Dissertations / Theses on the topic 'Meiosis'

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged student-submitted from PDF version of thesis.
Includes bibliographical references.
The vast majority of multicellular organisms reproduce using sexual reproduction, which requires the production of haploid gametes. These gametes are produced by meiosis, a specialized cell division during which one round of DNA replication is followed by two rounds of chromosome segregation, Meiosis I (MI) and Meiosis II (MII). This imbalance between rounds of DNA replication and chromosome segregation causes diploid cells to produce haploid gametes. In contrast, mitotically-dividing cells maintain ploidy by alternating between rounds of replication and segregation. It is unclear how meiosis accomplishes two sequential chromosome segregation events without an intervening round of DNA replication. In mitotic cells, both DNA replication and chromosome segregation are regulated by oscillations of cyclin-dependent kinase (CDK) activity. Both events initiate during G1 due to the associated low CDK-activity state, and both events are completed later in the cell cycle due to increased CDK activity. During meiosis, uncoupling replication and segregation presents a unique problem. After completion of MI, CDK activity decreases and then increases to drive MII chromosome segregation. However, DNA replication must remain inhibited between MI and MII. Given that an oscillation of CDK activity is sufficient for genome re-duplication in mitotic cells, I sought to understand how meiotic cells prevent DNA replication while resetting the chromosome segregation program. In this thesis, I show that meiotic cells inhibit two distinct steps of DNA replication: (1) loading of the replicative helicase onto replication origins, and (2) activation of the replicative helicase. CDK and the meiosis-specific kinase Ime2 cooperatively inhibit helicase loading during the meiotic divisions, and their simultaneous inhibition causes inappropriate helicase reloading. Further studies of Ime2 revealed two mechanisms by which it inhibits this process. First, I showed that Ime2-phosphorylation of the helicase directly inhibits its loading onto origins. Second, Ime2 cooperated with CDK to transcriptionally and proteolytically repress Cdc6, an essential helicase-loading protein. In addition, I found that meiotic cells use CDK and the polo-like kinase Cdc5 to promote degradation of Sld2, an essential helicase-activation protein. Together, these data demonstrate that multiple kinases inhibit both helicase loading and activation between MI and MII, thereby ensuring a reduction in ploidy.
by David V. Phizicky.
Ph. D.

Per tal de protegir les cèl·lules germinals de sofrir inestabilitat genòmica, diversos mecanismes de control s’encarreguen de que la progressió de la meiosis sigui correcte. En mamífers, els espermatòcits que presenten defectes de recombinació o de la formació de la vesícula sexual pateixen un bloqueig a l’estadi de paquitè. Estudis previs del nostre laboratori descriuen que la via complex MRE11-ATM-CHK2 activa l’arrest dependent de recombinació en presència de trencaments de doble cadena (DSBs) no reparats. L’objectiu d’aquest treball ha estat identificar si els membres de la família p53, els quals són possibles substrats de ATM i CHK2, participen en l’activació del arrest depenent de recombinació. En una aproximació genètica, hem obtingut ratolins doble mutants portadors d’una mutació de un membre de la família p53 (p53, Tap63 o p73) en un fons defectiu per Trip13. La mutació de Trip13 causa defectes de recombinació, el qual activa l’arrest depenent de recombinació en els espermatòcits a l’estadi de paquitè. Per tant, hem estudiat com l’absència d’algun membre de la família p53 afectava aquest fenotip d’arrest el espermatòcits Trip13mod/mod. Els nostres resultats demostren que tant la deficiència de p53 com Tap63, però no p73, permeten que els espermatòcits progressin més enllà i arribin a l’estadi de paquitè tardà tot i acumular nombrosos DSBs no reparats. Addicionalment, l’absència de p53 o Tap63 resulta en una disminució del nombre d’espermatòcits apoptòtics a l’estadi de paquitè primerenc. Així, els nostres resultats indiquen que p53 i TAp63 són responsables d’activar l’arrest dependent de recombinació en els espermatòcits de ratolí. Tot i així, els espermatòcits doble mutants encara presenten un bloqueig a l’estadi de paquitè. Per tal d’estudiar si els espermatòcits doble mutants arresten a causa de l’activació de l’arrest depenent de la correcta formació de la vesícula sexual, hem analitzat la funcionalitat del MSCI en els mutants Trip13. Per tant, el fet de saltar-se l’arrest dependent de recombinació ens ha permès elucidar el paper de TRIP13 en el silenciament meiòtic, de manera que al fallar la vesícula sexual es desencadena l’apoptosi i bloqueig dels mutants Trip13. Aquests resultats infereixen que el bloqueig depenent de recombinació i el depenent de la correcta formació de la vesícula sexual, són mecanismes que s’activen per mecanismes genèticament separats. A partir de l’observació que TRIP13 és necessari per implementar el silenciament del MSCI, he dut a terme un anàlisis exhaustiu de la transcripció en els mutants de Trip13. Els nostres resultats de marcatge de RNA amb EU i activació de la RNA polimerasa II fosforilada (S2) suggereixen que la expressió de RNA en els espermatòcits mutants per Trip13 es troba incrementada en els estadis inicials de la meiosis. Addicionalment, la seqüenciació del RNA ha permès observar que els gens dels cromosomes sexuals i gens pre-meiòtics es troben sobre expressats en els mutants de Trip13, suggerint que TRIP13 és necessari per mantenir l’expressió d’aquests gens a nivells baixos. En conjunt, els resultats presentats en aquest treball contribueixen a entendre com els mecanismes de control regulen diversos passes crucials de la progressió de la profase meiòtica en els espermatòcits de mamífer.
In order to protect germinal cells from genomic instability, surveillance mechanisms ensure that meiosis occurs properly. In mammals, spermatocytes that display recombination or sex body defects experience an arrest at pachytene stage. Previous studies from our lab described that the MRE11 complex-ATM-CHK2 pathway activates the recombination-dependent arrest in the presence of unrepaired double strand breaks (DSBs). In this work we aimed to identify if p53 family members, which are putative targets of ATM and CHK2, participate in the activation of the recombination-dependent arrest. As a genetic approach, we bred double mutant mice carrying a mutation of a member of the p53 family (p53, TAp63, p73) in a Trip13 defective background. Trip13 mutation causes recombination defects, which activate the recombination-dependent arrest in pachytene-stage spermatocytes. Thus, we studied how the absence of p53 family members affected the arrest phenotype of Trip13mod/mod spermatocytes. Our data showed that p53 and TAp63 deficiency, but not p73, allowed spermatocytes to progress further into late pachynema, despite accumulating numerous unrepaired DBSs. In addition, lack of p53 or TAp63 resulted in a decrease of apoptotic spermatocytes at early pachytene stage. Therefore, our results indicate that p53 and TAp63 are responsible to activate the recombination-dependent arrest in mouse spermatocytes. Even though, double mutant spermatocytes still arrested at pachytene stage. To study if double mutant spermatocytes were arresting due to the activation of the sex body deficient arrest we analyzed MSCI functionality in Trip13 mutants. Thus, by bypassing the recombination-dependent arrest has allowed us to elucidate a role for TRIP13 protein in meiotic silencing, which consequently triggers apoptosis in double mutants at late pachytene stage due to sex body impairment. These results infer that the recombination-dependent and the sex-body deficient arrest are activated by two genetically separated mechanisms. From the observation that TRIP13 is required to implement MSCI silencing, we performed an exhaustive analysis of transcription in Trip13 mutants. Our results suggested that RNA expression in Trip13 mutants was increased in early meiotic stage spermatocytes, assessed by EU-labeling RNA and phosphorylated(S2)-RNA polymerase II. Moreover, RNA sequencing data highlighted the observation that sex chromosome genes and pre-meiotic genes are overexpressed in Trip13 mutants, suggesting that TRIP13 is required to maintain the expression of these genes at low levels. Overall, the data presented in this work contributes to the understanding on how surveillance mechanisms control several crucial steps of meiotic prophase progression in mammalian spermatocytes.

The production of germ cells is an essential process in all sexually reproducing eukaryotes. During male meiosis, four haploid sperm cells are formed from one primary spermatocyte, thereby undergoing two consecutive cell divisions after only one round of chromosome duplication. This process was studied in the nematode Caenorhabditis elegans, as this model organism offers a number of experimental advantages to simultaneously analyze spindle dynamics and ultrastructure. The worm is easy to cultivate, completely sequenced and numerous mutants are available, the worm is small and thus ideal for light and electron microscopic investigations, and the transparent body allows live-cell imaging within living animals. Importantly, meiotic spindles in C. elegans males are organized by centrosomes and show a lagging X-chromosome, which is always segregated after the autosomes have been partitioned to the newly forming secondary spermatocytes. The aim of this thesis was to systematically investigate this characteristic feature of chromosome segregation in male meiotic spindles. For that, spindle dynamics in the first and second meiotic division was analyzed with fluorescence light microscopy. Furthermore, the spindle ultrastructure was investigated in spindles of various stages of meiosis I using electron tomography. Light microscopy revealed a shortening of the distance between centrosomes and chromosomes (anaphase A) and an increase in the pole-to-pole distance (anaphase B). Moreover, spindles in male meiosis I and II showed differences in certain aspects of spindle dynamics. In addition it was demonstrated that spindles in metaphase II in the presence of a single X-chromosome were shorter compared to spindles without the X-chromosome. In addition, it was found that the process of aging had an impact on spindle length in both metaphase I and II. By manipulating the number of unpaired chromosomes, it could be demonstrated that the lagging behavior of univalent chromosomes is caused by the incapability of pairing in meiotic prophase. After performing a quantitative analysis of the light microscopic data it was further shown that a dynamic microtubule bundle is connecting the X-chromosome to the spindle poles. Using laser microsurgery it could be demonstrated that this bundle exerts a pulling force to the univalent chromosome throughout anaphase. Unexpectedly, electron tomography showed that anaphase-type movements of the autosomes were not accompanied by a shortening of the kinetochore microtubules. Instead, three findings indicated a shortening of the centrosome-chromosome distance itself: (1) upon anaphase onset, tension is released on the beforehand stretched autosomes; (2) centrosomes shrink in preparation for meiosis II and (3) the attachment angle of end-on microtubules changes. Interestingly, microtubules connecting the X-chromosome to the spindle poles showed a high curvature around the kinetochore region of the X-chromosome, suggesting an involvement of motor proteins in the process of segregation. Taken together, this thesis gives the first detailed quantitative analysis of spindle dynamics and architecture during male meiosis in the nematode C. elegans. This wild-type data will serve as a basis for future mutant analyses and should help to further understand the complex dynamic and ultrastructural aspects of spindle organization in the meiotic divisions in C. elegans males.

