Au cours de la méiose, des centaines de cassures doubles brins (CDB) sont générées et réparées par recombinaison homologue. Ces CDB peuvent être réparés par deux voies différentes donnant lieu à des crossing-overs (CO) ou des non crossing-overs (NCO). Le choix entre les deux voies est finement régulé pour assurer un nombre suffisant de CO; les facteurs influençant ce choix n’ont pas encore été bien caractérisés. L’environnement chromatinien pourrait jouer un rôle important dans ce processus.Peu d’études ont été réalisées sur l’influence de la chromatine sur la recombinaison méiotique. Le but de ma thèse a été de caractériser les facteurs chromatiniens nécessaires au remaniement de la chromatine pendant la recombinaison méiotique chez Saccharomyces cerevisiae.J’ai pu montrer que CAF-1 (Chromatin Assembly Factor 1) et Hir (Histone Regulator), deux protéines chaperons capables de réassembler les histones, s’associent aux sites de cassures doubles brins méiotiques lors de la recombinaison. L’absence de CAF-1 et Hir n’a pas d’effet sur la progression et la formation de CO. Cependant, par des études de recombinaison sur l’ensemble du génome, j’ai pu observer que l’absence de CAF-1 tend à réduire l’interférence des CO. Ce résultat suggère que CAF-1 pourrait être un des facteurs régulant la réparation au cours de la recombinaison méiotique. Pour finir, je me suis aussi intéressée à un troisième chaperon d’histone H3/H4, Asf1. J’ai aussi pu montrer que la délétion d’un autre chaperon Asf1 (Anti-silencing Function 1) entraîne des défauts de progression méiotique et de formation des spores.Ce travail aide à mieux comprendre l'impact de la chromatine sur la réparation de la méiose et le rôle des facteurs d'assemblage de la chromatine. / During meiosis, hundreds of programmed double strand breaks (DSB) are generated and repaired by homologous recombination. Meiotic DSB can be repaired by two major alternative pathways, which generate either crossing-over (CO) or non-crossing-over (NCO) products. The choice between the two repair pathways is tightly controlled to ensure sufficient and accurate CO formation. The chromatin environment could play a crucial role in this process that has not been elucidated yet. Little information is available about the importance of chromatin factors for meiotic recombination. The aim of my PhD was to study chromatin factors necessary for chromatin dynamic during meiotic recombination. I have shown that CAF-1 (Chromatin Assembly Factor 1) and Hir (Histone Regulator), two chaperone proteins that are able to incorporate histones into chromatin, associate with DSB sites during meiotic recombination. CAF-1 and Hir deletion have no effect on the outcome of meiosis and CO formation. However, by genome-wide recombination studies, I have observed that the absence of CAF-1 histone chaperone results in a slight decrease in CO interference. The result suggests that CAF-1 could be one of the factors regulating DNA repair during meiotic recombination. Finally, I have also studied another H3/H4 chaperone, Asf1 (Anti-silencing Function1). Asf1 deletion gives rise to a defect in meiotic progression and spore formation. This work helps to better understand the impact of chromatin on meiotic repair and the role of chromatin assembly factors.

