Universiẗat, Diss., 2002--München.

Orientador: Marielle Cristina Schneider / Banca: Douglas de Araujo / Banca: Vanessa Bellini Bardella / Banca: Ana Paula de Moraes / Banca: Simone Lilian Gruber / Resumo: O subgênero Tityus (Archaeotityus) compreende escorpiões sedentários que vivem na serapilheira. Neste trabalho, seis espécies brasileiras de Archaeotityus (Tityus clathratus, Tityus maranhensis, Tityus mattogrossensis, Tityus paraguayensis, Tityus pusillus e Tityus silvestris) foram caracterizadas quanto ao número diploide e haploide, comportamento dos cromossomos durante a meiose, localização das regiões organizadoras nucleolares (NORs), sequências de DNA repetitivo, tais como de heterocromatina constitutiva, rDNA 28S e telomérica (TTAGG)n e marcadores epigenéticos (H3K9ac, H4K5ac, H3S10f, H3K4m2, H3K9m2, H3K9m3). Todas as espécies analisadas apresentaram cromossomos holocêntricos, meiose sináptica e aquiasmática e presença de cadeias cromossômicas na meiose I. Variação intraespecífica de número diploide e a presença de associações multivalentes formadas por um número variável de elementos foi visualizado dentro e entre as populações das cinco espécies. Metáfases espermatogoniais mostram: 2n=16, 2n=17 e 2n=18 em T. paraguayensis; 2n=16 e 2n=24 em T. silvestris; 2n=20 em T. maranhensis; 2n=19 e 2n=20 em T. clathratus, T. mattogrossensis e T. pusillus. Núcleos pós-paquitênicos revelaram uma alta variabilidade no número de bivalentes e/ou elementos envolvidos em associações multivalentes, dando destaque para T. clathratus, o qual mostrou 11 configurações meióticas distintas, e T. pusillus que exibiu células poliploides. Metáfases II indicaram que todos os cromossomos possuem disjunção regular e segregação balanceada. Metáfases mitóticas submetidas à impregnação por íon prata exibiram regiões organizadoras nucleolares localizadas na região terminal/subterminal de um par de cromossomos em todas as espécies. A heterocromatina constitutiva foi ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The subgenus Tityus (Archaeotityus) comprises sedentary scorpions that live in the upper layers of the leaf litter. In this work, six Brazilian Archaeotityus species (Tityus clathratus, Tityus maranhensis, Tityus mattogrossensis, Tityus paraguayensis, Tityus pusillus and Tityus silvestris) were examined regarding to the diploid/haploid number, chromosome behavior during meiosis, repetitive DNA sequences (constitutive heterochromatin, 28S rDNA and telomeric (TTAGG)n), and epigenetic marks (H3K9ac, H4K5ac, H3S10f, H3K4m2, H3K9m2, H3K9m3). All analyzed species showed holocentric chromosomes, synaptic and achiasmatic meiosis, and chromosome chains in meiosis I. Intraespecific variation of diploid number and the presence of multivalent associations formed by a variable number of elements were visualized within and among populations of five species. Spermatogonial metaphase cells exhibited: 2n=16, 2n=17 and 2n=18 in T. paraguayensis; 2n=16 and 2n=24 in T. silvestris; 2n=20 in T. maranhensis, 2n=19 and 2n=20 in T. clathratus, T. mattogrossensis and T. pusillus. In postpachytene nuclei, a high variability in the number of bivalents and/or chromosomes involved in multivalent associations was verified. The most extreme variability occurred in T. clathratus, which showed 11 distinct chromosome configurations in meiosis, and T. pusillus, which exhibited polyploid cells. Metaphase II cells indicated that all chromosomes possess regular disjunction and balanced segregation. In all species, mitotic metaphase cells submitted to silver impregnation revealed two nucleolar organizer regions localized on terminal/subterminal chromosome sites. The constitutive heterochromatin was localized in the terminal region of the one chromosome pair. Meiotic cells submitted to FISH with 28S rDNA probe showed ribosomal cistrons in the terminal region of one bivalent-like element ... (Complete abstract click electronic access below) / Doutor

Die Lamina ist ein dichtes Netzwerk aus Intermediär-Filamenten, den Laminen, an der nucleoplasmatischen Seite der inneren Kernmembran. Hier interagieren Lamine sowohl mit Transmembran-Proteinen der Kernhülle als auch mit dem Chromatin. Diese Wechselwirkungen mit Interaktionspartnern verschiedener zellulärer Kompartimente macht die Lamina, neben einer Gerüststruktur mit wichtigen mechanische Aufgaben, auch zu einer zentralen Schnittstelle von Signalwegen, die eine intrazelluläre Kommunikation zwischen Nucleus und Cytoplasma ermöglichen. Die Lamina ist somit ein entscheidender Regulator der funktionellen Organisation des Chromatins und der differentiellen Genexpression. Das Expressionsmuster der Lamine während der Spermatogenese von Säugern unterscheidet erheblich von der Lamin-Expression somatischer Zellen und weist einige Besonderheiten auf. Dies schließt unter anderem die spezifische Expression der verkürzten A-Typ Lamin-Spleißvariante C2 während der meiotischen Phase der Spermatogenese ein. Diese und andere Beobachtungen deuteten bereits länger darauf hin, dass der speziellen Zusammensetzung der Lamina und vor allem dem meiosespezifischen Lamin C2 während der Gametogenese im männlichen Organismus