Meiosis is a specialized cell division that produces haploid gametes from a diploid progenitor cell. It consists of one round of DNA replication followed by two consecutive rounds of chromosome segregation. Homologous chromosomes segregate in meiosis I and sister chromatids segregate in meiosis II. Failure to correctly regulate meiosis can result in aneuploidy, where daughter cells inherit an incorrect number of chromosomes. Aneuploidy is usually poorly tolerated in eukaryotes, and is associated with infertility, miscarriages and birth defects. At the meiosis I to meiosis II transition, DNA replication does not occur between chromosome segregation steps despite the need for Spindle Pole Bodies (SPBs) to be re-licensed in order to build meiosis II spindles. The mechanisms that make this distinction are not yet known. In budding yeast, the protein phosphatase Cdc14 is essential for the progression of cells into meiosis II. Cdc14 is sequestered for the majority of the cell cycle in the nucleolus by the inhibitor Cfi1/Net, and is only released in anaphase. We have observed Cdc14 localizing to and interacting with SPB components when nucleolar sequestration is inhibited. Through fluorescence microscopy and EM analysis, we have determined that Cdc14 is required for the re-duplication of SPBs after meiosis I. Our data implies a role for Cdc14 in the phospho-regulation of SPB half-bridge component Sfi1. Cdc14 is therefore essential for the relicensing of SPB duplication, a crucial step necessary to ensure accurate chromosome segregation in meiosis.

In vertebrate oocytes, meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3’ untranslated regions (3´ UTRs). Sequential, phase-specific translation of these maternal mRNAs is required to complete the two meiotic divisions. Although the earlier polyadenylation events in prophase I and metaphase I are driven by the CPE-binding protein 1 (CPEB1), 90% of this protein is degraded by the anaphase promoting complex in the first meiotic division. The low levels of CPEB1 during interkinesis and in metaphase II raise the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we demonstrate that CPEB1 activates the translation of the maternal mRNA encoding CPEB4, which, in turn, recruits the cytoplasmic poly(A) polymerase GLD2 to “late” CPE-regulated mRNAs driving the transition from metaphase I to metaphase II, and, therefore, replacing CPEB1 for “late” meiosis polyadenylation.

Missegregation of chromosomes during meiosis leads to formation of aneuploid eggs. Estimates suggest that in humans, about 10-30% of fertilised eggs and one-third of all miscarriages are aneuploid. Accurate chromosome segregation depends on the coordination between stepwise cohesion resolution and attachments of homologous chromosomes through kinetochores to microtubules, emanating from opposite poles of the cell. The Spindle Assembly Checkpoint (SAC) monitors microtubule-kinetochore attachments and prevents resolution of cohesin complexes by inhibiting the ubiquitin ligase APC/Ccdc2o until all aberrant microtubule-kinetochore attachments have been rectified by an Aurora Kinase-dependent error correction machinery. During meiosis, these pathways work in seamless coordination to achieve balanced segregation of the genome at the first meiotic division. The cross-talk between different cell cycle pathways requires members with shared affiliations. During my DPhil studies, I worked on understanding the role of two such proteins, namely Bub1 (budding uninhibited by benzimidazoles 1) and Sgol2 (Shugoshin-like protein 2) in mouse oocytes. During the first meiotic division, Bub1 maintains the SAC, and through its kinase activity, Bub1 recruits Sgol2 to kinetochores to protect centromeric cohesion. This recruitment is essential for two rounds of chromosomes segregation in meiosis. Thus, Bub1localisation at kineto chores can coordinate the timing of anaphase with the centromeric cohesion protection.

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.

Les crossing-overs (CO) sont issus d’échange réciproque de matériel génétique entre les chromosomes homologues. Les COs produisent de la diversité génétique et sont essentiels chez la plupart des eucaryotes, pour la distribution équilibrée des chromosomes lors de la méiose. Malgré leur importance, et un large excès de précurseurs moléculaires, le nombre de CO est très limité dans la grande majorité des espèces (Typiquement 1 à 4 par paire de chromosomes). Cela suggère que les COs sont étroitement régulés, mais les mécanismes sous-jacents sont mal connus. Pour identifier les gènes qui limitent la formation des CO, l’équipe a mené un crible génétique chez Arabidopsis thaliana. Ces travaux ont mené à l’identification de plusieurs facteurs anti-CO, définissant trois voies : (i) L’hélicase FANCM et ses co-facteurs ; (ii) L’AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) ; (iii) Le complexe RECQ4-Topoisomerase 3α-RMI1.Le premier objectif de ma thèse est d’explorer les relations entre ces trois voies en s’attachant aux questions suivantes ; (1) Jusqu’où peut-on augmenter la recombinaison en combinant les mutations dans FANCM, FIGL1 et RECQ4 ? Nous avons montré que la plus forte augmentation de recombinaison était obtenue dans recq4 figl1, atteignant 7,5 fois la fréquence du sauvage en moyenne sur le génome. (2) Quel est la distribution de ces extra-COs ? L’augmentation de recombinaison n’est pas homogène le long du génome : Les fréquences de CO augmente fortement des centromères vers les télomères, avec les plus hautes fréquences observées dans les régions distales. (3) La modification des fréquences de recombinaison est-elle identique lors des méioses mâles et femelle ? Chez le sauvage, la fréquence de recombinaison est plus élevée lors de la méiose mâle que femelle. Au contraire, la recombinaison femelle devient plus élevée que la recombinaison mâle chez les mutants recq4 et recq4 figl1. Ceci suggère que des contraintes qui s’appliquent sur la formation des CO lors de la méiose femelle sont relâchées chez ces mutants. En poursuivant le crible génétique, un nouveau mutant hyper-recombinant a été identifié. Le second objectif de ma thèse fut d’identifier et de caractériser fonctionnellement le gène correspondant. Une cartographie génétique et des études d’interactions protéine-protéine, ont mené à l’identification d’un facteur qui interagit directement avec FIGL1 et semble former un complexe conservé depuis les plantes jusqu’au mammifères. Nous avons baptisé cette protéine FLIP (Fidgetin-like-1 interacting protein). Les fréquences de recombinaisons sont augmentées dans flip-1, confirmant que FLIP1 limite la formation des COs. Des études d’épistasie ont montré que FLIP et FIGL1 agissent dans la même voie. De plus les protéines FIGL1/FLIP d’Arabidopsis ou humaine, interagissent avec RAD51 et DMC1, les deux protéines qui catalyse une étape clef de la recombinaison, l’invasion d’un ADN homologue. Finalement, dans flip comme dans figl1, la dynamique de DMC1 est modifiée. Nous proposons donc un modèle dans lequel le complexe FLIP-FIGL1 régule négativement l’activité de RAD51/DMC1 pour limiter la formation des COs. L’étude du complexe conservé FLIP-FIGL1 a mis en évidence un nouveau mode de régulation de la recombinaison, qui agit vraisemblablement à l’étape clé de l’échange de brin homologue. De plus, l’augmentation des CO sans précédent obtenues chez recq4 figl1 peut être d’un grand intérêt pour l’amélioration des plantes en permettant de diversité de nouvelles combinaisons alléliques
Meiotic crossovers (CO) are formed by reciprocal exchange of genetic material between the homologous chromosomes. CO generate genetic diversity and are essential for the proper segregation of chromosomes during meiosis in most eukaryotes. Despite their significance and a large excess of CO precursors, CO number is very low in vast majority of species (typically one to three per chromosome pair). This indicates that COs are tightly regulated but the underlying mechanisms of this limit remain elusive. In order to identify genes that limit COs, a genetic screen was performed in Arabidopsis thaliana. This led to the identification and characterization of several anti-CO factors belonging to three different pathways: (i) The FANCM helicase and its cofactors (ii) The AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) (iii) The RECQ4 -Topoisomerase 3α-RMI1 complex. The first objective was to understand the functional relationship between these three pathways and to address following questions: (1) how far can we increase recombination when combining mutations in FANCM, FIGL1 and RECQ4? We show that the highest increase in recombination was obtained in figl1 recq4, reaching to 7.5 fold the wild type level, on average genome wide. (2) How is the distribution of recombination events genome wide in mutants? The increased CO frequency in the mutants was not uniform throughout the genome. CO frequency rises from the centromere to telomeres, with distal intervals having highest COs (3) is the recombination frequency increase same in both male and female? In Arabidopsis wild type, male has higher recombination than female meiosis. In contrast, in recq4 and recq4 figl1, female recombination was higher than male. This suggests that certain constraints that apply to CO formation in wild type females are relieved in the mutant. By continuing the same genetic screen, a novel anti-CO mutant was identified. The second objective was to identify and functionally characterize the corresponding gene. Genetic mapping and protein interaction studies led to the identification of a factor that directly interacts with FIGL1 and appears to form a conserved complex both in Arabidopsis and humans. Hence, the factor was named FLIP (Fidgetin-like-1 interacting protein). Recombination frequency is increased in flip, confirming that FLIP limit COs. Epistasis studies showed that FLIP and FIGL1 act in same pathway. Further, FIGL1/FLIP proteins of Arabidopsis and humans directly interact with the recombinases RAD51 and DMC1 which catalyze a crucial step of homologous recombination, the inter homolog strand invasion. In addition flip like figl1 modifies dynamics of DMC1. We thus propose a model wherein the FLIP-FIGL1 complex negatively regulates RAD51/DMC1 to limit CO formation. Studying the conserved FIGL1-FLIP complex led to the identification of a novel mode of regulation of recombination, that likely acts at the key step of homologous strand invasion. Further the unprecedented level of CO increase in recq4figl1 in hybrids could be of great interest for crop improvement, allowing the production of novel allele combinations

Meiosis is a fundamental part in the life cycle of sexual species. It denotes a specialised cell division that halves chromosome numbers to generate haploid gametes for reproduction. Cells unable to competently progress through meiotic prophase activate cell surveillance mechanisms causing their elimination. Given the importance of DNA damage kinases like ATR in facilitating mitotic cell surveillance mechanisms, I characterized Atr-deficient spermatocytes to determine the importance of ATR for mammalian meiosis. I found that ATR ensures efficient chromosome synapsis, and that that is partially independent of meiotic recombination. In addition, ATR has three distinct roles in meiotic recombination. Firstly, during nucleolytic processing, it acts to regulate SPO11-oligonucleotide size when ATM is deleted. Secondly, it is required for accurate RAD51 and DMC1 recruitment to DSBs. Thirdly, it regulates the timing of DNA DSB repair on both unsynapsed and synapsed chromosomes. Finally I found that the loss of ATR is unable to rescue meiotic arrest in multiple meiotic mutants, including mice deficient for the other DNA damage PIKKs ATM and DNA-PK. My findings reveal multiple roles for ATR in male mouse meiosis.