En phase somatique, plusieurs mécanismes de réparations de l’ADNinterviennent pour réparer les cassures double brin (CDB) de l’ADN. Enphase méiotique, les CDB de l’ADN engendrées de façon programmées parSpo11 sont réparées par la recombinaison homologue (RH) dont les acteursprincipaux sont Rad51 et Dmc1 aidés de Brca2. Chez Arabidopsis, en absencede Brca2, le déroulement méiotique est perturbé, les chromosomes nes’associent pas en bivalents, ils apparaissent emmêlés. Ainsi, en absencede Brca2, la recombinaison homologue pourrait ne plus être fonctionnelleet les anomalies chromosomiques observées pourraient être le résultat deréparations aberrantes des CDB de l’ADN effectuée par d’autres mécanismesde réparation de l’ADN. Nous avons montrés, chez Arabidopsis, que le Nonhomologous End Joining (NHEJ) et/ou le Single Strand Annealing (SSA),mécanismes de réparation des CDB de l’ADN en phase somatique,n’intervenaient pas en phase méiotique dans la formation des anomaliesobservées en absence de Brca2. Toujours dans l’hypothèse où ces figuresméiotiques soient le résultat de liaisons covalentes, nous avons regardési les ADN-ligases ne pourraient pas être impliquées. Ainsi, nous avons pumontrer que la Ligase 6, ADN-ligase spécifique des plantes, n’avait pas derôle dans les anomalies chromosomiques observées en méiose en absence deBrca2. D’ailleurs la Ligase 6 ne semble pas non plus intervenir dans lesfigures chromosomiques observées chez les mutants rad51 et mnd1. Le rôlede la Ligase 6 n’ayant pas été déterminé lorsque nous avons démarré cetravail, nous avons voulu identifier son le rôle en étudiant le mutantcorrespondant. Le mutant ligase 6 ne présente pas de sensibilité auxstress génotoxiques utilisés ce qui indique que la Ligase 6 ne semble pasintervenir dans la réparation de l’ADN. La mutation dans le gène LIGASE Iest létal à l’état homozygote, de plus nous avons pu observer uneségrégation anormale chez l’hétérozygote mutant pour le gène LIGASE I. Lalétalité du mutant ligase I a été contournée par l’utilisation d’unsystème ARNi pour éteindre l’expression du gène LIGASE I uniquement enméiose. Cependant, l’implication de la Ligase I, dans les anomaliesméiotiques observées en absence de Brca2 n’a pas pu être déterminée.Enfin, nous avons confirmé que, chez Arabidopsis, Xrcc4 avait un rôle dansle NHEJ via son interaction avec la Ligase IV et via la sensibilité dumutant xrcc4 à différents stress génotoxiques. En revanche, Xrcc4-like nesemble pas interagir avec les acteurs du complexe de ligation du NHEJ etle mutant ne présente pas de sensibilité aux stress génotoxique, indiquantque cette protéine n’est pas impliquée dans le NHEJ et plus généralementdans les mécanismes de réparation de l’ADN. / In somatic cells, several mechanisms are involved in the repair of DNAdouble strand breaks (DSB). In meiotic cells, programmed DSBs are causedby Spo11 and repaired by homologous recombination (HR), whose main playersare Rad51 and Dmc1 aided by Brca2. In Arabidopsis, in the absence ofBrca2, meiosis is disturbed, chromosomes do not organize into bivalents,they appear stuck and entangled together. Thus, in the absence of Brca2,HR may be no functional and the chromosomal anomalies we observecouldresult from the aberrant repair of the DNA DSBs due to other mechanisms ofDNA repair. We have shown in Arabidopsis that the homologous end joining(NHEJ) and/or Single Strand Annealing (SSA), mechanisms of DNA DSB repairthat are active in the somatic phase, were not involved in the formationof the meiotic anomalies observed in the absence of Brca2 in meioticcells. Still assuming that these figures are the result of meioticcovalent bond, we checked whether DNA ligases could be involved. Thus, wehave shown that 6 Ligase, DNA ligase specific plants, had no role in thechromosomal abnormalities observed in meiosis in the absence of Brca2.Besides the Ligase 6 does not seem to interfere with the meiotic figuresobserved in rad51 and mnd1 mutants. We wanted to identify the Ligase 6role in studying its mutant. Ligase 6 mutant did not show sensitivity togenotoxic stress. The Ligase 6 does not seem to be involved in DNA repair.The lethality of the ligase I mutant was bypassed with a RNAi constructaimed at extinguishing the gene expression of LIGASE I atmeiosis only.However, the involvement of Ligase I in the meiotic anomalies observed inthe absence of Brca2 could not be determined. Finally, we confirmed that,in Arabidopsis, Xrcc4 has a role in NHEJ through its interaction withligase IV and the sensitivity of the xrcc4 mutant to different genotoxicstress. In contrast, Xrcc4-like does not appear to interact with playersin the NHEJ ligation complex and the mutant shows no sensitivity togenotoxic stress. These result indicated that this protein is not involvedin NHEJ and, more generally in the mechanisms of DNA repair.