eine entscheidende Rolle zukommen könnte. Neuere Studien im Mausmodell bekräftigen diese Hypothese und leisten darüber hinaus einen entscheidenden Betrag dazu, die Funktion der Lamina während der Meiose auf molekularer Ebene präzise zu definieren. Im deutlichen Gegensatz zu den weitreichenden Kenntnissen zur Situation in Männchen lagen zu Beginn der vorliegenden Arbeit keine Daten über die Zusammensetzung der Lamina in weiblichen Keimzellen vor. Konsequenterweise existierten auch keine funktionellen Untersuchungen zur Relevanz der Lamina für die Oogenese. In der vorliegenden Arbeit wurden diese reproduktionsbiologisch hoch interessanten Fragestellungen detailliert untersucht. Dabei zeigte sich unter anderem, dass Lamin C2 auch in weiblichen Keimzellen spezifisch während der Meiose exprimiert wird. Durch Studien an einer Lamin C2-defizienten Mauslinie wurde die Funktion von Lamin C2 in der Meiose in Weibchen genau untersucht. Dabei wurde eine erhebliche Beeinträchtigung der strukturellen Paarung der homologen Chromosomen und der homologen Rekombination in Lamin C2-defizienten Weibchen festgestellt. Da die genannten Prozesse Schlüsselereignisse für die korrekte Segregation der Homologen in späteren Stadien der Meiose sind, deuten die erzielten Ergebnisse auf eine erhebliche qualitative Beeinträchtigung der reifen Gameten in Lamin C2-defizienten Weibchen hin. Ein weiterer zentraler Aspekt der Arbeit war die Analyse der molekularen Eigenschaften des meiosespezifischen Lamin C2 in vitro. Diese Experimente definieren wichtige Unterschiede hinsichtlich seiner Polymerisationseigenschaften im Vergleich zu Laminen somatischer Zellen und tragen, zusammen mit anderen Studien, dadurch erheblich dazu bei, die Funktion von Lamin C2 in der Meiose im mechanistischen Sinne besser zu verstehen. Zudem deckt die vorliegende Arbeit erstmals einen funktionellen Zusammenhang zwischen der Lamina-Zusammensetzung und der Qualität der Keimzellen weiblicher Säuger auf und ermöglicht dadurch zukünftige Studien zur Rolle der Lamine in der Oogenese, die möglicherweise auch für die menschliche Fertilität sehr interessant sein könnte. Der zweite Teil der Dissertation beschäftigt sich mit der Beschreibung einer trunkierten A-Typ Lamin-Spleißvariante in einer Mauslinie, die bislang als A-Typ Lamin-defizient angesehen wurde (Lmna-/-). Die durchgeführten Untersuchungen besitzen vor allem dadurch hohe Relevanz, dass die untersuchte Lmna-/- Mauslinie seit Jahren als das wichtigste Modell zur funktionellen Untersuchung der A-Typ Lamine gilt und bereits in einer Vielzahl von Publikationen eingesetzt wurde. In den hierzu durchgeführten Versuchen konnte das in der Lmna-/- Mauslinie persistierende A-Typ Lamin mittels diverser methodischer Ansätze als C-terminale Deletionsmutante definiert werden, der die Exons 8-11 der insgesamt 12 Exons des Lmna-Gens fehlen. Daher wurde diese Lamin A-Mutante als Lamin AΔ8-11 bezeichnet. Die Konsequenzen der C-terminalen Deletion für die physiologischen Eigenschaften des Lamin Adelta8-11 sowie die Auswirkungen seiner Expression in der Lmna-/- Mauslinie auf aktuelle Modellvorstellungen zur Funktion der A-Typ Lamine und zur Entstehung Lamin-assoziierter, humaner Erkrankungen (Laminopathien) werden in der Arbeit ausführlich diskutiert. / The nuclear lamina is a dense meshwork of intermediate filament proteins termed lamins that lines the nucleoplasmatic face of the inner nuclear membrane. By its interactions with both integral membrane proteins and chromatin the lamina constitutes a cellular hub at the nucleo-cytoplasmatic interface that serves both structural and regulatory functions. With regard to this, lamins have been implicated in basic cellular processes such as transcription, signaling and chromatin organization and are therefore currently considered as major regulators of differential gene expression. Compared to somatic cells, nuclear lamina composition of male mammalian meiocytes is remarkably different. This includes the specific temporal expression of the short A-type lamin splice variant lamin C2 at meiotic stages of male germ cell development. Several lines of evidence indicated that meiosis-specific lamin C2 could serve an essential role for meiosis in males. Recently, this hypothesis has found strong support by in vivo studies in lamin C2-deficient males and these studies substantially contributed to the precise definition of lamin C2 function on a molecular level. In contrast to this, nuclear lamina subtype composition in female meiocytes has never been analyzed and, consequently, its contribution to the formation of viable gametes in females remained elusive. In the present study, these issues have systematically been addressed. Here, detailed immunohistochemical analyses demonstrate that, as in the male, female germ cells (oocytes) undergoing prophase I of meiosis specifically express lamin C2. Following functional studies performed in lamin C2-deficient mice disclosed a pivotal role of lamin C2 for central meiotic processes in females. These include the requirement of lamin C2 for precise pairing of the homologous chromosomes that occurs in meiotic prophase I as well as its