- Introducció: La meiosi és un tipus de divisió cel·lular íntimament lligat a la gametogènesi en eucariotes superiors, la finalitat és la reducció del nombre cromosòmic de diploide a haploide (és a dir, de 2n a n) en el nucli dels gàmetes. En la present tesi s'han analitzat els processos de sinapsi i recombinació en tres espècies de mamífers: humans, moixos i cans. - Contingut de la investigació: Per a l’anàlisi dels processos de sinapsi i recombinació en tres espècies de mamífers (humans, moixos i cans) s'ha utilitzat la tècnica d’immunocitogenètica, la qual ha permès determinar els valors quantitatius i les característiques qualitatives d'aquests processos. - Conclusió: La infertilitat atribuïble a alteracions en processos de recombinació i sinapsi cromosòmica en autosomes i cos sexual es restringeix a aquells individus que presenten desviacions extremes respecte del rang control d'aquests paràmetres. Existeix un fenotip meiòtic recurrent, caracteritzat per bloqueig en la transició zigotépaquité. L'aplicació de tècniques de NGS (anàlisi de l'exoma) en aquest individu infèrtil ha permès localitzar una mutació en el gen TEX11, probablement patogènica i responsable de les característiques associades a aquest fenotip. L'anàlisi de la relació de les variants al·lèliques del gen PRDM9 amb problemes de fertilitat i generació de síndromes de novo no va detectar una associació significativa en cap cas. La influència del gen RNF212 sobre la variabilitat interindividual de la recombinació observada en el present estudi està associada a la presència de polimorfismes genètics en aquest gen. Així, cada còpia de l'al·lel T en l'SNP rs3796619 d'aquest gen suposa una disminució mitjana de la taxa de recombinació de 132,5 cM en comparació amb l'al·lel C. Hi ha una relació complexa entre els genotips de PRDM9 i la taxa de recombinació, que sembla estar determinada per la longitud dels al·lels, per l'estat de homozigositat o heterozigositat dels mateixos i per efectes de dominància. Hi ha una gran variabilitat intraindividual dels aspectes quantitatius i qualitatius de la recombinació i sinapsi en les tres espècies analitzades. Aquesta variabilitat suggereix que, en mamífers, aquests processos i els factors que els controlen han de ser prou flexibles per permetre generar diversitat, encara que dins d'uns marges que garanteixin l'estabilitat genòmica. S'han observat diferències notables en els processos de recombinació entre humans i gats. Així, en gats s'ha determinat l'absència de pics de recombinació en les regions subtelomèriques, un menor efecte inhibidor del centròmer i una distribució més uniforme al llarg dels braços cromosòmics. Aquestes dues últimes observacions estan relacionades amb la menor interferència observada en aquesta espècie. S'han observat característiques sinàptiques pròpies de gats mascle, no descrites fins al moment, com la presència d'un reservori de proteïna SYCP3 des de leptoté fins paquité inicial, la separació prematura dels elements laterals a partir de paquitè tardà o la morfologia pròpia i canviant del cos sexual que, igual que en humans, pot associar-se al subestadi d'aquesta fase meiòtica. S'han detectat anomalies sinàptiques en humans, gats i gossos, encara que la incidència de asinapsis, gaps i MSUC va variar entre espècies. A més, s'ha descrit per primera vegada en aquestes tres espècies el fenomen de MSUC en individus sense alteracions cromosòmiques de poblacions salvatges. En el cos sexual, s'ha detectat presència de proteïna SYCP1 més enllà de la regió PAR a les tres espècies analitzades. Aquesta presència podria relacionar-se amb un procés de polimerització per defecte de la proteïna SYCP1, que ajudaria a la reparació dels DSBs situats al cromosoma X.
- Introducción: La meiosis es un tipo de división celular íntimamente ligado a la gametogénesis en eucariotas superiores, la finalidad es la reducción del número cromosómico de diploide a haploide (es decir, de 2n a n) en el núcleo de los gametos. En la presente tesis se han analizado los procesos de sinapsis y recombinación en tres especies de mamíferos: humanos, gatos y perros. - Contenido de la investigación: Para el análisis de los procesos de sinapsis y recombinación en tres especies de mamíferos (humanos, gatos y perros) se ha utilizado la técnica de inmunocitogenética, la cual ha permitido determinar los valores cuantitativos y las características cualitativas de estos procesos. - Conclusión: La infertilidad atribuible a alteraciones en procesos de recombinación y sinapsis cromosómica en autosomas y cuerpo sexual se restringe a aquellos individuos que presentan desviaciones extremas respecto del rango control de estos parámetros. Existe un fenotipo meiótico recurrente, caracterizado por bloqueo en la transición zigoteno-paquiteno. La aplicación de técnicas de NGS (análisis del exoma) en este individuo infértil ha permitido localizar una mutación en el gen TEX11, probablemente patogénica y responsable de las características asociadas a este fenotipo. El análisis de la relación de las variantes alélicas del gen PRDM9 con problemas de fertilidad y generación de síndromes de novo no detectó una asociación significativa en ningún caso. La influencia del gen RNF212 sobre la variabilidad interindividual de la recombinación observada en el presente estudio está asociada a la presencia de polimorfismos genéticos en ese gen. Así, cada copia del alelo T en el SNP rs3796619 de este gen supone una disminución media de la tasa de recombinación de 132,5 cM en comparación con el alelo C. Existe una relación compleja entre los genotipos de PRDM9 y la tasa de recombinación, que parece estar determinada por la longitud de los alelos, por el estado de homozigosidad o heterozigosidad de los mismos y por efectos de dominancia. Hay una gran variabilidad intraindividual de los aspectos cuantitativos y cualitativos de la recombinación y sinapsis en las tres especies analizadas. Esta variabilidad sugiere que, en mamíferos, estos procesos y los factores que los controlan deben ser lo suficientemente flexibles para permitir generar diversidad, aunque dentro de unos márgenes que garanticen la estabilidad genómica. Se han observado diferencias notables en los procesos de recombinación entre humanos y gatos. Así, en gatos se ha determinado la ausencia de picos de recombinación en las regiones subteloméricas, un menor efecto inhibidor del centrómero y una distribución más uniforme a lo largo de los brazos cromosómicos. Estas dos últimas observaciones están relacionadas con la menor interferencia observada en esta especie. Se han observado características sinápticas propias de gatos macho, no descritas hasta el momento, como la presencia de un reservorio de proteína SYCP3 desde leptoteno hasta paquiteno inicial, la separación prematura de los elementos laterales a partir del paquiteno tardío o la morfología propia y cambiante del cuerpo sexual que, al igual que en humanos, puede asociarse al subestadio de esta fase meiótica. Se han detectado anomalías sinápticas en humanos, gatos y perros, aunque la incidencia de asinapsis, gaps y MSUC varió entre especies. Además, se ha descrito por primera vez en estas tres especies el fenómeno de MSUC en individuos sin alteraciones cromosómicas de poblaciones salvajes. En el cuerpo sexual, se ha detectado presencia de proteína SYCP1 más allá de la región PAR en las tres especies analizadas. Esta presencia podría relacionarse con un proceso de polimerización por defecto de la proteína SYCP1, que ayudaría a la reparación de los DSBs situados en el cromosoma X.
- Introduction: Meiosis is a type of cell division intimately linked to gametogenesis in higher eukaryotes, the aim is the reduction of the chromosome number from diploid to haploid (from 2n to n) in the nucleus of the gametes. In the present thesis, we have analysed the processes of synapsis and recombination in three species of mammals: humans, cats and dogs. - Content of the research: For the analysis of synapsis and recombination in three species of mammals (humans, cats and dogs) the immunocytogenetic technique has been used. It allowed to determine the quantitative values and the qualitative characteristics of these processes. - Conclusion: Infertility attributable to alterations in processes of recombination and chromosomal synapsis in autosomes and the sexual body is restricted to those individuals who present extreme deviations from the control range of these parameters. There is a recurrent meiotic phenotype characterized by an arrest in the zygotene to pachytene transition. The application of NGS techniques (exome analysis) in this infertile individual has allowed to locate a mutation in the TEX11 gene, probably pathogenic and responsible for the characteristics associated with this phenotype. The analysis of the relationship of the allelic variants of the PRDM9 gene with fertility problems and generation of de novo syndromes did not detect a significant association in any case. The influence of the RNF212 gene on the interindividual variability of recombination observed in the present study is associated with the presence of a genetic polymorphisms in that gene. Thus, each copy of the T allele in the SNP rs3796619 of this gene implies a mean decrease in the recombination rate of 132.5 cM compared to the C allele. There is a complex relationship between the PRDM9 genotypes and the rate of recombination, which appears to be determined by the length of the alleles, by the homozygosity or heterozygosity, and by dominance effects. There is significant intraindividual variability of the quantitative and qualitative aspects of recombination and synapsis in the three species analysed. This variability suggests that, in mammals, these processes and the factors controlling them must be flexible enough to generate diversity, albeit within margins that guarantee genomic stability. Significant differences have been observed in recombination processes between humans and cats. Thus, in cats the absence of recombination peaks has been determined in the subtelomeric regions, a lower inhibitory effect of the centromere and a more uniform distribution along the chromosome arms. These last two observations are related to the lower interference observed in this species. Synaptic characteristics of male cats, not previously described, have been observed. These include the presence of a reservoir of SYCP3 protein from leptotene to initial pachytene, premature separation of lateral elements from late pachytene or the changing morphology of the sexual body, which, as in humans, may be associated with the substage of this meiotic phase. Synaptic abnormalities have been detected in humans, cats and dogs, although the incidence of asynapsis, gaps and MSUC varied between species. In addition, the MSUC phenomenon has been described for the first time in these three species in individuals without chromosomal alterations of wild populations. In the sexual body, SYCP1 protein has been detected beyond the PAR region in the three species analysed. This presence could be related to a default polymerization process of SYCP1 protein, which would aid in the repair of DSBs located on the X chromosome.

The spindle assembly checkpoint (SAC) functions to prevent anaphase onset until all chromosomes are correctly bi-oriented on the mitotic spindle and aligned at the metaphase plate. Cohesion between sister chromatids is essential for this biorientation. In animal cells, most cohesin is removed from chromosome arms during prophase and prometaphase. Cohesin at centromeres is refractory to removal at this stage and persists until metaphase, whereupon its Sccl subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? 1 show that depletion of Sgol by RNA interference in HeLa cells causes precocious loss of centromeric cohesin from chromosomes in prometaphase and a permanent cell cycle arrest, presumably due to inactivation or the spindle checkpoint.

This thesis presents ultrastructural and biochemical information on meiotic reinitiation during oocyte maturation in the polychaetes, Arenicola marina, A. defodiens and Nereis virens. The ultrastructural changes during meiotic maturation was characterised in the oocytes of Arenicola marina and Nereis virens using transmission electron microscopy in addition to germinal vesicle breakdown, release of the prophase I block was signified by major cortical changes in both species. The ultrastructure of fertilization in A. marina was independent of whether the oocytes were matured in vivo and spawned or matured in vitro by CMF. Oocyte maturation in Arenicola marina is controlled by a hormonal cascade that is initiated by the prostomial maturation hormone, PMH, and followed by the coelomic maturation factor, CMF (Watson and Bentley, 1997). Results presented here demonstrated that PMH has a molecular mass greater than 10 kDa, yet how this molecule triggers CMF activity remains unknown. M-phase promoting factor (MPF) consists of two subunits, cdkl and cyclin B, and is responsible for the control of mitosis and meiosis. The cytoplasmic "second messenger" that transduces the hormone signal to the activation of MPF in the oocyte cytoplasm was investigated in the two Arenicola species and is discussed. MPF regulation was investigated in Arenicola marina and Nereis virens oocytes. MPF activation was driven by the dephosphorylation of cdkl and phosphorylation of cyclin B. The results indicate that as with all other higher eukaryotes, the precursor of MPF in A. marina oocytes was maintained inactive by the phosphorylation of threonine 14 and tyrosine 15 (or equivalent residues) on the cdkl subunit. In contrast to other organisms, however, only a fraction of the cdkl present was complexed to cyclin B and utilised during meiotic reinitiation. All the cdkl in N. virens oocytes was joined with cyclin B but results suggest that the inactive complex contained tyrosine-only phosphorylated cdk1.

