Mutation of the Mouse Syce1 Gene Disrupts Synapsis and Suggests a Link between Synaptonemal Complex Structural Components and DNA Repair

In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination

Genome-Wide Control of the Distribution of Meiotic Recombination

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects

Les cellules souches embryonnaires de souris (un modèle de cardiogenèse physiopathologique)

Le muscle cardiaque est constitué de cellules contractiles, ou cardiomyocytes, majoritairement composées de myofibrilles. De nombreuses mutations affectant les protéines myofibrillaires sont responsables de cardiomyopathies congénitales. Jusqu'alors le processus de différenciation des cardiomyocytes en présence d'une telle mutation était mal connu. Dans la première partie de ce travail de thèse, nous avons établi un modèle in vitro de cellules souches embryonnaires de souris génétiquement modifiées reproduisant les premières étapes de la différenciation des cardiomyocytes en présence d'une mutation myofibrillaire. Dans les cellules mutées nous avons mis en évidence un défaut sévère dans la formation des myofibrilles et dans la gestion du calcium intracellulaire. La répartition du calcium intracellulaire en microdomaines finement régulés semble avoir un rôle primordial pour le bon déroulement de la myofibrillogenèse. Dans la deuxième partie de ce travail de thèse nous nous sommes intéressés à l'origine des battements cardiaques dans le cœur embryonnaire. En effet, comme le cœur adulte, le cœur embryonnaire se contracte en réponse à des oscillations calciques. Cependant, dans ces derniers, ces oscillations sont dues à l'activation d'une cascade intracellulaire impliquant l'IP3. Dans ce travail nous avons pu mettre en évidence l'implication de la voie FGF dans la génération des oscillations calciques induites par l'IP3. Ces résultats ont été obtenus par des approches pharmacologiques et génétiques sur des cœurs embryonnaires de souris en comparaison avec le modèle de différenciation in vitro. Notre modèle in vitro de cellules souches génétiquement modifiées est un excellent outil pour disséquer les processus physiopathologiques au cours des étapes précoces de la cardiogenèse.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

PRDM9, a driver of the genetic map.

During meiosis, maternal and paternal chromosomes undergo exchanges by homologous recombination. This is essential for fertility and contributes to genome evolution. In many eukaryotes, sites of meiotic recombination, also called hotspots, are regions of accessible chromatin, but in many vertebrates, their location follows a distinct pattern and is specified by PR domain-containing protein 9 (PRDM9). The specification of meiotic recombination hotspots is achieved by the different activities of PRDM9: DNA binding, histone methyltransferase, and interaction with other proteins. Remarkably, PRDM9 activity leads to the erosion of its own binding sites and the rapid evolution of its DNA-binding domain. PRDM9 may also contribute to reproductive isolation, as it is involved in hybrid sterility potentially due to a reduction of its activity in specific heterozygous contexts

Déterminants de la carte génétique

Les événements d’échanges réciproques entre chromosomes, ou crossing-over, qui se produisent lors de la méiose et qui définissent la carte génétique ne sont pas distribués de manière aléatoire dans le génome. Des données récentes chez les levures et chez les mammifères révèlent différents mécanismes impliqués dans cette distribution et mettant en jeu des modifications post-traductionnelles d’histones en des sites précis du génome, appelés points chauds de recombinaison. Ces sites sont certains promoteurs de transcription chez S. cerevisiae ou des sites de fixation de facteurs de transcription chez S. pombe, où des activités de modification de la chromatine sont recrutées. Chez les mammifères, la majorité des points chauds correspondent à des régions qui contiennent des séquences d’ADN reconnues par la protéine PRDM9 (PR domain zinc finger protein 9) qui possède une activité de triméthylation sur la lysine 4 de l’histone H3. Cette modification, ou d’autres qui lui sont associées, permettent de manière directe ou indirecte le recrutement des protéines impliquées dans la formation des cassures double brin qui déclenchent la recombinaison méiotique