contribution to the timely and efficient progression of homologous recombination events in oocytes. Since accurate segregation of paternal chromosomes during first meiotic division is known to be critically dependent on both precise homologous pairing and recombination, these data point to an as yet unanticipated role of lamin C2 for the development of viable gametes in females. Moreover, detailed in vitro experiments were performed to analyze the molecular properties of meiosis-specific lamin C2 compared to somatic lamin variants. This revealed distinct properties of lamin C2 that are compatible with models suggesting that this short lamin variant significantly modifies the structural properties of the nuclear envelope (NE) of mammalian meiocytes. These structural modifications, in turn, are considered to facilitate dynamic repositioning of NE-attached meiotic telomeres which, besides homologous pairing and recombination, is a further most central and evolutionarily conserved hallmark of meiotic prophase I with an essential relevance for accurate genome haploidization. Thus, together with other important observations made in the lamin C2-deficient mouse model, the data presented in this thesis significantly contribute to our understanding of lamin C2 function on a molecular level. The second part of the study is based on a rather unexpected finding in another lamin-deficient mouse model. This finding concerns the Lmna-/- mouse line that was established in 1999 by Sullivan and others by gene targeting of the Lmna gene encoding A-type lamins. Since then, these mice have frequently been used in a multitude of important studies that aimed to analyze various different aspects of A-type lamin function. This thesis now provides compelling evidence that, unexpectedly and contradicting previous reports, a truncated lamin A mutant persist in the Lmna-/- mouse line that was considered as completely devoid of A-type lamins thus far. By the use of various technical approaches, including detailed mass spectrometry, the presented data precisely define the lamin A variant present in Lmna-/- mice (designated lamin Adelta8-11) as a C-terminal lamin deletion mutant that lacks domains with known important functions for protein interactions and posttranslational processing. Based on these findings, implications for the interpretation of previous reports using Lmna-/- mice as well as for our current models of A-type lamin function and the pathophysiology of lamin-associated disorders in humans (laminopathies) are considered in the discussion of the thesis.

Sex determination (SD) is a complex and diverse developmental process that leads to the decision whether the bipotential gonad anlage will become a testis or an ovary. This mechanism is regulated by gene cascades, networks and/or chromosomal systems, and can be influenced by fluctuations of extrinsic factors like temperature, exposure to hormones and pollution. Within vertebrates, the group of fish show the widest variety of sex determination mechanism. This whole diversity of processes and mechanisms converges to the formation of two different gametes, the eggs and the sperm, the first bigger and static, and the second smaller and motile. Meiosis is crucial for the formation of both types of gametes, and the timing of meiosis entry is one of the first recognizable differences between male and female in vertebrates. The germ cells go into meiosis first in female than in male, and in mammals, this event has been shown to be regulated by retinoic acid (RA). This small polar molecule induces in the germ cells the expression of the pre-meiotic marker Stra8 (stimulated by retinoic acid gene 8), which is necessary for meiosis initiation. Interestingly, genome analyzes have shown that the majority of fish (including medaka) lack the stra8 gene, adding a question mark to the role of RA in meiosis induction in this group. Since a role of RA in entry of meiosis and sexual development of fish is still far from being understood, I investigated in medaka (Oryzias latipes) a possible signaling function of RA during the SD period in embryos and in reproductively active gonads of adults. I generated a transgenic medaka line that reports responsiveness to RA in vivo. With this tool, I compared RA responsiveness with the expression of the main gene involved in the synthesis of RA. My results show that there is a de-correlation between the action of RA with its source. In adults, expression of the RA metabolizing enzymes show sexually dimorphic RA levels, with aldh1a2 levels being higher in testis, and cyp26a1 stronger in female gonad. In ovary, the responsiveness is restricted to the early meiotic oocytes. In testis, RA is acting directly in the pre-meiotic cells, but also in Sertoli and Leydig cells. Treatment experiments on testis organ culture showed that RA pathway activation leads to a decrease in meiosis markers expression levels. During the development, RA responsiveness in the germ cells was observed in both sexes much earlier than the first female meiosis entry. Treatments with RA-synthesis inhibitor show a decrease in meiosis markers expression levels only after the sex