Meiosis as a general process is prevalent across the eukaryotes, as are the orthologs of many genes encoding proteins known to function in meiosis. However, many organisms have experienced derived losses of otherwise well-conserved meiosis genes without losing meiosis and sexual reproduction. Although this general conservation of meiosis genes and precedent for derived meiosis gene losses has been previously established, questions remain about the frequency of and evolutionary forces contributing to these trends. This work sought (i) to characterize the phylogenetic distribution of 15 meiosis genes (most of which are known to function only in meiosis) in the exemplar eukaryotic kingdom Fungi and (ii) to use this dataset to investigate evolutionary processes contributing to the loss and retention of these genes. Orthologs of 15 meiosis genes (Rad51, Rad21, Spo11, Rec8, Dmc1, Hop2, Mnd1, Sae3/Swi5, Mei5/Sfr1, Pch2, Hop1, Msh4, Msh5, Mer3, Zip3) were identified by BLAST-based techniques and phylogenetically validated in most of the 109 publicly available sequenced fungal genomes investigated, but numerous putative derived losses were also detected. Rad51, Rad21, Rec8, and Spo11 were nearly universally conserved; the remaining genes were each undetectable or independently pseudogenized multiple times within fungi, particularly often for Pch2. Genes with previously known functional interactions tended to show parallel presence, absence, or pseudogenization patterns. Although this work primarily established the conserved presence of meiosis gene orthologs at the DNA level, examination of expressed sequence tags (ESTs) showed that many species--including some not previously known to undergo sexual reproduction--were competent to transcribe (and often splice) mRNA from the identified meiosis genes. Factors potentially influencing derived meiosis gene losses were investigated in two ways. First, degenerate PCR was used to amplify loci expected to contain orthologs of Msh4, Msh5, Pch2, and Zip3 in various Aspergillus species closely related to Aspergillus nidulans (a species with undetected or pseudogenized orthologs of these four genes.) The loss of Pch2 substantially predated the pseudogenization of Msh4, Msh5, and Zip3. Evolutionary rate analyses using the Ka/Ks ratio found no change in nonsynonymous substitution patterns in Msh4 and Msh5 in species that had lost Pch2 compared to those retaining Pch2. Elevated Zip3 Ka/Ks values were found in species with pseudogenized Msh4 and Msh5, suggesting possible obligate functional interactions of Zip3 with Msh4 and Msh5. Second, phylogenetically independent contrasts (PIC) analyses were performed on species from the 109-taxon inventory with published chromosome number and chromosome size estimates to investigate whether changes in either parameter were consistently associated with changes in the presence or absence of meiosis genes. Many analyses had low statistical power, neither detecting nor being able to exclude an association between gene loss and the tested variables. However, several comparisons did detect significant or nearly significant trends: for example, fungi that had lost genes related to crossover interference (Msh4, Msh5, or Pch2) tended to have fewer and/or larger chromosomes than their closest relatives without gene loss. A final objective was to determine the distribution of meiosis genes in lichenized fungi and green algae to see whether this form of symbiosis was associated with differences in the presence or molecular evolution of meiosis genes. Rad51, Dmc1, and Mnd1 were each amplified by degenerate PCR from multiple lichenized fungi that lacked sequenced genomes, and no systematic difference in evolutionary rate was found between examined lichenized fungi compared to other examined classes in phylum Ascomycota. Bioinformatic analyses of meiosis gene distribution in green algae revealed not only no obvious increased tendency for derived gene losses in examined lichenized green algae but also very few derived meiosis gene losses in green algae in general. This suggests that lichenization may not be associated with consistent differences in the evolution of meiosis genes in either fungal or green algal symbionts. The green algal results also illustrate the need to investigate the extent to which eukaryotes as a whole exhibit the same trends of meiosis gene evolution described here for fungi: frequent derived losses of meiosis genes, genes encoding proteins with function interactions showing similar distributions, likely roles for post-transcriptional regulation of meiosis gene transcripts, and loss of crossover distribution-related genes potentially being associated with constraints on chromosome size and/or haploid chromosome number.

Durant la oogènesi dels mamífers, les oogònies proliferen forman els anomenats cists. Les oogònies entren en meiosis progressant en la profase I i els cists es trenquen al mateix temps que es produeix una mort massiva perinatal dels oòcits. En la profase I, s’indueixen trencaments de doble cadena (DSBs) per tot el genoma, que son reparats per recombinació homòloga per a promoure la sinapsi dels cromosomes homòlegs. Existeixen diferents mecanismes que s’activen en resposta a errors en aquests processos i que aturen el cicle cel·lular i produeixen l’apoptosi de les cèl·lules danyades. La resposta al dany al DNA (DDR) es activada en presència d’oòcits i d’espermatòcits amb errors de recombinació en l’anomenat checkpoint de recombinació. Per l’altre banda, errors en la sinapsi activen el checkpoint de sinapsi. El nostre objectiu era caracteritzar les funcions de la DDR i del checkpoint de sinapsi durant l’oogènesi en mamífers. Contràriament al que succeeix en espermatòcits, els oòcits presenten un alt número de DSBs no reparats a l’estadi de paquitè en el moment en que es produeix la mort oocitària massiva i el trencament del cists. Per tal d’esbrinar si el checkpoint de recombinació participa en la regulació del número d’oòcits en mamífers, hem analitzat el número de DSBs, el número d’oòcits en femelles perinatals i adultes, el trencament dels cists, la formació de fol·licles i la vida reproductiva de femelles de ratolí control i mutants per a la quinasa efectora de la via de la DDR, la proteïna CHK2. Les nostres dades han revelat la implicació de CHK2 en la regulació del número d’oòcits, però només en ovaris fetals, obrint la possibilitat de l’existència d’una via alternativa regulant el número d’oòcits després del naixement. Els nostres estudis utilitzant ovaris cultivats in vitro en presència d’inhibidors, suggereixen que CHK1 podria compensar l’absència de CHK2 in vivo. Per tant, la via de la DDR controlaria el número d’oòcits en mamífers. A més, hem trobat un augment del número d’oòcits en adultes velles mutants per CHK2 suggerint que la DDR controla la llargada de la vida reproductiva en mamífers. Finalment, hem estudiat el possible paper de TRIP13 en el checkpoint de sinapsi. La proteïna TRIP13 es necessària per a la recombinació, però també per a la sinapsi dels cromosomes sexuals i per a la formació de la vesícula sexual, suggerint un possible rol al checkpoint de sinapsi. Hem analitzat el número d’oòcits en ovaris Spo11-/- Trip13mod/mod i Dmc1-/- Chk2-/- Trip13mod/mod per a esbrinar si TRIP13 es necessària per a activar el checkpoint de sinapsi en femelles. Les nostres dades han revelat un rescat en el número d’oòcits en el triple mutant, però no en el doble. Aquest resultats obren la possibilitat de que TRIP13 participi en el checkpoint de sinapsis, però com a alternativa, proposem que aquesta participació podria ser compatible amb una possible regulació per part de TRIP13 de la elecció de la via de reparació dels DSBs.
During mammalian oogenesis, oogonia proliferate forming the so-called cysts. The oogonia enter meiosis progressing through prophase I and the cysts break down concomitantly to massive perinatal oocyte death. During meiotic prophase I, double strand breaks (DSBs) are induced throughout the genome and repaired by homologous recombination to promote the synapsis of the homologous chromosomes. In response to errors in these processes, different response pathways are activated triggering cell cycle arrest or even apoptosis. The DNA damage response (DDR) is activated in response of meiocytes with recombination failure in the recombination checkpoint; while errors in synapsis trigger the synapsis checkpoint. We aimed to characterize the roles of the DDR and synapsis checkpoint in mammalian oogenesis. Contrary to what occurs in spermatocytes, oocytes present high numbers of unrepaired DSBs at pachynema, at the time of the massive oocyte death and cyst breakdown. In order to know if the recombination checkpoint participates in the regulation of the oocyte number in mammals, we analyzed the presence of DSBs, the oocyte number in both perinatal and adult females, the cyst breakdown, the formation of follicles and the reproductive lifespan using control and mutant mice for the effector kinase of the DNA damage response pathway, CHK2. Our data revealed the involvement of CHK2 in the regulation of the oocyte number but only in fetal ovaries prior to birth, raising the question of a possible alternative regulator acting just after birth. Our studies using in vitro ovarian cultures using inhibitors, suggest that CHK1 may compensate the loss of CHK2 perinatally in vivo. Thus, revealing that the DDR pathway controls the oocyte number in mammals. Furthermore, we found an increased number of oocytes in elder Chk2 mutant females suggesting that the DDR controls the reproductive lifespan extension in mammals. Finally, we studied the possible involvement of TRIP13 in the synapsis checkpoint. The protein TRIP13 is required for recombination, but it is also needed for the synapsis of sex chromosomes and the sex body formation. Thus, suggesting a possible role in the synapsis checkpoint. We analyzed the oocyte number in females from Spo11-/- Trip13mod/mod and Dmc1-/- Chk2-/- Trip13mod/mod ovaries in order to infer if TRIP13 is required to implement the synapsis checkpoint in females. Our data revealed a rescue in the number of oocytes in the triple mutant, but not in the double mutant. These results leave open the possibility of a participation of TRIP13 in the synapsis checkpoint, but as an alternative, they could be compatible with a possible role of TRIP13 regulating the DSB repair pathway choice.