Chez la femme, le risque de concevoir un embryon aneuploïde augmente de façon exponentielle dès 35 ans. La majorité de ces aneuploïdies sont dues à de mauvaises ségrégations des chromosomes lors des deux divisions de méiose dans l'ovocyte. Afin de limiter les erreurs de ségrégation, les divisions méiotiques doivent impérativement se dérouler en deux temps et de manière contrôlée. Un premier aspect de ma thèse a consisté à étudier les mécanismes qui régulent la séparation des chromosomes dans l'ovocyte de souris. J'ai montré que la Cycline A2 joue un rôle essentiel pour la séparation des chromatides s¿urs en méiose II. Au contraire, cette protéine doit impérativement être absente des régions centromériques en méiose I afin d'éviter une séparation précoce des chromatides s¿urs qui conduirait à la formation d'un ovocyte aneuploïde. Dans un seconde temps, mes travaux de thèse ont visé à étudier le mécanisme de surveillance de la première transition métaphase-anaphase appelé SAC (Spindle Assembly Checkpoint). Il a été montré que l'expression d'une protéine du SAC appelée BubR1 décroît naturellement dans les ovocytes avec l'âge maternel. Afin d'analyser les conséquences de cette diminution, j'ai utilisé une lignée de souris totalement délétée pour la protéine BubR1 spécifiquement dans les ovocytes. J'ai montré que la perte totale de BubR1 entraîne plus de 80% d'aneuploïdies dans l'ovocyte dès la première division de méiose. La méiose I est accélérée et les chromosomes homologues se séparent de façon anarchique. De plus, le fuseau méiotique devient instable. La diminution naturelle de BubR1 pourrait ainsi expliquer l'augmentation du nombre d'aneuploïdie avec l'âge maternel. / Women in industrialized countries tend to postpone childbearing, leading to a 70% increase intrisomic pregnancies over 20 years. Meiosis in females is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that deterioration of spindle assembly checkpoint fidelity contributes to age-related aneuploidization. However, this idea has remained controversial since transient knock-down of BubR1 was found to prevent meiotic prophase arrest and chromosome segregation in a checkpoint independent manner. We employed a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I, and establishment of robust kinetochore-microtubule attachments in a meiosis specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multi-faceted role in chromosome segregation during the first meiotic division and suggest that age-related loss of BubR1 is a key determinant of formation of aneuploid oocytes as women approach menopause. Using mouse oocytes, a second aspect of my thesis reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role, namely, its requirement for separase- dependent sister chromatid separation in meiosis II.

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluído o sistema reprodutor. O sistema NOS/NO parece desempenhar papel importante na maturação oocitária entre outras funções. No entanto, apesar das evidências, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar os níveis de outro nucleotídeo, o AMPc, que é um importante elemento da via de sinalização das gonadotrofinas nos oócitos e no controle da maturação oocitária. O objetivo do presente estudo foi de investigar o envolvimento da via do GMPc na ação do sistema NOS/NO na maturação in vitro (MIV) de oócitos bovinos e seu efeito sobre a via do AMPc. Com a maior concentração estudada do doador de NO (10-7M de SNAP), apenas 36% dos oócitos conseguiram alcançar o estágio de RVG (P< 0,05), após 9 horas de maturação. Esse atraso também foi observado com diferentes concentrações do estimulador de GC (5, 10 ou 50&mu;M de Proptoporfirina IX) e pelo análogo de GMPc (1, 2 e 4mM de 8-Br-GMPc ), que apresentaram uma taxa média de RVG de 50% para os tratamentos e 70% para os grupos controles sem as drogas (P<0,05). No início da maturação (0h), os níveis de GMPc foram de 5,29 pmol/oócito sofrendo uma queda logo na primeira hora de cultivo para 2,97 pmol nos oócitos do grupo controle e 1,54 pmol nos cultivados com associação de 10-7M de