differentiation period in female. Expression analyzes of embryos treated with exogenous RA showed induction of dmrt1a at the gonad levels and an increase of amh levels. Both genes are not only involved in male formation, but also in the regulation of germ cell proliferation and differentiation. RA is important in meiosis induction and gametogenesis in adult medaka. However, there is no evidence for a similar role of RA in initiating the first meiosis in female germ cells at the SD stage. Moreover, contrary to common expectation, RA seems to induce sex related genes that are involved indirectly in meiosis inhibition. In this thesis, I showed for the first time that RA can be involved in both induction and inhibition of meiosis entry, depending on the sex and the developmental stage in a stra8-independent model organism. / Geschlechtsbestimmung ist ein komplexer und vielfältiger Entwicklungsprozess, der zu der Entscheidung führt, ob sich aus der bipotenten Gonadenanlage Hoden oder Ovarien entwickeln. Dieser Mechanismus ist durch Genkaskaden, Netzwerke und/oder chromosomale Systeme reguliert, kann aber auch durch Fluktuation äußerer Faktoren wie beispielsweise Temperatur, durch Hormonexposition oder durch Umweltverschmutzung beeinflusst werden. Innerhalb der Wirbeltiere zeigen Fische die größte Vielfalt in Bezug auf die Mechanismen der Geschlechtsbestimmung. Die unterschiedlichen Mechanismen der Geschlechtsbestimmung konvergieren in der Entstehung von der beiden unterschiedlichen Geschlechtszellen, der Eizelle und des Spermiums. Die Eizelle ist groß und statisch, das Spermium hingegen ist kleiner und beweglich. Die entscheidende Rolle für die Entstehung der Geschlechtzellen spielt die Meiose. Der Zeitpunkt, an dem zum ersten Mal in der Entwicklung die Meiose einsetzt, ist der erste erkennbare Unterschied in der Gonadenentwicklung zwischen männlichen und weiblichen Wirbeltieren. Die Meiose der Keimzellen beginnt bei Weibchen früher als bei Männchen. Bei Säugetieren reguliert Retinsäure (RA) diesen Prozess. Dieses kleine polare Molekül induziert die Expression des Prä-Meiose-Markers Stra8 (stimulated by retinoic acid gene 8) in den Keimzellen, welcher für den Eintritt in die Meiose essentiell ist. Interessanterweise haben Genomanalyzen gezeigt, dass das stra8 Gen in Medaka sowie in den meisten anderen Fischarten nicht vorhanden ist. Dies stellt eine vergleichbare Rolle von RA für die Induktion der Meiose wie bei Säugetieren in diesen Fischen in Frage. Da die Rolle von RA für den Eintritt in die Meiose sowie für die Geschlechtsentwicklung in Fischen bisher nur unzureichend untersucht und verstanden ist, habe ich bei Medaka (Oryzias latipes) eine mögliche Funktion von RA für die Geschlechtsdetermination in Embryonen sowie in Gonaden geschlechtsreifer Tiere untersucht. Ich habe im Rahmen dieser Arbet eine transgene Medakalinie generiert, die in vivo eine RA induzierte Genexpression durch ein GFP Reportergen anzeigt. Mit Hilfe dieser Linie wurde die transkriptionsreulierende Aktivität von RAmit der Expression der wichtigsten Gene, die in die RA Synthese involviert sind, verglichen. Meine Ergebnisse zeigen eine Diskrepanz zwischen dem Wirkungs- und Syntheseort von RA. Die RA metabolisierenden Enzyme zeigten eine geschlechtsdimorphe Expression in adulten Medakas, mit einer höheren aldh1a2 Expression im Hoden sowie einer stärkeren cyp26a1 Expression in weiblichen Gonaden. Im Ovar sind lediglich frühe meiotische Eizellen RA-sensitiv. Im Hoden wirkt RA direkt in prä-meiotische Zellen, aber auch in Sertoli und Leydig Zellen. Stimulations-Experimente an Hoden Organkulturen ergaben, dass eine Aktivierung des RA Signalwegs zu einer Abnahme des Expressionslevels von Meiose-Markern führt. Während der Embryonalentwicklung konnte in den Keimzellen beider Geschlechter eine transkriptions-induziernende Aktivität von RA bereits zu einem Zeitpunkt beobachtet werden, der deutlich vor dem ersten Meiose Eintritt in Weibchen liegt. Behandlungen mit einem RA-Synthese Inhibitor zeigten lediglich nach der Geschlechtsdifferenzierung in Weibchen eine verminderte Expression der Meiose-Marker. Expressionsanalysen von Embryonen, die mit exogener RA behandelt wurden, ergaben eine Induktion von dmrt1a in den Gonaden und ein Anstieg von amh. Beide Gene sind sowohl in die männliche Geschlechtsentwicklung involviert, als auch in die Regulation der Keimzellproliferation und –differenzierung. Zusammen ergaben meine Untersuchungen, dass RA für die Induktion der Meiose und der Gametogenese in adulten Medakas wichtig ist. Allerdings gibt es keinen Hinweis für eine ähnliche Rolle 11 von RA bei der Initiierung der ersten Meiose in weiblichen Keimzellen während der Geschlechtsdetermination. Im Gegensatz zur bisher beschriebenen Situation, scheint darüber hinaus RA die Expression geschlechtsspezifischer Gene zu induzieren, die indirekt in die Inhibition der Meiose involviert sind. In der vorliegenden Arbeit konnte in einem stra8-unabhängigen Modelorganismus das erste Mal gezeigt werden, dass RA – abhängig vom Geschlecht und vom Stadium der Entwicklung - sowohl in die Induktion als auch in die Inhibition des Meiose-Eintritts involviert ist.