Eukaryotes have developed stringent regulatory mechanisms that control cell division and ensure proper chromosome segregation. Maintaining genome integrity is especially important during meiosis, the specialised cell division programme in the germline that generates haploid gametes. As these cells transmit genetic information to the next generation, the consequences of meiotic errors are not restricted to an organismal level, but can directly impact the fitness of the offspring. Mammals display a high degree of sexual dimorphism in meiosis with regard to the stringency of regulatory mechanisms. This manifests in a relatively high degree of maternally-derived aneuploidies due to weaker checkpoint control in females, whereas more rigorous checkpoints in males frequently perturb fertility. Mouse models of aneuploidy often exhibit complete male sterility and early germ cell arrest, preventing the study of aneuploidy during late and post-meiotic stages in males. In this thesis, we have used the trans-chromosomic mouse model, Tc1, which carries a single copy of human chromosome 21 (HsChr21) and show that, unlike other aneuploid mouse strains, the Tc1 mouse can successfully passage the exogenous human chromosome through male meiosis and generate aneuploid offspring. Our investigations have shown that the presence of the aneuploid human chromosome causes spermatogenic defects due to an arrest at the first meiotic division. Despite this impairment, we found an unexpectedly high number of aneuploid gametes in Tc1 males and the majority of males were able to produce aneuploid offspring, albeit at a lower frequency. Transmission of HsChr21 through the male germline was less efficient compared to female germline transmission, but allowed us to study the impact of male germline-associated chromatin remodelling on the transcriptional deployment of HsChr21 in the offspring. This revealed that, despite fundamentally different developmental dynamics, male- versus female-germline passage result in indistinguishable transcriptional and regulatory phenotypes. An important pathway in the male germline involves the expression of piRNAs, a class of small non-coding RNAs that are commonly found in the germline of animals where they defend cells against transposable elements. Profiling the expression of small RNAs in the Tc1 mouse showed that conserved human piRNA clusters can be successfully transcribed by the mouse piRNA machinery. In addition, we detected Tc1-specific piRNA sequences that were neither present in human nor mouse, mapping to a human-specific repeat element. In line with the previously observed activation of human-specific repeat elements in the Tc1 mouse, this suggests that novel transcripts arising from human repeats can trigger an adaptive piRNA response, thereby demonstrating the plasticity of this pathway to newly invading repeat elements. Transcriptional profiling of spermatogenic cell populations on a single-cell level allowed us to generate an atlas of gene expression over the course of spermatogenesis and dissect meiotic silencing dynamics in the presence of aneuploidy. Transcriptional silencing during meiosis occurs in response to unpaired chromosomes and, in male germ cells, affects the sex chromosomes due to their largely unpaired nature. We found that the presence of HsChr21 has no impact on the silencing of chromosome X, however, the two chromosomes display drastically different silencing patterns with HsChr21 showing a much weaker repression. Taken together, this study revealed a higher than expected tolerance for aneuploidy in the mouse male germline thus allowing the characterisation of meiotic checkpoint mechanisms, the meiotic silencing response to unpaired chromosomes as well as piRNA expression in the presence of an exogenous human chromosome.

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.

Meiosis is a specialized cell cycle, which generates haploid gametes from diploid parental cells. During meiosis one round of cohesion establishment during premeiotic DNA replication mediates two rounds of chromosome segregation. During meiosis I homologous chromosomes separate, whereas sister chromatids segregate during the second meiotic division without an intervening round of DNA replication. Both rounds of chromosome segregation are triggered by an ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC/C). APC/C-dependent destruction of securin/Pds1 is required to activate separase, a thiol protease that mediates chromosome segregation by cleavage of the cohesin complex. The first meiotic division is preceded by an extended prophase I, during which maternal and paternal chromatids undergo recombination. The persistence of cohesion during premeiotic S- and prophase I is essential for recombination and both meiotic nuclear divisions. In order to prevent premature loss of cohesion, the APC/C has to be inactivated during early meiosis. How the APC/C is kept inactive during premeiotic S- and prophase I was unknown. This question has been addressed by studying the APC/C subunit Mnd2 from the budding yeast Saccharomyces cerevisiae. This work demonstrates that Mnd2 is required for the persistence of cohesion during premeiotic S- and prophase I. Mnd2 prevents premature activation of the APC/C by the meiosis-specific substrate recognition factor Ama1. In cells lacking Mnd2, the APC/C-Ama1 enzyme triggers premature ubiquitin-dependent degradation of Pds1, which leads to premature separation of sister chromatids due to an unrestrained activity of separase. Thus, chromosome segregation during meiosis depends on both inhibition of a meiosis-specific APC/C and timely activation of APC/C- dependent proteolysis
Die Meiose ist ein spezialisierter Zellzyklus, der zum Ziel hat haploide Gameten aus diploiden Vorläuferzellen zu produzieren. Dafür erfolgen nach der prä-meiotischen DNA Replikation zwei aufeinanderfolgende Kernteilungen. In der ersten meiotischen Teilung erfolgt die Trennung der homologen Chromosomen. In einer zweiten meiotischen Teilung werden dann die Schwesterchromatiden getrennt. Die Trennung der Chromosomen wird durch den Anaphase-Promoting Complex oder Cyclosome (APC/C), einer Ubiquitin Ligase, reguliert. Der APC/C initiiert den Abbau von Securin/Pds1, einem Inhibitor der Thiol-Protease Separase, welche für die Trennung der Chromosomen zum Beginn der Anaphase verantwortlich ist. In einer im Vergleich zur Mitose extrem langen meiotischen Prophase I findet Rekombination zwischen maternalen und paternalen Chromosomen statt. Für diesen Vorgang, sowie für die beiden folgenden meiotischen Teilungen, wird Kohäsion zwischen den Schwesterchromatiden benötigt. Ein frühzeitiger Verlust der Kohäsion führt zur frühzeitigen Trennnung der Schwesterchromatiden, wodurch aneuploide Gameten produziert werden können. Daher muss die Aktivität des APC/C während der meiotischen Prophase I inhibiert werden. Wie der APC/C während der Prophase I inaktiviert wird, war bisher unbekannt. Einsicht in dieses Problem ergab sich aus der Untersuchung der APC/C Untereinheit Mnd2 aus der Bäckerhefe Saccharomyces cerevisiae. Es wird gezeigt, dass Mnd2 für den Verbleib der Kohäsion zwischen den Schwesterchromatiden während der meiotischen S- und Prophase I benötigt wird. Während dieser Phase verhindert Mnd2 die frühzeitige Aktivierung der Meiose-spezifischen Form des APC/C-Ama1. In meiotischen Zellen, die kein Mnd2 besitzen, löst das APC/C-Ama1 Enzym die Ubiquitin-abhängige Zerstörung von Pds1 aus. Dies führt zu einer frühzeitigen Aktivierung von Separase, welches die Trennung der Schwesterchromatiden schon während der meiotischen S- und Prophase I zur Folge hat. Die korrekte Verteilung der Chromosomen hängt daher sowohl von der Inhibierung als auch der Aktivierung des APC/C ab

SYN1 is a meiosis-specific Arabidopsis homologue of yeast REC8. REC8 is an important component of the meiotic cohesion complex which maintains cohesion between sister chromatids. Cytological analysis of syn1\(^{-/-}\) has shown chromosome fragmentation at metaphase I. To determine the basis of chromosome fragmentation in the syn1\(^{-/-}\), three double mutants were constructed. I have demonstrated that chromosome fragmentation in syn1 is AtSPO11-1-dependent. Moreover, I have also shown that SYN1 has a role in DSB repair by analysing Atdmc1\(^{-/-}\)/syn1\(^{-/-}\) meiocytes. To investigate this further, immunolocalization studies in wild-type and syn1\(^{-/-}\) were conducted. Distribution of ASY1 and AtZYP1 was affected in syn1\(^{-/-}\). Both proteins appeared as aggregates, developing into an abnormal short linear signal in early prophase I, suggesting that both axis formation and synapsis are compromised. Distribution of the recombination proteins AtRAD51 and AtMLH1 was also aberrant. Localization of SYN1 in wild-type nuclei revealed a continuous signal along the chromosome axes. However, careful inspection revealed that this was accompanied by patches of more intense signals, possibly corresponding to DSB regions. To investigate this further I analysed SYN1 distribution in an Atspo11-1-4\(^{-/-}\) mutant. Whilst faint SYN1 signals were apparent along the axis, no patches of intense signals were visible. Cisplatin-induced DSBs restored AtZYP1 foci in Atspo11-1-4\(^{-/-}\) and also resulted in restoration of intense patches of the SYN1 signals. This is consistent with the recruitment of SYN1 to DSB sites.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Meiosis is the process by which haploid gametes are produced from a diploid progenitor cell. Accurate completion of the meiotic divisions requires a variety of modifications to the mitotic chromosome segregation machinery, which allow the reductional meiotic chromosome segregation program to occur. Oscillations in the activity of Cyclin- Dependent Kinases (CDKs) drive virtually every event in the mitotic cell cycle, including events such as cell cycle entry, DNA replication, and chromosome segregation. While much is known about the activity of CDKs, the regulation of CDK activity, and the mechanisms by which CDK activity promotes cell cycle events during vegetative growth in Saccharomyces cerevisiae, relatively little is known about the roles of CDKs during the meiotic divisions. This work examines CDK activity during meiosis, the regulation of CDK activity during meiosis, and mechanisms by which CDKs regulate proper meiotic chromosome segregation. First, a striking diversity in Clb-CDK activity is observed during meiosis, including the identification of Clb1-CDK, and Clb3-CDK as meiosis I and meiosis II specific Clb-CDKs respectively. Second, Clb3 protein is shown to be restricted to meiosis II by translational control mediated by the 5'UTR of the CLB3 message. Finally, premature production of Clb3 results in the premature separation of sister-chromatids during meiosis I.
by Thomas M. Carlile.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Vita.
Includes bibliographical references.
Numerous DNA double-strand breaks (DSBs) are introduced into the genome in the course of meiotic recombination. This poses a significant hazard to the genomic integrity of the cell. Studies in a number of organisms have unveiled the existence of surveillance mechanisms or checkpoints that couple DNA repair and microtubule integrity to meiotic cell cycle progression. Through their action, aberrant meiocytes are delayed in their meiotic progression to facilitate repair of meiotic DSBs, or are culled through programmed cell death, thereby protecting the germline from aneuploidies that could lead to spontaneous abortions, birth defects and cancer predisposition in the offspring. Two such surveillance mechanisms are analyzed in this thesis. The first is the meiotic recombination checkpoint, which delays meiotic cells in G2/prophase if recombination intermediates remain unrepaired. The extent of the delay is modulated by protein phosphatase 1 (PP1), whose activity allows cells to overcome the checkpoint dependent delay in a process called adaptation. In this work, experiments in the budding yeast Saccharomyces cerevisiae are described that show that premature adaptation is prevented by the FK506-binding protein Fpr3, which associates with and counteracts PP1 in vivo.
(cont.) The checkpoint activity of Fpr3 can be inhibited by the small molecule inhibitor rapamycin and requires the proline isomerase domain of Fpr3, but not its catalytic activity. The second surveillance mechanism analyzed here is a spindle checkpoint independent arrest response of meiotic cells to microtubule perturbation. This arrest is caused by down-regulation of the meiotic transcriptional program and occurs at one of two possible stages, in meiotic G1 prior to entry into the meiotic program, or in meiotic G2/prophase after pre-meiotic DNA replication. Both mechanisms described in this work may be conserved in other organisms, including mammals. The findings presented herein are incorporated into a general model of the surveillance mechanisms of meiotic recombination.
by Andreas Hochwagen.
Ph.D.