SNAP (doador de NO) e 100&mu;M de OQD (inibidor de GC (P<0,05). No grupo de oócitos cultivados apenas com SNAP, os níveis de GMPc se mantiveram em 4,51 pmol/oócito, semelhante ao grupo imaturo (0h de cultivo, P>0,05). O doador de NO manteve estável o nível de GMPc somente na primeira hora de maturação. Após 3 e 6 h de MIV, os níveis de GMPc permaneceram baixos e similares (0,07 a 2,46 pmol/oócito, P>0,05) nos grupos controle (sem drogas), tratado com doador de NO (10-7M de SNAP) associado ou não ao inibidor de guanilato ciclase (100&mu;M de OQD). Também foi observada uma queda nos níveis de AMPc em relação ao grupo imaturo (32,42 fmol de AMPc/oócito) para os demais grupos (P<0,05), que apresentaram aproximadamente, 12,0 a 16,0 fmol de AMPc/oócito durante a primeira hora, 3,3 a 8,0 fmol/oócito durante a terceira hora e 7,4 a 18,3 durante a sexta hora de maturação (P>0,05). O NO afetou os níveis de GMPc no início da maturação, mas não os níveis de AMPc. O NO e o GMPc podem atuar no controle da expressão gênica de uma série de proteínas envolvidas no controles dos níveis de AMPc e GMPc ou suas funções. Esse controle pode ser efeito direto do NO (PKG2, PDE3A, PDE4D e PDE8A), do GMPc (ADCY6) ou do NO via GMPc (PKA1) e varia com o compartimento considerado (oócito ou células do cumulus). Esses resultados demonstraram a inter-relação das vias NO/GMPc/AMPc e toda a sua complexidade dependendo do tipo celular e da fase da maturação de oócitos bovinos. / The NOS/NO system seems to play an important role in oocyte maturation besides other functions. However, despite the evidence, there are few studies on the possible role of this system in bovine oocytes. It is known that NO acts via guanylate cyclase (GC) by stimulating the production of the nucleotide cGMP, which in turn can influence the levels of another nucleotide, cAMP, which is an important element of the signaling pathway of gonadotropins in oocytes and in the control of oocyte maturation. The aim of the present study was to investigate the involvement of the cGMP pathway in the action of the NOS/NO system on the in vitro maturation (IVM) of bovine oocytes and its effect on the cAMP pathway. The highest studied concentration of the NO donor (10-7M SNAP), only 36% of oocytes were able to undergo GVBD (P<0.05) after 9 hours of maturation. This delay was also observed with different concentrations of the GC stimulator (5, 10 or 50&mu;M Proptoporfirin IX) and the cGMP analogue (1, 2 and 4 mM 8-Br-cGMP), which had an average of 50% GVBD for treatment groups and 70% for control groups without drugs (P<0.05). At the beginning of maturation (0 h) cGMP levels were 5.29 pmol/oocyte and decreased within the first hour of culture to 2.97 pmol and 1.54 pmol in the control group and in oocytes cultured in 10-7M SNAP (NO donor) associated with 100&mu;M OQD (GC inhibitor; P<0.05). In the group of oocytes cultured only with SNAP, cGMP levels remained at 4.51 pmol/oocyte similar to the immature group (0 h culture, P> 0.05). The increase of NO maintained cGMP levels stable only during the first hours of maturation. After 3 and 6 h IVM, cGMP levels remained low and similar (0.01 to 2.5 pmol/ ocyte, P>0.05) in control (without drugs), treated with NO donor (SNAP 10-7M) with or without the guanylate cyclase inhibitor (100&mu;M OQD). A decrease in cAMP levels was also observed when compared with the immature group (32.42 fmol cAMP/oocyte) for the other groups (P <0.05), which showed 12.0 to 16.0 fmol cAMP/oocyte after the first hour, 3.3 to 8.0 fmol/oocyte after the third hour and 7.4 to 18.3 after the sixth hour of IVM (P>0.05). NO and cGMP may act to control gene expression in a series of proteins involved in control of the levels of cAMP and cGMP or their functions. The control may be a direct effect of NO (PKG2, PDE3, PDE4D and PDE8A), cGMP (ADCY6) or NO via cGMP (PKA1) and varies with the compartment considered (oocyte or cumulus cells). The results showed the interrelationship of the NO/cGMP/cAMP pathway and all its complexity depending on the cell type and the stage of maturation in bovine oocytes.