University, Diss., 2003--München.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-07-04T18:14:17Z No. of bitstreams: 1 texto completo.pdf: 492073 bytes, checksum: 6ad56958fb94f4a0587a049b848c5bd2 (MD5) / Made available in DSpace on 2017-07-04T18:14:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 492073 bytes, checksum: 6ad56958fb94f4a0587a049b848c5bd2 (MD5) Previous issue date: 2001-03-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A família Coccinellidae (Coleoptera) apresenta, aproximadamente, 4.000 espécies. Muitas delas têm sido utilizadas com eficiência tanto em programas de manejo de pragas quanto em estudos relacionados com impacto ambiental, causado por plantas transgênicas. As análises citogenéticas, na família Coccinellidae, espécies, foram sendo que realizadas a maioria somente em, aproximadamente, 4,3% das restringiu-se à descrição do número de cromossomos. Neste trabalho, foram realizadas análises citogenéticas em Eriopis connexa, Olla v-nigrum e Cycloneda sanguinea, visando: 1. adaptar a técnica para obtenção de cromossomos em células mitóticas e meióticas; 2. determinar o número e morfologia dos cromossomos metafásicos por coloração convencional; 3. avaliar o comportamento dos cromossomos durante a meiose, descrevendo o sistema de determinação sexual; 4. caracterizar a distribuição da heterocromatina constitutiva utilizando banda-C, banda HSS (Hot Saline Solution), enzimas de restrição (Msp-I e Hae-III) e fluorocromo (CMA 3 ); 5. aplicar metodologia de banda Ag-NOR para localizar a atividade gênica da região organizadora de nucléolo; 6. mapear, por meio de FISH, os cromossomos carreadores de genes rDNA. O número cromossômico foi 2n=18+XX para fêmeas e 2n=18+Xy para os machos, em todas as espécies. A única espécie que apresentou um cromossomo B eucromático restrito a fêmeas foi E. connexa. A meiose dos machos revelou a meio-fórmula n=9+Xy p , e o modo de associação dos cromossomos sexuais foram do tipo pára-quedas, em todas as espécies. Em C. sanguinea, durante a prófase I (paquíteno), a associação dos cromossomos sexuais ocorreu de maneira ponta-a- ponta linearmente até formar um pseudo-anel. Os resultados das metáfases mitóticas avaliadas por banda NOR, tanto em E. connexa quanto em C.sanguinea , mapearam a região organizadora de nucléolo ativa em um par autossômico. A análise com fluorocromo CMA 3 , realizada em E. connexa, foi consistente com a banda NOR sendo positiva nesta região. Em células meióticas de O. v-nigrum, a região NOR ativa esteve presente na vesícula sexual, sendo confirmada por FISH com genes rDNA. Entretanto, os resultados das análises da mitose e meiose de C. sanguinea , por banda NOR e coloração seqüencial FISH/AgNOR, foram controversos. Os genes ativos localizaram-se em um par autossômico (metáfase espermatogonial), porém a vesícula sexual (prófase I) e o lúmen do pára-quedas (metáfase I) ficaram fortemente impregnados por prata. O FISH mapeou os genes rDNA fora da vesícula sexual. Com base nestes resultados sugeriu-se que essas substâncias sejam proteínas nucleolares, sintetizadas pelo par autossômico e importadas durante a associação dos cromossomos sexuais (prófase I). Tanto em C. sanguinea quanto em E. connexa, a heterocromatina constitutiva avaliada por banda-C, localizou-se principalmente na região pericentromérica de todos os cromossomos. Esta região foi rica em pares de base GC (digerida pela Hae III). Entretanto, alguns blocos de heterocromatina constitutiva (telômeros) não foram digeridos. A banda HSS (Hot Saline Solution) extraiu toda a heterocromatina, sugerindo-se que a heterocromatina seja rica, também, em seqüências repetitivas de bases AT, conferindo, portanto, uma natureza molecular heterogênea. A utilização da enzima Msp-I forneceu resultado negativo. / The family Coccinellidae (Coleoptera) comprises about 4,000 species in which some environmental of them disturbance are used studies in pest caused by management transgenic programs plants. and in Cytogenetic analyses of Eriopis connexa, Olla v-nigrum and Cycloneda sanguinea were made with the following purposes: 1. Modification of methodology with the purpose of obtaining best mitotic and meiotic chromosome preparations; 2. Establishment of number and morphology of mitotic chromosomes obtained by conventional staining; 3. Evaluation of meiotic chromosome behavior and description of sexual determination system; 4. Characterization of constitutive heterochromatin by C-band, HSS-band (Hot Saline Solution), restriction enzymes (Msp-I and Hae III), and CMA 3 fluorochrome; 5. Determination of genetic activity of NOR-region by Ag-NOR band; 6. Application of FISH methodology with the purpose of locating the chromosomes with rDNA genes. All the species studied showed 2n = 18 + XX for females and 2n = 18 + Xy for males. E. connexa was the only species with an euchromatic B-chromosome in female individuals. The male meiosis showed n= 9 + Xy p , with a parachute association of sexual chromosomes in all species studied. In prophase I of C. sanguinea , the association of sexual chromosomes were initially end -to-end, finishing in a pseudo-ring formation. The NOR-band technique in E. connexa and C. sanguinea showed the nucleolar organizing region in a pair of autosomic chromosomes. These chromosomes in E. connexa were also stained by CMA 3 fluorochrome. Both NOR and CMA 3 bands stained the same chromosome region. In meiotic cells of O. v -nigrum, an active NOR-region was observed in sexual vesicle, which was confirmed by FISH/rDNA probe. However, the results