Meiosis is a specialised form of cell division in which diploid cells divide to form four non-identical spores containing half the genetic complement of the parent. During this cell division program, much of the usual machinery regulating cell division is put to alternate use to allow the cells to undergo an extra round of division without an intervening phase of DNA synthesis. In particular, the end of the first division, meiosis I, must be regulated differently than the end of the mitotic division. We used the model organism Saccharomyces cerevisiae to determine some of these differences in regulation. The cell division program is driven by the sequential association of cyclins with the CDK (cyclin dependent kinase), leading to waves of kinase activity. Exit from mitosis requires the downregulation of CDK activity, and is coordinated by two signalling networks, the FEAR (Cdc14 Early Anaphase Release) network and the MEN (Mitotic Exit Network). Both networks initiate the release of the phosphatase Cdc14 from its inhibitor, Net1, to counter CDK activity. Exit from meiosis I similarly relies on Cdc14 activity, but is driven only by the FEAR network. Experimental results showed that the phosphorylation state and subcellular localisation of the meiotic cyclin, C1b1, are altered in meiosis I. We investigated this relationship and aimed to determine the kinase responsible. We used modelling techniques to explore several rationales for the specific regulation of C1b1. We examined the functional significance of C1b1 localisation, using localisation mutants, and made an investigation into Cdc14 release in meiosis I.

Meiosis is a specialized cell cycle, which generates haploid gametes from diploid parental cells. During meiosis one round of cohesion establishment during premeiotic DNA replication mediates two rounds of chromosome segregation. During meiosis I homologous chromosomes separate, whereas sister chromatids segregate during the second meiotic division without an intervening round of DNA replication. Both rounds of chromosome segregation are triggered by an ubiquitin ligase called the Anaphase-Promoting Complex or Cyclosome (APC/C). APC/C-dependent destruction of securin/Pds1 is required to activate separase, a thiol protease that mediates chromosome segregation by cleavage of the cohesin complex. The first meiotic division is preceded by an extended prophase I, during which maternal and paternal chromatids undergo recombination. The persistence of cohesion during premeiotic S- and prophase I is essential for recombination and both meiotic nuclear divisions. In order to prevent premature loss of cohesion, the APC/C has to be inactivated during early meiosis. How the APC/C is kept inactive during premeiotic S- and prophase I was unknown. This question has been addressed by studying the APC/C subunit Mnd2 from the budding yeast Saccharomyces cerevisiae. This work demonstrates that Mnd2 is required for the persistence of cohesion during premeiotic S- and prophase I. Mnd2 prevents premature activation of the APC/C by the meiosis-specific substrate recognition factor Ama1. In cells lacking Mnd2, the APC/C-Ama1 enzyme triggers premature ubiquitin-dependent degradation of Pds1, which leads to premature separation of sister chromatids due to an unrestrained activity of separase. Thus, chromosome segregation during meiosis depends on both inhibition of a meiosis-specific APC/C and timely activation of APC/C- dependent proteolysis.
Die Meiose ist ein spezialisierter Zellzyklus, der zum Ziel hat haploide Gameten aus diploiden Vorläuferzellen zu produzieren. Dafür erfolgen nach der prä-meiotischen DNA Replikation zwei aufeinanderfolgende Kernteilungen. In der ersten meiotischen Teilung erfolgt die Trennung der homologen Chromosomen. In einer zweiten meiotischen Teilung werden dann die Schwesterchromatiden getrennt. Die Trennung der Chromosomen wird durch den Anaphase-Promoting Complex oder Cyclosome (APC/C), einer Ubiquitin Ligase, reguliert. Der APC/C initiiert den Abbau von Securin/Pds1, einem Inhibitor der Thiol-Protease Separase, welche für die Trennung der Chromosomen zum Beginn der Anaphase verantwortlich ist. In einer im Vergleich zur Mitose extrem langen meiotischen Prophase I findet Rekombination zwischen maternalen und paternalen Chromosomen statt. Für diesen Vorgang, sowie für die beiden folgenden meiotischen Teilungen, wird Kohäsion zwischen den Schwesterchromatiden benötigt. Ein frühzeitiger Verlust der Kohäsion führt zur frühzeitigen Trennnung der Schwesterchromatiden, wodurch aneuploide Gameten produziert werden können. Daher muss die Aktivität des APC/C während der meiotischen Prophase I inhibiert werden. Wie der APC/C während der Prophase I inaktiviert wird, war bisher unbekannt. Einsicht in dieses Problem ergab sich aus der Untersuchung der APC/C Untereinheit Mnd2 aus der Bäckerhefe Saccharomyces cerevisiae. Es wird gezeigt, dass Mnd2 für den Verbleib der Kohäsion zwischen den Schwesterchromatiden während der meiotischen S- und Prophase I benötigt wird. Während dieser Phase verhindert Mnd2 die frühzeitige Aktivierung der Meiose-spezifischen Form des APC/C-Ama1. In meiotischen Zellen, die kein Mnd2 besitzen, löst das APC/C-Ama1 Enzym die Ubiquitin-abhängige Zerstörung von Pds1 aus. Dies führt zu einer frühzeitigen Aktivierung von Separase, welches die Trennung der Schwesterchromatiden schon während der meiotischen S- und Prophase I zur Folge hat. Die korrekte Verteilung der Chromosomen hängt daher sowohl von der Inhibierung als auch der Aktivierung des APC/C ab.

Correct chromosome segregation during meiosis requires that the paternal and maternal copies of each chromosome, known as homologues, recognise and pair with one another before they can undergo meiotic recombination. Defects in this process lead to sterility and the formation of aneuploid gametes, which is the leading cause of birth defects in humans. In this study the process of homologue pairing during meiosis has been investigated in C. elegans, an organism especially well suited for meiotic studies. During a genetic screen for meiotic mutants, several mutants with defects in meiotic chromosome segregation were isolated. One of these mutants, me85, was identified as a new allele of the spd-3 gene, which had previously been shown to be required for mitotic divisions in the early embryo. spd-3(me85) mutants display defects in homologue pairing similar to those observed in mutants lacking SUN-1 or ZYG-12, two proteins that form a bridge across the nuclear envelope (NE). This bridge transmits cytoskeletal forces generated outside the nucleus to meiotic chromosomes inside the nucleus, thereby facilitating chromosome clustering, a process that is though to facilitate homology search. The localisation of SUN-1 and ZYG-12 to the NE is not affected in spd-3(me85) mutants and chromosomes remain tethered to the NE. However, live imaging experiments in spd-3(me85) mutants demonstrate that the movement of chromosomes through the NE is severely impaired, which results in lack of chromosome clustering. Knocking down the activity of the dynein-dynactin complex by RNAi resulted in a phenocopy of the chromosome clustering defects observed in spd-3(me85) mutants, although dynein localisation is not affected in spd-3(me85) mutants. Interestingly, the SPD-3 protein localizes outside the nucleus in the germline. These observations suggest that SPD-3 affects the earliest steps of homologue pairing by regulating the cytoskeletal forces outside the nucleus.

Drosophila melanogaster is unique amongst model organisms in that males utilize achiasmatic meiosis, where formation of the synaptonemal complex (SC) and recombination are absent. Most organisms require the SC and chiasmata for the successful completion of meiosis and production of viable gametes, making D. melanogaster an ideal system for the study of meiotic variation. The goal of my research was to examine in detail the origin and evolution of male achiasmatic meiosis in Diptera. This was done in three parts: 1) assessing the presence and absence of meiosis genes across dipteran species, 2) analyzing the rate of evolution of Drosophila achiasmatic meiosis genes, and 3) evaluating differences in expression and splicing of meiosis genes between D. melanogaster males and females. I queried genome and transcriptome data from eleven dipteran species for both canonical and achiasmatic meiosis genes. Surprisingly, I found that a set of meiosis-specific genes was lost prior to the gain of Drosophila male achiasmy genes, suggesting that the latter were a later addition to an already non-canonical meiotic process. To assess the evolution of fourteen Drosophila achiasmatic meiosis genes, I performed phylogenetic, rate, selection and co-evolution analyses. My results show that, although these genes appear to be evolving under purifying selection, they are all evolving rapidly compared to their paralogs and paralogous genes throughout the Drosophila genome. Some groups of these genes are also co-evolving, supporting their potential for encoding members of protein complexes. These results suggest that male achiasmy is globally influencing the rapid evolution of these genes, even though their functions within meiosis vary greatly. Lastly, I investigated the expression and splicing of meiosis genes between male and female D. melanogaster. As expected, many meiosis genes with sex-limited roles showed biased expression for the sex that utilized them. However, some genes were expressed equally in both sexes or higher in the opposite sex. I also found evidence that sex-biased splicing may have a role in regulating protein production for some meiosis genes. These results indicate that the regulation of meiotic gene expression is more complex than originally thought and that multiple mechanisms, including alternative splicing, are utilized to control protein production. The combination of results from all parts of this work highlight some of the major events that occurred prior to and during the evolution of Drosophila male achiasmy and lay groundwork for future studies examining the details of this unusual evolutionary path.