La recombinaison méiotique produite par les Crossing Overs (COs) est un facteur limitant pour l’efficacité de la sélection variétale. Une possibilité pour produire des plantes hyper-recombinantes serait d’exploiter la variabilité intraspécifique pour les fréquences de recombinaison. L’identification des polymorphismes causaux, liés à la séquence ou l’expression, représente un travail de longue haleine. Une approche alternative serait de produire des mutants pour des régulateurs négatifs des fréquences de recombinaison. Chez le colza, jeune allotétraploïde (AACC, 2n=38), il est possible de jouer sur ces 2 approches. Dans un premier temps j’ai cherché à vérifier dans quelle mesure pouvait varier le transcriptome méiotique entre 2 variétés ayant servi à cartographier un QTL pour le contrôle de la recombinaison entre chromosome homoéologues (hérités des génomes parentaux). Ce transcriptome méiotique s’est révélé de façon inattendue très variable ; les principales sources de cette variation étant notamment la nature du génome (A ou C) ainsi que l’effet variété. J’ai montré que les HEs (le remplacement d’une région chromosomique par la duplication de la région homoéologue) contribuent de façon importante aux différences d’expression observées à la fois entre variétés ou au sein d’un même génotype. Dans un second temps, j’ai vérifié que FANCM décrit chez Arabidopis thaliana comme un régulateur négatif pour les fréquences de recombinaison avait bien la même fonction chez les Brassica. Chez Brassica rapa j’ai vérifié qu’un mutant fancm complémente comme attendu un mutant déficient pour la voie majoritaire de formation des COs. Chez Brassica napus j’ai observé une faible augmentation à la fois des fréquences de recombinaison entre chromosomes homologues et homoéologues. Ce travail souligne l’importance de la caractérisation des HEs chez les allopolyploïdes. Au-delà de leurs impacts sur le contenu et l’expression génique, les HEs ont très certainement des conséquences phénotypiques. Cette étude présente aussi un exemple de biologie translationnelle pour un trait important en amélioration des plantes. / Meiotic recombination driven by Crossing-Over (CO) is a limiting factor for the efficiency of plant breeding. One way to produce hyper-recombinant plants is to use the existing interspecific variability for recombination frequencies. Identification of the causal polymorphisms, either link to gene sequence or expression, represents a long-term endeavour. Another possibility is to mutate anti-meiotic CO genes. In rapeseed, a young allotetraploid species (AACC, 2n=38), both of these approaches are possible. First I wanted to check how much varies the meiotic transcriptome between 2 varieties that differ in term of recombination between homoeologous chromosomes (inherited from parental genomes). Unexpectedly, the meiotic transcriptome turned out to be very variable, the main source of this variation being notably the origin of the genome (A or C) and the variety. I also showed that homoeologous exchanges (HEs; the replacement of one chromosomal region with a duplicate of the homeologous region) contributed to this variation and led to large changes in expression both between and within varieties. Then I assessed whether FANCM, an anti-CO protein identified in Arabidopis thaliana had the same function in the Brassica genus. In Brassica rapa, a fancm mutant complements as expected a meiosis mutant defective in the main formation pathway for the formation of meiotic COs. In Brassica napus, I observed a slight increase in both homologous and homoeologous recombination frequencies. This work emphasizes the importance of characterizing HEs in allopolyploids species. Beyond their impact on gene content and expression, HEs most have likely phenotypic consequences. This study also presents an example of translational biology for an important trait in crop breeding.