of C-band and sequential staining by FISH/agNOR were controversial. The functional genes were located in an autosomic pair (spermatogonial metaphase); however, the sexual vesicle (prophase I) and the parachute lumen (metaphase I) were staining strongly with Ag. The FISH methodology showed the rDNA genes out of the sexual vesicle. These results suggested that nucleolar proteins made in the autosomic pair were imported during the association of sexual chromosomes (prophase I). Both in C. sanguinea and E. connexa, constitutive heterochromatin showed by C-band was found mainly in pericentromeric regions of all chromosomes. These regions were GC-rich (digested by Hae III), but some heterochromatic blocks (telomere) were not digested by Hae III). The HSS band extracted all the heterochromatin, suggesting that this chromosome region was also AT-rich with a heterogeneous molecular nature. A negative result was seen with digestion by Msp I restriction enzyme. / Tese importada do Alexandria

BEZERRA, F. T. G. Efeito da cilostamida e das metades foliculares no bloqueio da retomada da meiose e no nível de AMPc em oócitos bovinos. 2014. 67 f. Dissertação (Mestrado em Biotecnologia) - Campus de Sobral, Universidade Federal do Ceará, Sobral, 2014. / Submitted by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:05:58Z No. of bitstreams: 1 2014_dis_ftgbezerra.pdf: 1009970 bytes, checksum: c13e81b55e7bd82edc9f4a494ecdb1ba (MD5) / Approved for entry into archive by Djeanne Costa (djeannecosta@gmail.com) on 2016-06-29T14:06:50Z (GMT) No. of bitstreams: 1 2014_dis_ftgbezerra.pdf: 1009970 bytes, checksum: c13e81b55e7bd82edc9f4a494ecdb1ba (MD5) / Made available in DSpace on 2016-06-29T14:06:50Z (GMT). No. of bitstreams: 1 2014_dis_ftgbezerra.pdf: 1009970 bytes, checksum: c13e81b55e7bd82edc9f4a494ecdb1ba (MD5) Previous issue date: 2014 / The aims of this study were (1) to evaluate the effects of cilostamide and follicular hemisections on the blockage of in vitro oocyte meiotic resumption in bovine cumulus oocyte complexs (COCs), (2) to investigate the reversibility of cilostamide effect on meiotic resumption, and (3) to quantify the levels of cAMP in COCs, denuded oocytes (DO) and cumulus cells (CC) after culturing COCs in presence of cilostamide and follicular hemisections. To this end, bovine oocytes were subjected to a pre-maturation period of 12 h in medium containing 10 µM cilostamide, follicular hemisections or combination of both and,then, submitted to in vitro maturation. To evaluate the reversibility of cilostamide effect, at the end of this culture period, COCs were washed and placed in maturation medium without cilostamide for 12, 14, 16, 18, 20, 22 and 24 hours. To investigate the levels of cAMPc, COCs subject to pre-maturation for 6 h were used to measure the levels of cAMP. After 12 hours of culture, groups COCs were fixed to assess chromatin configuration and meiotic rogression. The results showed that COCs cultured in presence of cilostamide and follicular hemisections had significantly higher percentages of oocytes at germinal vesicle stage (94%), after a period of 12 h of pre-maturation, than COCs cultured in other treatments. After blocking treatment, 75% of oocytes reached metaphase II after 16 h of maturation, emphasizing that the treatment is not toxic to oocytes. Moreover, oocytes cultured in medium containing cilostamide and follicular hemisctions had significantly higher levels of cAMP when compared to other treatments. It is concluded that cilostamide and follicular hemisections interact and promotes the maintenance of oocytes at germinal vesicle stage by increasing the levels cAMP in cultured COCs. / Os objetivos deste estudo foram: (1) avaliar os efeitos da cilostamida e das metades foliculares sobre o bloqueio da retomada da meiose de oócitos in vitro no complexo cumulus oócito bovinos (COC) , (2) investigar a reversibilidade do efeito da cilostamida na retomada da meiose, e (3) quantificar os níveis de AMPc em COCs, oócitos desnudos (OD) e células do cumulus (CC) após o cultivo de COCs na presença de cilostamida e metades foliculares. Para este fim, os oócitos de bovino foram submetidos a um período de pré-maturação de 12 h em meio contendo 10 μM cilostamida, metades foliculares ou combinação de ambos e, em seguida, submetidos a maturação in vitro. Para avaliar a reversibilidade do efeito da cilostamida, no final deste período de cultura, COC foram lavados e colocados em meio de maturação, sem cilostamida por 12, 14, 16, 18, 20, 22 e 24 horas. Para avaliar os níveis de AMPc, COCs submetidos a pré-maturação por 6 h foram utilizados para a quantificação do nível de AMPc. Após 12 horas de cultivo, grupos de COCs foram fixados para avaliar a configuração da cromatina e progressão da meiose. Os resultados mostraram que COCs cultivados em presença de cilostamida e metades foliculares apresentaram percentuais significativamente maiores de oócitos em vesícula germinativa (94%), depois de um período de 12 h de pré-maturação, do que COCs cultivados em outros tratamentos. Após o bloqueio, 75% dos oócitos atingiram metáfase II após 16 horas de maturação, enfatizando que o tratamento não é tóxico para os oócitos. Além disso, os oócitos cultivados em meio contendo cilostamida e metades foliculares tinham níveis significativamente mais elevados de AMPc quando comparado com outros tratamentos. Concluiu-se que a cilostamida e metades foliculares interagem e promovem a manutenção de oócitos no estágio de vesícula germinativa, aumentando os níveis de AMPc em COCs cultivados.