La méiose est une étape essentielle de la reproduction chez tous les organismes sexués. En effet, celle-ci permet l’obtention de quatre gamètes haploïdes à partir d’une seule cellule diploïde grâce à la réalisation deux divisions successives suivant une seule étape de réplication. Un des éléments essentiels permettant une bonne ségrégation en première division méiotique est la création d’un échange physique entre les chromosomes homologues parentaux. Ce lien physique, plus communément appelé crossing-over (CO), est produit par un mécanisme de recombinaison entre les chromosomes homologues au cours de la prophase I méiotique. La recombinaison homologue est initiée par la formation simultanée de nombreuses cassures double-brin au sein du génome. Chez la levure de boulanger, la formation des COs est dépendante de la famille protéique ZMM (un acronyme pour Zip1/2/3/4-Msh4/5-Mer3-Spo16) composée de huit protéines hautement conservées, et impliquées dans la reconnaissance et la stabilisation des intermédiaires d’ADN formés au cours de la recombinaison homologue. Nous avons montré que la protéine Zip4 forme un complexe stable avec deux autres protéines ZMM, Zip2 et Spo16. Le complexe Zip2-Zip4-Spo16 (ZZS), de type XPF-ERCC1, serait capable de reconnaitre et de stabiliser les intermédiaires de recombinaison afin de promouvoir leur réparation en tant que CO. Chez les mammifères, Zip2 et Zip4 possèdent des homologues décrits, SHOC1 et TEX11 respectivement, mais aucun homologue n’a été découvert pour Spo16. Nous avons réalisé une analyse in silico et pu déterminer un homologue de Spo16 chez les mammifères, MmSPO16. Par la suite, j’ai pu co-purifier MmSPO16 avec le domaine XPF de SHOC1, ce qui suggère la conservation du complexe ZZS chez les mammifères. De plus, le processus de formation des COs est corrélé́ à la mise en place d’un complexe protéique formé entre les deux chromosomes homologues, appelé complexe synaptonémal (CS). Le CS est composé de deux éléments axiaux, accolés entre eux à une distance précise de 100 nm par la région centrale. La région centrale comprend un élément central, composé de l’hétérodimère Ecm11-Gmc2, et d’un élément transversal formé par la protéine Zip1. Les éléments transversaux partant des axes opposés se lient tête-bêche au niveau de l’élément central. Malgré des liens fonctionnels évidents entre la formation des COs et l’assemblage du CS entre les chromosomes homologues, aucun lien physique direct n’a été établi à ce jour. Au cours de mon doctorat, j’ai pu démontrer l’existence d’une interaction physique entre la protéine du CS Ecm11 et la protéine ZMM Zip4. Cette interaction est nécessaire pour la localisation et la polymérisation d’Ecm11 sur les chromosomes, l’assemblage correct du CS et la ségrégation des chromosomes homologues en première division méiotique
Meiosis is a highly conserved mechanism among organisms with sexual development. This process consists in producing four haploid gametes from one diploid cell by executing two successive rounds of cell division. During the first meiotic division, reciprocal exchanges of parental DNA strands, also known as crossing-overs (COs), ensure the faithful segregation of homologous chromosomes. COs arise from a specific type of DNA repair, homologous recombination. This pathway is initiated by the simultaneous induction of hundreds of double strand breaks (DSBs) in the genome. In budding yeast, the major CO pathway is promoted by a family of eight conserved proteins, named ZMMs (acronym for Zip1/2/3/4-Msh4/5-Mer3-Spo16), involved in recognizing and stabilizing DNA intermediates formed during homologous recombination. We showed that the Zip4 protein forms a stable tripartite complex with two other ZMM proteins, Zip2 and Spo16. Our data suggests that the Zip2-Zip4-Spo16 (ZZS) complex binds recombination intermediates through its XPF-ERCC1-like domain and drives them towards a CO fate. The homologs of Zip2 and Zip4 in mammals, SHOC1 and TEX11 respectively, have been described, but no Spo16 homolog has been found so far. We could identify the homolog of Spo16 in mammals by an in silico screen, MmSPO16. In addition, I could co-purify MmSPO16 with the XPF domain of SHOC1, thus revealing the potential conservation of the entire ZZS complex in mammals. ZMM-dependent COs are formed within the context of a meiosis-specific structure, named synaptonemal complex (SC). The SC is a proteinaceous structure composed of two axial elements physically maintained together at a precise distance of 100 nm by a central region. The central region encompasses a central element, composed of the two proteins Ecm11 and Gmc2, and the transverse filaments composed of Zip1. The transverse filaments from opposing axial elements overlap and bind head-to-head in the central element. However, despite evidence of a close relationship between SC assembly and CO formation, nothing is known about a direct link that could coordinate these two events spatially and temporally. During my PhD, I found a new interaction between the SC protein Ecm11 and the ZMM protein Zip4. This newly discovered interaction is necessary for Ecm11 association and polymerization on chromosomes, the SC assembly and the homolog disjunction in meiosis I. Our results suggest a direct connection that ensures SC assembly from CO sites through the Zip4-Ecm11 interaction. This way, ensuring SC polymerization from emerging CO sites could be a way of fine-tuning CO distribution, by participating to CO interference and/or by regulating nearby DSB formation. Moreover, I could identify an interaction between the mammalian ortholog of Zip4, TEX11, and one of the five members composing the SC central element, TEX12, raising the possibility that this mechanism synchronizing CO formation and SC polymerization could be conserved

La recombinaison méiotique est au cœur de l'hérédité Mendélienne, de l'évolution et de l'amélioration des plantes, car elle assure, grâce aux crossovers, une transmission fidèle des chromosomes et le brassage de l’information génétique au fil des générations. Deux voies de formation des crossovers coexistent chez les plantes. La voie principale (voie I) dépend de la protéine MSH4 (et de quelques autres). La voie secondaire ne produit que quelques crossovers (dits de voie II) au cours de la méiose d’une plante de type sauvage ; ils sont indépendants de MSH4 et leur nombre est limité par des protéines telles que FANCM. Si ces deux voies ont été bien décrites chez des espèces diploïdes, ce n’est pas le cas chez des plantes allopolyploïdes, pourtant très fréquentes parmi les plantes cultivées. Il s'agit là d'une lacune importante, car la présence de plusieurs jeux de chromosomes apparentés conduit à augmenter le nombre de partenaires susceptibles de former des crossovers et le nombre de copies de tous les gènes méiotiques, rendant la recombinaison méiotique plus complexe. Cette thèse vise à explorer l'interaction entre les voies de formation des crossovers et la polyploïdie en utilisant des mutants de colza (Brassica napus; AACC) et d’un de ces parents diploïdes (B. rapa; AA) pour deux gènes de la recombinaison méiotique.J'ai tout d'abord testé dans quelle mesure la formation de crossovers entre chromosomes homologues et entre homéologues (chromosomes A et C) est tributaire des voies I et II en évaluant l’effet d’une diminution du nombre de copies fonctionnelles de MSH4 sur le nombre de crossovers. J'ai montré que ce dernier n'est altéré que lorsque les deux copies MSH4 sont inactivées, toute autre combinaison de mutations conduisant au même nombre de crossovers inter-homologues que chez le sauvage. J'ai également montré que la proportion de crossovers de voie II chez des mutants msh4 de colza est bien supérieure à celles observées chez d’autres plantes mutantes pour msh4. Cette observation reste vraie chez des mutants msh4 de B. rapa, suggérant que la proportion accrue de crossovers de voie II n’est pas spécifique au colza, mais probablement une caractéristique des Brassicaceae. Chez des plantes allohaploïdes (AC) de colza, chez lesquelles les crossovers ne peuvent se former qu’entre homéologues, les copies MSH4 ne se compensent plus complétement ; le nombre de crossovers de voie I fluctue au contraire proportionnellement au dosage de MSH4, devenant presque nul lorsque toutes les copies sont inactivées. Mes résultats illustrent deux nouvelles propriétés spécifiques des crossovers entre homéologues: une plus grande sensibilité vis-à-vis du dosage MSH4 pour les crossovers de voie I et une plus faible efficacité des crossovers de voie II.Dans un second temps, j'ai caractérisé cytologiquement des mutants fancm de colza pour vérifier que l'augmentation des crossovers de voie II ne nuit pas à au bon déroulement de sa méiose. Cette question est restée en suspens, les mutants fancm de colza n’étant pas complètement nuls. Cet écueil m'a incité à développer une approche de TILLING par séquençage pour identifier de nouveaux mutants de recombinaison chez le colza. J'ai alors combiné les mutations fancm et msh4 chez B. rapa pour vérifier si les premières suffisent à corriger les défauts méiotiques induits par les secondes. J'ai montré que, conformément à ce qui avait été observé chez Arabidopsis thaliana, la mutation fancm augmente le nombre de crossovers à un point tel qu’elle restaure la formation de bivalents dans un mutant msh4. La fonction de FANCM est donc conservée chez B. rapa.Mes résultats ont fait progresser la compréhension des voies de formation des crossovers lors d’une méiose allopolyploïde. Ils indiquent que la transmission des chromosomes chez ces espèces implique principalement des crossovers de voie I, et qu’elle pourrait être assurée en limitant l’efficacité de cette voie (e.g. en diminuant le nombre de copies de gènes)
Meiotic recombination ensures, through the formation of crossovers (COs), both faithful chromosome transmission and allelic shuffling over generations; it is at the heart of Mendelian heredity, evolution and plant breeding. Two crossover pathways co-exist in plants. The main pathway (class I) is dependent on MSH4 (and additional proteins). The secondary pathway produces only a few MSH4-independent (class II) crossovers during wild-type meiosis that are limited in number by anti-crossover proteins such as FANCM. These pathways have been extensively described in diploid species, disregarding one of the most pervasive features of crop genomes: polyploidy. This is a major gap in our understanding because the presence of more than two related sets of chromosomes leads both to extra partners for crossover formation and additional copies for all meiotic genes, which make meiotic recombination more intricate. This thesis aims at exploring the interplay between meiotic recombination pathways and polyploidy using mutants for two recombination genes in allotetraploid Brassica napus (AACC; 2n=38) and its diploid progenitor, B. rapa (AA; 2n=20). I have first tested the extent to which class I and class II pathways contribute to inter-homolog and inter-homoeolog (between A and C chromosomes) crossover formation by analyzing how crossovers are affected as the number of functional MSH4 copies decreases. I showed that inter-homolog crossover formation is impaired only when the two MSH4 copies are lost, any other combination of msh4 mutations resulting in wild-type crossover numbers. I also observed that, when class I crossovers are completely abolished in B. napus, the highest frequency of class II crossover ever reported among plant msh4 mutants is observed. I reproduced this result using B. rapa msh4 mutants, thereby demonstrating that increased class II crossover frequencies is not specific to B.napus, but could instead be a general feature of the Brassicaceae. In B. napus allohaploids (AC), where crossovers are forced to occur between homeologs, MSH4 copies no longer complement each other perfectly; counter to the situation in euploids, the number of MSH4-dependent crossovers formed between homoeologs fluctuates with MSH4 dosage in these plants, and approximate zero when all MSH4 copies are depleted. Altogether, my results illustrate two novel specific properties of inter-homeolog crossovers: a greater sensitivity to MSH4 dosage for class I pathway and a lower efficiency for class II.Next, I characterized cytologically B. napus fancm mutants to confirm that boosting class II crossovers would not be detrimental to B. napus meiosis. However, a prudential interpretation of these results is demanded since the B. napus fancm alleles retained residual anti-crossover activity. This has prompted me to set up a TILLING-by-sequencing procedure in order to produce new recombination mutants in B. napus. I also combined the B. rapa fancm and msh4 mutations to test whether the former is sufficient to fix the meiotic defects resulting from the latter. I showed that, similarly to what had been observed in Arabidopsis thaliana, fancm mutation boost COs to such a point that it restores bivalent formation in B. rapa msh4 background. My results therefore confirmed that the function of FANCM is conserved in B. rapa. Overall, the findings and achievements of this thesis make a step forward dissection of CO pathways during allopolyploid meiosis. They indicate that meiotic adaptation to allopolyploidy mainly involve the class I crossover pathway and could be achieved by limiting its efficiency (e.g. by decreasing gene copy number)