La méiose est essentielle pour tous les organismes à reproduction sexuée car cette division cellulaire spécialisée conduit à la formation de gamètes. Au cours de la méiose, la formation de bivalents est une étape clé dans la répartition équilibrée des chromosomes homologues. Dans la majorité des espèces, la formation de ces bivalents repose sur le mécanisme de la recombinaison homologue qui est un mécanisme de réparation des cassures double brin (CDB) de l’ADN. En méiose, la cassure est programmée et provoquée par l’action de Spo11. A.thaliana contient deux homologues SPO11-1 et SPO11-2 qui ne sont pas redondants dans la formation des CDB. Spo11 est une protéine apparentée à la sous-unité A des topoVI d’Archaea. Or, les topoVI d’Archaea fonctionnent en hétérotétramère composé de deux sous-unités A et deux sous-unités B pour former une cassure double brin (CDB) mais jusqu'à mon travail de thèse, aucun homologue méiotique de sous unité B n'avait été identifié. Au cours de ma thèse, j’ai caractérisé la fonction méiotique de la protéine MTOPVIB et montré que c’est un homologue structural de la sous-unité B des TopoVI d’Archaea. Par différentes approches, j’ai montré que MTOPVIB est nécessaire à l’hétérodimérisation de SPO11-1 avec SPO11-2 et je propose que chez A. thaliana, un complexe catalytique de type TopoVI composé de MTOPVIB, SPO11-1, et SPO11-2 est nécessaire à la formation des CDB méiotiques. Chez A. thaliana, en plus de SPO11-1, SPO11-2 et MTOPVIB, quatre autres protéines sont nécessaires à la formation des CDB : PRD1, PRD2, PRD3 et DFO. Par des approches double hybride, j’ai analysé le réseau d’interaction entre ces protéines de « cassure ». Les résultats suggèrent que ces protéines interagiraient au sein d’un « super » complexe essentiel à la formation des CDB méiotiques. / Meiosis is an essential step in sexual reproduction because it leads to the formation of haploid gametes. During meiosis, the formation of bivalents is a key step for the balanced chromosome distribution. In most species, the formation of bivalents lies on the mechanism of homologous recombination that is a repair mechanism for double stranded DNA breaks (DSB). In meiosis, DSB formation is programmed and provoked by the action of Spo11. A.thaliana contains two SPO11-1 and SPO11-2 counterparts which are not redundant in the formation of DSB. Spo11 is related to the A subunit of Archaea topoVI. However, Archaea topoVI operate through a heterotetramer composed of two A subunits and two B subunits but until my thesis work, no meiotic homolog of the B subunit had been identified. During my thesis, I characterized the meiotic function of the new protein MTOPVIB and showed that it shares structural similarities with the B subunit of Archaea TopoVI. Using different strategies, I also demonstrated that MTOPVIB is necessary to the SPO11-1/ SPO11-2 heterodimerization strongly suggesting that in A. thaliana, a catalytic TopoVI like complex is necessary for the formation of meiotic DSB. In addition to SPO11-1, SPO11-2, and MTOPVIB, four other proteins are necessary for the formation of meiotic DSB in A. thaliana : PRD1, PRD2, PRD3 and DFO. By yeast two hybrid approach, I analysed the interaction network between the "DSB" proteins. The results suggest that these proteins could act in a "super" complex which would be essential to the formation of DSBs.

Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-­‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search. Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knock­out oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search.

The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis-specific STAG component of cohesin, STAG3. Newly generated STAG3-deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot-like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3-devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis-specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.