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-08-09Bitstream added on 2014-06-13T19:45:10Z : No. of bitstreams: 1 000735059.pdf: 4335227 bytes, checksum: 7137c59ad39313bbb60a087cc2ad7500 (MD5) / O forskolin eleva as concentrações de adenosina monofosfato cíclica (cAMP) por meio da ativação da adenilato ciclase. Esse experimento possuiu o objetivo de utilizar o forskolin para melhorar a produção in vitro (PIV) de embriões bovinos. No Experimento I o forskolin foi adicionado no meio de maturação in vitro (MIV) com o objetivo de bloquear a meiose nuclear induzindo sua sincronização com a maturação citoplasmática. O Experimento II possuiu o objetivo de induzir uma lipólise química em embriões do D 6 a partir da utilização do forskolin, diminuindo sua sensibilidade após a vitrificação. No Experimento I, os oócitos permaneceram em meio MIV durante 6 horas na presença de três concentrações de forskolin: 0,025 mM; 0,05 mM; 0,1 mM e em seguida foram cultivados em meio MIV livre de forskolin por 18 horas. Após esse tempo os oócitos foram fertilizados e cultivados in vitro. Foi avaliado o estágio de maturação nuclear, a atividade e distribuição mitocondrial, a ultraestrutura oocitária, contagem do número de células e a apoptose celular. O tratamento com forskolin por 6 horas não resultou em atraso na quebra da vesícula germinativa em relação ao controle. Entretanto, após a retirada do bloqueio e maturação por 18 horas um maior número de oócitos tratados com 0,1 mM de forskolin se encontrou em MI (P<0,05). Não houve diferença com relação a atividade e distribuição mitocondrial nos oócitos, no entanto a maior concentração da droga levou a alterações ultraestruturais nos oócitos, com excesso de metabolização lipídica e desorganização do citoesqueleto. Apesar disso, não se observou diferença na taxa de produção de blastocistos avaliados no D 7, bem como no número total de células íntegras dos embriões. Entretanto, observou-se uma maior taxa de apoptose no grupo de blastocistos produzidos a partir de oócitos bloqueados com a maior concentração... / The forskolin raises concentrations of cyclic adenosine monophosphate (cAMP) through the activation of adenylate cyclase. This experiment aimed the use of forskolin to enhance in vitro production (IVP) of bovine embryos. In Experiment I, forskolin was added in the medium of in vitro maturation (IVM) with the objective of blocking nuclear meiosis inducing your synchronization with cytoplasmic maturation. Experiment II aimed to induce lipolysis chemical in D 6 embryos from the use of forskolin, decreasing its sensitivity after vitrification. In the first experiment, the oocytes remained in IVM medium for 6 hours in the presence of three concentrations of forskolin: 0.025 mM, 0.05 mM, 0.1 mM and then were cultured in medium free forskolin for 18 hours. After this time the oocytes were in vitro fertilized and cultured. We assessed the stage of nuclear maturation, activity and distribution mitochondrial, ultrastructure oocyte, total number of cells and apoptosis. The treatment with forskolin for 6 hours did not resulted in delay in VCBD in relation to control group. However, after the removal of the drug and maturation for 18 hours a higher number of oocytes treated with 0.1 mM of forskolin were still in MI (P<0.05). No differences in relation to mitochondrial activity and distribution were observed between groups. However the higher concentration of the drug induced ultrastructural damage to oocytes, with excess of lipid metabolization and cytoskeleton disorganization. Nevertheless, no statistical differences were observed on blastocyst production and on the number of total cells. Despite of that, a higher rate of ...