The cell cycle is orchestrated by the periodic activation of Cyclin-dependent kinases (CDKs). The enzymatic activity of CDKs depends on their association with cyclins, however, in some cases these kinases can be activated by non-cyclin proteins. RINGO is an atypical CDK activator which regulates the meiotic maturation of Xenopus oocytes and has been recently described as essential for meiotic prophase in mouse. As an activator of CDKs, RINGO has a potential role in cell cycle regulation. CDK regulation by RINGO has been extensively studied in vitro. However, the implication of RINGO in particular cellular processes is poorly understood. Moreover, very little is known about RINGO functions in vivo beyond meiosis. This thesis has addressed the functional relevance of mammalian RINGO proteins in somatic cells, both during homeostasis and in cancer. We have characterized the effects of RingoA knock-down in human cells and found changes in cell cycle progression as well as reduced cell viability. In an attempt to reveal the RingoA interactome, an unbiased proteomic approach was used, which allowed the identification of the cohesin complex and ANKRD11 as new RingoA interactors. Moreover, we describe the expression of endogenous RingoA during the cell cycle of human cells and show that it is present in nuclear speckles. The study of RingoA expression in vivo using a reporter system and gene expression analysis pointed to the brain as the somatic tissue where RingoA is most expressed. We have also analyzed genetically modified mice and found that RingoA and RingoB are not essential for somatic tissue homeostasis. Nevertheless, RingoA is expressed in the sub-ventricular zone of the adult mouse brain and is important for neural stem cell self-renewal ex vivo. Finally, using the Polyoma middle T mammary tumorigenesis model, we showed that RingoA and RingoB are required for tumor growth.
El cicle cel·lular és orquestrat per l’activació periòdica de les quinases dependents de ciclines (CDKs). L’activitat enzimàtica de les CDKs depèn de la seva associació amb ciclines, no obstant hi ha excepcions on aquestes quinases poden ser activades per proteïnes no englobades en la família de les ciclines. RINGO n’és un exemple; aquesta proteïna és un activador atípic de CDKs que regula la maduració meiòtica dels oòcits de Xenopus. A més, recentment també s’ha descrit com a essencial en la profase meiòtica i progrés meiòtic en ratolins. RINGO, com a activador de CDKs, té un rol potencial en la regulació del cicle cel·lular. La regulació d’aquestes quinases per RINGO s’ha estudiat en detall in vitro però poc se sap de la implicació de RINGO en processos cel·lulars. A més no se sap gairebé res de la funció de RINGO in vivo més enllà de la meiosi. En aquesta tesi s’estudia la rellevància funcional de les proteïnes RINGO de mamífers en cèl·lules somàtiques, durant condicions homeostàtiques i càncer. S’han caracteritzat els efectes del knock-down de RingoA en cèl·lules humanes i trobat canvis en la viabilitat i cicle cel·lular d’aquestes. Amb l’objectiu de revelar l’interactoma de RINGO, s’ha utilitzat un cribratge de proteòmica que ha permès la identificació del complex de cohesines i la proteïna ANKRD11 com interactors de RingoA. A més, s’ha descrit l’expressió de RingoA durant el cicle cel·lular de cèl·lules humanes i descobert que està present en nuclear speckles. L’estudi de l’expressió de RingoA utilitzant un sistema reporter i l’anàlisis de l’expressió gènica ha permès la identificació del cervell com el teixit somàtic amb més expressió de RingoA. Mitjançant l’estudi de models de ratolí modificats genèticament s’ha demostrat que RingoA i RingoB no són essencials per la homeòstasi de teixits somàtics. No obstant, RingoA s’expressa en la zona sub-ventricular del cervell adult i és important per la renovació de cèl·lules mare ex vivo. Per últim, utilitzant el model tumoral Polyoma middle T, que permet la generació de tumors mamaris en ratolí, s’ha demostrat que RingoA i RingoB són importants en el creixement tumoral.

Breeding programs have the objectives to develop more productive and more stable varieties. Hybridization and selection are frequently employed in plant breeding and the success of introgression of special traits such as disease resistance relies on genetic recombination between the host and alien chromosomes. During meiosis, homologous chromosomes recognized each other, align and pair which ensure their recombination and correct segregation at metaphase. This process controls aberrant chromosome number within the gametes, and ensures that genes are shuffled by recombination. 70% of flowering plants are polyploids including bread (hexaploid) and pasta wheat (tetraploid), and strict homologous pairing in species containing more than one genome is even more important. Wheat homologous chromosomes and their relative homoeologues are genetically close enough to pair during meiosis, however, the Ph1 locus ensures that only true homologues pair and recombine, stabilizing the wheat genome. Because the pairing is exclusive to homologues, alien chromosomes cannot recombine with wheat chromosome and are eliminated. Deletion lines for the Ph1 locus, allowing recombination of wheat and its relatives, are used for new wheat variety production.

Cohesin mediates sister chromatid cohesion (SCC), and its regulated association with chromatin is required to promote faithful chromosome segregation during mitosis and meiosis, as well as for the efficient repair of DNA double strand breaks (DSBs). In the mitotic cell cycle loading of cohesin requires a conserved complex containing the Scc2INipbl protein, which has also been proposed to promote binding of the cohesin-related complexes condensin and SMC-5/6. However, little is known about the factors that promote loading of cohesin and related SMC (structural maintenance of chromosomes) complexes during meiosis. During a screen for meiotic mutants in C. elegans, I isolated an allele of sec- 2, scc-2 (jql), that has allowed me to determine the roles that SCC-2 plays during meiosis. I show that during C. elegans meiosis loading of cohesin, but not condensin 11 or SMC-5/6, requires SCC-2, demonstrating that loading of condensin 11 and SMC-5/6 can be achieved by mechanisms independent of both SCC-2 and cohesin. The lack of cohesin in scc-2 mutants impairs the repair of meiotic DSBs and recombination intermediates accumulate extensively. Surprisingly, these accumulated intermediates fail to induce an apoptotic response, which is the normal outcome when persistent DNA lesions are detected by the conserved pachytene DNA damage checkpoint. I observed that this defect is caused by a failure to load the DNA damage sensor 9-1 ~ I complex onto persistent recombination intermediates in scc-2 mutants. A lack of meiotic cohesin also impairs the timely loading of the RAD-51 recombinase to irradiation-induced DSBs. These findings suggest that meiotic cohesin is required in the early steps of DSB processing and for the recruitment of checkpoint proteins to sites of DNA damage, thus revealing novel roles for cohesin.

The organisation of microtubule networks into a bipolar spindle is essential for reliable chromosome segregation during cell division. A pair of centrioles surrounded by pericentriolar material (PCM), define the canonical centrosome that acts as the main microtubule organising centre (MTOC) during mitosis. In mammalian meiosis, centrioles are eliminated early on during oogenesis. Despite the absence of centrosomes, a large number of centrosomal proteins are highly expressed in mouse oocytes. Here, I characterise the localisation and function of centrosomal proteins at a previously undescribed meiotic spindle pole domain (MSPD). An initial protein screen identified a group of pericentriolar satellite proteins that localised to a previously undescribed spindle pole domain throughout meiotic maturation in mouse oocytes, including Pericentriolar material 1 protein (PCM1). This domain was distinct from spindle microtubules and the acentrosomal microtubule organising centres (aMTOCs). Initial characterisation focused on PCM1, the main centriolar satellite scaffold protein in somatic cells. Depletion of PCM1 revealed interdependence with the essential aMTOC component, Pericentrin. In the absence of PCM1, aMTOCs could no longer assemble or maintain their structural integrity. PCM1 degradation and disassembly of aMTOCs disrupted spindle assembly and reduced the total amount of nucleated microtubules throughout meiosis. In the absence of the main microtubule nucleating aMTOCs, oocytes relied on the Ran GTPase activity to form a small bipolar spindle. A similar mechanism was previously reported in human oocytes that lack prominent MTOCs. The extended centrosomal protein screen identified additional components of the MSPD. TACC3, under the regulation of Aurora-A at aMTOCs, drive assembly of the MSPD. This domain was absent in MTOC free human oocytes but a second population of TACC3 (identified in mouse oocytes) localised to the meiotic spindle and K-fibres was essential for maintaining spindle pole integrity. Establishing the Lightsheet Z.1 system for live cell imaging of human oocytes enabled us to observe the dynamic distribution of TACC3 in these oocytes. In the absence of prominent MTOCs and the MSPD, human oocytes likely rely on other spindle assembly factors and motor proteins to organise their spindle. Future work to address if the absence of the MSPD could account (in part) for the observed spindle instability in human oocytes is an exciting outlook.

The interval between meiotic nuclear divisions can be regarded as a modified mitotic cell cycle where DNA replication is blocked. Mechanisms regulating this critical aspect of meiosis that allows haploid cells to be generated from a diploid progenitor were investigated in this project. Licensing is restricted after meiosis I due to down-regulation of Cdc18 and Cdt1. Late meiotic expression of Cdc18 and Cdt1, which load the MCM helicase onto replication origins, can lead to partial DNA replication after meiosis I. This implies that block to initiation via licensing forms an important component of this regulation. As detecting any minor DNA re-replication after meiosis I requires a technique more sensitive than flow cytometry for detection of total cell DNA contents, I also investigated a procedure to allow incorporation and detection of 5-ethynyl-2'-deoxyuridine (EdU) in fission yeast. Additional inactivation of Spd1 or stabilization of Dfp1 after MI when Cdc18 and Cdt1 are also expressed does not enhance re-replication, but cyclin-dependent kinase Cdc2 plays a role in preventing re-replication during the MI-MII interval. Unexpectedly, when the licensing block is subverted, replication forks only move a short distance in the interval between meiosis I and II, implying that the elongation step of DNA replication is also inefficient. In addition, I show that the regulation of entry into meiosis II is not delayed by a partial round of DNA replication or DNA damage, indicating that replication and DNA damage checkpoints do not operate in late meiosis.

The meiotic cell cycle is the process by which diploid organisms divide to produce haploid gametes and consists of one round of DNA replication followed by two successive nuclear divisions. In the fission yeast, Schizosaccharomyces pombe, meiosis is initiated from G1 phase and involves a complex series of cellular events that lead to the production of four haploid ascospores. The periodic regulation of gene expression is an important mechanism of control of both mitotic- and meiotic-cell-cycle progression. During the mitotic cell cycle of fission yeast a number of genes, including cdc22+, cdc18+ and cdt1+, are expressed specifically at the G1-S phase boundary. These genes are known to be under the control of MCB (MluI cell-cycle box) upstream-activating-sequence motifs and the MBF (MCB binding factor; also known as DSC1) complex. Here we show that control of gene expression during pre-meiotic G1-S-phase is mediated by an MBF-related transcription-factor complex acting upon similar MCB promoter motifs. Several genes, including rec11+, rec11+, cdc18+, and cdc22+, which contain MCB motifs in their promoter regions, are shown to be coordinately regulated during pre-meiotic S-phase. These genes can be divided into 'mitotic and meiotic' and 'meiotic specific' groups, which contain related but distinct arrangements of MCB motifs. MCB motifs are shown to be physiologically relevant during the meiotic cell cycle and to confer meiotic-specific transcription to a heterologous reporter gene. An MBF-like transcription factor complex that binds to MCB motifs is identified in meiotic cells. The effect of mutating and over-expressing individual components of MBF (cdc10+, res1+, res2+, rep1+ and rep2+) on cdc22+, rec8+ and ree11+ transcription during meiosis was examined. We found that cdc10+, res2+ and rep1+ are required for meiotic transcription, while rest has no role in this process. Surprisingly, manipulation of the mitotic-specific rep2+ gene had an effect on 'meiotic specific' but not 'mitotic and meiotic' MCB-regulated transcription during the meiotic cell cycle. This indicates that Rep2p might have a role in allowing the MBF complex to distinguish between 'mitotic and meiotic' and 'meiotic specific' MCB motifs. This work is the first demonstration in yeast of a role for MCB motifs in control of transcription during meiosis, and identifies a meiotic MBF-like transcription-factor complex.