Orientador: Lilian Madi-Ravazzi / Coorientador: Maria Tercília Vilela de Azeredo-Oliveira / Banca: Blanche Christine Pires de Bitner-Mathé Leal / Banca: Maria Izabel Camargo Mathias / Banca: Hermione Elly Melara de Campos Bicudo / Banca: Patrícia Vilamaior / Resumo: Zaprionus indianus é um drosofilídeo nativo da região Afrotropical que colonizou o continente Sul Americano, apresentando uma ampla distribuição geográfica enquanto Z. sepsoides é restrita a algumas regiões africanas. As duas espécies diferem em relação ao tamanho dos testículos e dos espermatozoides que é maior em Z. indianus do que em Z. sepsoides. Com o intuito de conhecer aspectos da biologia e o grau de diferenciação destas espécies, o presente estudo avaliou a espermatogênese de machos de diferentes idades (1, 3, 5 e 8 dias) de ambas as espécies por meio de técnicas de coloração convencional e de ultraestrutura. A espermatogênese e ultraestrutura dos espermatozoides foram semelhantes nas espécies em que foi confirmado o número diploide de cromossomos com 2n = 12. Entretanto, foi observada uma quantidade maior de espermatozoides em machos jovens (1 a 3 dias de idade) em Z. indianus do que em Z. sepsoides, o qual apresentou maior frequência de estágios iniciais da espermatogênese nestas idades. A porção da cabeça dos espermatozoides foi fortemente marcada nas duas espécies pela coloração por prata (AgNOR), orceína lacto-acética e pela reação de Feulgen. Quando submetidos à reação de P.A.S., os testículos de Z. sepsoides e Z. indianus apresentaram grânulos de glicogênio. As espécies possuem a mesma ultraestrutura flagelar, em que o axonema mostra um arranjo de 9+9+2 microtúbulos, com a presença de dois derivados mitocondriais de diferentes tamanhos e o número de 64 espermatozoides por feixe, em ambas as espécies. A grande semelhança observada no padrão do arranjo de microtúbulos do axonema e nos derivados mitocondriais com diferentes tamanhos nas espécies de Zaprionus, comparadas com outras espécies de Drosophila, é indicativa da conservação destas estruturas na família Drosophilidae... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America. Z. indianus exhibits a wide geographical distribution, whereas Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages (1, 3, 5 and 8 days old) from both species by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, for which the diploid number was confirmed to be 2n = 12 chromosomes. However, a greater number of spermatozoa were observed in young Z. indianus males (1-3 days old) than in young Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis at this age. A portion of the head of the sperm was strongly marked in both species by silver staining (AgNOR), lacto-acetic orcein and the Feulgen reaction. Additionally, when submitted to P.A.S. reaction, the testes of both Z. sepsoides and Z. indianus exhibited glycogen granules. The two species also presented the same flagellar ultrastructure, in which the axoneme includes a 9+9+2 arrangement of microtubules, two mitochondrial derivatives of different sizes are present and the number of spermatozoa per bundle is 64. The great similarity in the pattern of microtubule arrangement in the axoneme and in the mitochondrial derivatives of the species Zaprionus, as compared with other species of Drosophila, indicates that these structures are preserved in the family Drosophilidae. The differences observed between the young males of Z. indianus and Z. sepsoides, including the number and frequency of sperm... (Complete abstract click electronic access below) / Doutor

(eng) The phenomenon of hybrid sterility represents one of the evolutionary mechanisms that enables speciation. Only a few speciation genes have been uncovered. The only one found in mammals is Prdm9 (PR-domain 9). Data in the literature on the involvement of Prdm9 in decreased fertility of various semifertile hybrid males of house mouse subspecies were scarce before the results of this thesis were completed, despite that such males are much more frequent in nature than the fully sterile ones. Utilizing a panel of genetic tools and a battery of phenotyping tests, this thesis shows a central role of Prdm9 in fecundity of hybrids, including many fertility disorders and age dependency. Both increasing and reducing the Prdm9 gene dosage significantly elevated fertility parameters. Surprisingly, even the allele that in one copy causes full hybrid sterility increased F1 hybrid fertility when present in multiple copies. The PRDM9 protein also plays a role in identifying the sites of meiotic recombination. This study also points out the principles of allelic competition in determination of the sites of preferred recombination (hotspots), which suggests a possible link between both previously described Prdm9 roles. This thesis summarizes a set of three logically interconnected publications with the ambition...