Orientador: Fernanda da Cruz Landim / Banca Eunice Oba / Banca: Maria Denise Lopes / Banca: Claudia Barbosa Fernandes / Banca: Gisele Zoccal Mingoti / Resumo: O forskolin eleva as concentrações de adenosina monofosfato cíclica (cAMP) por meio da ativação da adenilato ciclase. Esse experimento possuiu o objetivo de utilizar o forskolin para melhorar a produção in vitro (PIV) de embriões bovinos. No Experimento I o forskolin foi adicionado no meio de maturação in vitro (MIV) com o objetivo de bloquear a meiose nuclear induzindo sua sincronização com a maturação citoplasmática. O Experimento II possuiu o objetivo de induzir uma lipólise química em embriões do D 6 a partir da utilização do forskolin, diminuindo sua sensibilidade após a vitrificação. No Experimento I, os oócitos permaneceram em meio MIV durante 6 horas na presença de três concentrações de forskolin: 0,025 mM; 0,05 mM; 0,1 mM e em seguida foram cultivados em meio MIV livre de forskolin por 18 horas. Após esse tempo os oócitos foram fertilizados e cultivados in vitro. Foi avaliado o estágio de maturação nuclear, a atividade e distribuição mitocondrial, a ultraestrutura oocitária, contagem do número de células e a apoptose celular. O tratamento com forskolin por 6 horas não resultou em atraso na quebra da vesícula germinativa em relação ao controle. Entretanto, após a retirada do bloqueio e maturação por 18 horas um maior número de oócitos tratados com 0,1 mM de forskolin se encontrou em MI (P<0,05). Não houve diferença com relação a atividade e distribuição mitocondrial nos oócitos, no entanto a maior concentração da droga levou a alterações ultraestruturais nos oócitos, com excesso de metabolização lipídica e desorganização do citoesqueleto. Apesar disso, não se observou diferença na taxa de produção de blastocistos avaliados no D 7, bem como no número total de células íntegras dos embriões. Entretanto, observou-se uma maior taxa de apoptose no grupo de blastocistos produzidos a partir de oócitos bloqueados com a maior concentração ... / Abstract: The forskolin raises concentrations of cyclic adenosine monophosphate (cAMP) through the activation of adenylate cyclase. This experiment aimed the use of forskolin to enhance in vitro production (IVP) of bovine embryos. In Experiment I, forskolin was added in the medium of in vitro maturation (IVM) with the objective of blocking nuclear meiosis inducing your synchronization with cytoplasmic maturation. Experiment II aimed to induce lipolysis chemical in D 6 embryos from the use of forskolin, decreasing its sensitivity after vitrification. In the first experiment, the oocytes remained in IVM medium for 6 hours in the presence of three concentrations of forskolin: 0.025 mM, 0.05 mM, 0.1 mM and then were cultured in medium free forskolin for 18 hours. After this time the oocytes were in vitro fertilized and cultured. We assessed the stage of nuclear maturation, activity and distribution mitochondrial, ultrastructure oocyte, total number of cells and apoptosis. The treatment with forskolin for 6 hours did not resulted in delay in VCBD in relation to control group. However, after the removal of the drug and maturation for 18 hours a higher number of oocytes treated with 0.1 mM of forskolin were still in MI (P<0.05). No differences in relation to mitochondrial activity and distribution were observed between groups. However the higher concentration of the drug induced ultrastructural damage to oocytes, with excess of lipid metabolization and cytoskeleton disorganization. Nevertheless, no statistical differences were observed on blastocyst production and on the number of total cells. Despite of that, a higher rate of ... / Doutor

Orientador: Gisele Zoccal Mingoti / Banca: Fernanda da Cruz Landim / Banca: Joaquim Mansano Garcia / Resumo: O objetivo desse estudo foi avaliar os efeitos da suplementação do meio com diferentes fontes de macromoléculas, com bloqueadores da meiose e com antioxidantes durante o transporte de oócitos bovinos por 6 horas sobre: 1) progressão da maturação nuclear; 2) maturação citoplasmática e 3) competência no desenvolvimento e criotolerância dos embriões produzidos. Para tanto, o meio de transporte de oócitos foi suplementado com bloqueadores da meiose (forscolina e IBMX; Experimento 1) ou com diferentes tipos de macromoléculas (SFB ou BSA; Experimento 2), sendo que estes tratamentos ainda receberam ou não a suplementação com antioxidantes (mistura de cisteína, cisteamina e catalase). Os oócitos foram incubados em incubadora portátil (Minitub®) para simulação de transporte. Posteriormente, foram submetidos à maturação in vitro (MIV) em incubadora a 5% de CO2 em ar até completar 24h e, em seguida, foram fecundados e os prováveis zigotos foram cultivados in vitro durante 7 dias. Foi feito um grupo controle adicional no experimento I: MIV em incubadora com 10% de SFB por 24h. No experimento II foram feitos dois grupos controle adicionais MIV em incubadora com 10% de SFB por 24h sem e com antioxidantes (cisteína, cisteamina e catalase). Nos experimentos 1 e 2 foi avaliada a cinética da maturação nuclear e a maturação citoplasmática (através do posicionamento de mitocôndrias, do potencial de membrana mitocondrial e do conteúdo intracelular de espécies reativas do oxigênio) após o transpor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the effects of supplementation of the medium with different sources of macromolecules with blockers of meiosis and antioxidants during transport of bovine oocytes for 6 hours on: 1) progression of nuclear maturation; 2) cytoplasmic maturation and 3) competence in the development and cryotolerance of embryos produced. Therefore, the medium of transport oocytes was supplemented with blocking of meiosis (forskolin and IBMX; Experiment 1) or with different types of macromolecules (FCS or BSA; Experiment 2), and these treatments yet received or not antioxidant supplementation (mixture of cysteine, cysteamine and catalase). Oocytes were incubated in a portable incubator (Minitub®) for transport simulation. Posteriorly were submitted in vitro maturation (IVM) in incubator at 5% CO2 in air until to complete 24 hours and then were fertilized and presumptive zygotes were cultured in vitro for 7 days. Has been made an additional control group in the experiment I: MIV incubator with 10% FCS for 24 hours (Control). In the second experiment were performed two additional control groups: IVM incubator with 10% FCS for 24 hours (Control); and IVM in an incubator with 10% FCS and antioxidants (cysteine, cysteamine and catalase) for 24 hours (Contr+Atx). In Experiments 1 and 2 were evaluated after nuclear maturation kinetics and cytoplasmic maturation (made by positioning mitochondria, the mitochondrial membrane potential and intracellular content of ... (Complete abstract click electronic access below) / Mestre