the expression and nuclear localization of cell cycle inhibitor P21cip1 and by repressing pluripotency markers and PGD2 has negative effects on Stra8 expression. In addition PGD2 supports activation of male specific gene Nanos2. Hence these data suggest that PGD2 signalling through DP2 receptor is required for proper male germ cell differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(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 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cytokine induced transcription of the germline immunoglobulin heavy chain gene directs isotype switch recombination to that gene. Therefore, understanding the regulation of germline transcription is an important first step in understanding the class switching process. Treatment of human B cells with IL-4 results in germline epsilon transcription. Subsequent activation of a second signal is necessary for these cells to undergo class switch recombination and express surface IgE. In contrast, treatment of splenic murine B-cells with IL-4 alone does not induce germline epsilon transcription. However, treatment with IL-4 plus LPS does induces germline epsilon transcription, followed by class switching to the IgE isotype. In both human and mouse, IL-4 is absolutely required for induction of germline transcripts and expression of IgE. Therefore, IL-4 is considered to be an IgE switch factor. The murine B lymphoma line, I.29μ is an IgM+ B cell line which can be induced to switch to the IgE isotype by treatment with IL-4 plus LPS. In these cells, germline epsilon transcription is constitutive and can be further induced 5-20 fold with IL-4, whereas LPS has no effect at the RNA level. Thus, the I.29μ cell line provides a model system to study the regulatory effects of IL-4 on the murine germline epsilon promoter. The aim of this thesis is to characterize the murine germline epsilon promoter and identify the minimal DNA elements necessary and sufficient for IL-4 induction. To identify the promoter elements, two kb of the 5' flanking region to the first exon (Iε) of the germline epsilon transcript was cloned into a Luciferase reporter plasmid and assayed for promoter activity. Assay of successive 5' deletion mutations by transfections into two B cell lines, I.29μ and M12.4.1, identified the 213 bp promoter construct, -162Luc, as containing sufficient sequence to confer full promoter function. Assay of the linker scanning mutations in the -162Luc plasmid localized the IL-4 responsive effect to a 46 bp region of the promoter. This region contains three nuclear factor binding elements: a C/EBP site, a recently identified NF-IL-4 site and a NFкB/p50 site. In order to detect protein complexes that specifically interact with this active region of DNA, electrophoretic mobility shift assays were performed using double stranded, oligonucleotide probes of this IL-4 responsive region. An IL-4 inducible complex was identified in nuclear extracts of I.29μ as well as murine splenic B-cells. Competition experiments with mutant probes mapped this inducible complex to the NF-IL-4 site. Constitutive binding of both C/EBP and NFкB/p50 was demonstrated by cold competition and supershift experiments. Transfection experiments using a series of linker scanning mutations allowed identification of DNA elements necessary for IL-4 induction. In order to test if these elements are sufficent for IL-4 induction, double stranded oligonucleotides containing these elements were transfered to a minimal fos promoter plasmid and assayed for IL-4 responsiveness. A 27 bp fragment containing two DNA elements, a C/EBP and a NF-IL-4 site were sufficient to confer IL-4 inducibility to a minimal c-fos promoter. This study defined a different IL-4 response element in the murine germline epsilon promoter from that previously published. This IL-4 response element is identical to the IL-4 response element in the human germline epsilon promoter. The NF-IL-4 site is also present in the promoter of the IL-4 responsive gene, CD23b (FcεRII), and this element binds an IL-4 inducible complex present in the human monocytic cell line U937. Various reports demonstrate the presence of an IL-4 inducible complex by gel shift assays and indeed the binding activity of NF-IL-4 has been mapped to a 9 bp consensus sequence within a 19 bp fragment. However, the transfer of IL-4 inducibility has not been reported using fragments smaller than 123 bp, the importance of which is underscored by the fact that more than one factor is involved in this induction. The contribution of this thesis to the understanding of transcriptional induction by IL-4 is in the delineation of the factors involved - namely, a member of the C/EBP family and NF-IL-4 are required for IL-4 induction and NFкB/p5O modulates this induction.

Proteins, which are T cell-dependent antigens, preferentially induce antibodies of the IgG1 class in mouse, whereas LPS, which is a T-independent antigen, preferentially induces IgG3 and IgG2b. Interaction between CD40 on B cells and CD40 ligand (CD40L) on T cells has been shown to mediate T cell contact help for B cell proliferation, differentiation and immunoglobulin isotype switching. In addition, it has been shown that membranes from activated T cells induce germline γ1 transcripts, and that CD40 signaling induces germline γ1 transcripts. These results indicate that T cell contact help mediated by CD40 ligand (CD40L)-CD40 interaction may contribute to this preferential IgG1 isotype selection in response to T-dependent antigens by inducing transcription of germline Ig γ1 transcripts. Here we show that signaling via CD40 increases expression of a transiently transfected luciferase reporter plasmid driven by the germline γ1 promoter in M12.4.1 B lymphoma cells. By linker scanning mutation analysis of the promoter, we have identified a CD40 responsive region (CD40RR) which is able to confer inducibility by CD40L to a minimal c-fos promoter. The CD40RR contains three NF-кB-binding sites, each of which is required for maximal induction of the γ1 promoter activity by CD40L. Binding of the NF-кB/Re1 proteins p50, Re1A, c-Re1 and Re1B to the CD40RR can be induced by CD40 signaling in M12.4.1 cells or in splenic B cells. Co-transfection of expression plasmids for p50 together with Re1A or Re1B, but not p50 alone or p50 and c-Re1, transactivates the CD40RR in transient transfection assays in M12.4.1 cells. These data demonstrate NFкB/Re1 proteins activated by CD40 engagement play an important role in regulation of the germline γ1 promoter. Further support for this conclusion is provided by the finding that treatment of splenic B cells with NF-кB inhibitors prevents induction of germline γ1 transcripts by CD40L. Although LPS also induces NF-кB activation, it poorly induces germline γ1 promoter activity in M12.4.1 cells and it also poorly induces germline γ1 transcripts in splenic B cells and in the mouse B cell line, 1B4.B6. Western blot analyses show that LPS predominantly activates p50 and c-Re1, whereas CD40L induces all NF-кB/Re1 proteins (Re1A, Re1B, c-Re1 and p50). Likewise, in nuclear extracts from LPS-treated cells, p50/cRe1 and p50/p50 dimers are the major NF-кB/Re1 proteins which bind to the promoter for germline γ1 transcripts in electrophoretic mobility shift assays, whereas in nuclear extracts from CD40L-treated cells, p50/Re1A and p50/Re1B dimers are the major complexes. Reporter gene assay by over expressing NF-кB/Re1 fusion proteins indicates that p50/Re1A and p50/Re1B dimers, but not p50/c-Re1 or p50/p50 dimer, can transactivate the germline γ1 promoter. Despite their inability to activate the promoter, p50/c-Re1 and p50/p50 can bind to the promoter and suppress the transactivation activity of p50/Re1A and p50/Re1B. Therefore, the effect of NF-кB activation on the germline γ1 promoter depends on the Relative amounts of transactivating and non-trans activating NF-кB/Re1 dimers. The inability of LPS to induce germline γ1 transcripts can be explained by induction of non-transactivating NF-кB/Re1 dimers and the ability of CD40L to activate the promoter by a greater induction of Re1A and Re1B Re1ative to c-Re1.

L’anémie de Fanconi (FANC) est une maladie génétique humaine causant chez les patients une anémie sévère, une susceptibilité accrue à certains cancers, ainsi que des anomalies du développement dont un hypogonadisme. La voie FANC est impliquée dans la réponse au stress réplicatif et la réparation de l’ADN, et joue un rôle potentiel dans la régulation physiologique des cellules souches fœtales et adultes. Dans les modèles murins de la voie FANC, le phénotype majeur est une infertilité liée à un problème de développement du lignage germinal, qui est un paradigme pour l’étude des mécanismes contribuant à la pathologie. Notre étude a porté sur la caractérisation du défaut des cellules germinales primordiales (CGP) dans les embryons murins fancg-/-. Nous avons mis en évidence un défaut numérique des CGP très tôt dans le développement du lignage germinal dès les stades 9,5-10,5 jours post coïtum (jpc). Les CGP présentent un léger défaut de la prolifération à 10,5-11,5 jpc, mais aucun blocage de cycle. L’atteinte proliférative semble trop faible pour expliquer à elle seule la diminution drastique du nombre de CGP, ainsi que le profil mosaïque des gonades embryonnaires avec la présence de cordons dépourvus de CGP observé à 13,5 jpc. L’étude in vivo et ex vivo du comportement migratoire des CGP fancg-/- a permis de mettre en évidence des anomalies de la motilité des CGP, probablement liée à une activation anormale de la GTPase RAC1. Nous avons observé à 11,5 jpc une diminution du nombre de cellules au niveau du front de migration de la population de CGP. Ces anomalies entraîneraient ainsi un retard de migration et l’incapacité pour une fraction des GCP fancg-/- d’atteindre correctement les crêtes génitales à 11,5 jpc. La déplétion des CGP semble ainsi liée principalement à un défaut migratoire conduisant à une augmentation de la mort des CGP à 11,5 jpc, associé au défaut prolifératif intrinsèque des CPG fancg-/-. / Fanconi Anemia (FANC) is a genetic human disease, causing in patient a severe anemia, a higher risk to develop some cancers, and developmental anomalies including hypogonadism. The FANC pathway is involved in the replicative stress response and DNA repair, and has a potential role in the physiological regulation of fetal and adult stem cells. In mice models of the FANC pathway, the main phenotype is the sterility issue, associated to a developmental defect of the germ cell lineage, and is so a paradigm to study the pathology. Our study aimed to characterize the primordial germ cell (PGC) defect in fancg-/- mouse embryos. We showed a numerical defect of PGC early in the germ cell lineage development, as soon as 9.5–10.5 days post coitum (dpc). PGC show a mild defect of proliferation at 10.5–11.5 dpc, but no cell cycle arrest. This low proliferative defect can neither fully explain the drastic decrease of the PGC number, nor the mosaic profile of fetal gonads displaying cords without PGC at 13.5 dpc. In vitro and ex vivo studies of the migration behavior of fancg-/- PGC highlight abnormalities of the PGC’s motility, probably linked to abberant RAC1 GTPase activity. We also observed at 11.5 dpc a decrease of the cell number at the front of migration of the PGC population in 11.5 dpc fancg-/- embryos. Those motility defects could induce a migration delay, preventing a fraction of the fancg-/- PGC population to reach correctly the genital crests by 11.5 dpc. Hence, PGC depletion seems mainly linked to a migratory defect leading to increased cell death in PGC at 11.5 dpc, associated to an intrinsic proliferation defect of fancg-/- PGC.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17793号 / 農博第2014号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4784(農学部図書室) / 30600 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM