ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en SURVEILLANCE MECHANISMS IN MAMMALIAN MEIOSIS Thesis submitted by Marina Marcet Ortega To opt for the title of Doctor in Cellular Biology Doctoral thesis directed by Dr. Ignasi Roig Navarro Departament de Biologia Cel·lular, Fisiologia i Immunologia Institut de Biomedicina i Biotecnologia (IBB) Universitat Autònoma de Barcelona Director PhD candidate Dr. Ignasi Roig Navarro Marina Marcet Ortega Bellaterra (Cerdanyola del Vallès), 2016 This work received financial support from: . Ministerio de Ciencia e innovación grant (BFU2010-18965) . FPI program fellowship (BES-2011-045381) . Estancias breves FPI (EEBB-I-13-06647, EEBB-I-14-08517, EEBB-I-15-09556) A la meva família, INDEX ABSTRACT ACCRONYMS AND ABBREVIATIONS 1. INTRODUCTION ....................................................................................................................................... 13 1.1 Gametogenesis .................................................................................................................................. 13 1.1.1 Oogenesis .................................................................................................................................. 14 1.1.2 Spermatogenesis .................................................................................................................... 15 1.1.3 Meiosis ........................................................................................................................................ 16 1.1.4 Meiotic recombination ......................................................................................................... 18 1.2 Control of meiotic recombination............................................................................................. 20 1.2.1 Homologous chromosome synapsis .............................................................................. 21 1.2.2 Checkpoint mechanisms control meiotic progression ........................................... 25 1.3 Pachytene checkpoint 2 protein / TRIP13 ............................................................................ 29 1.3.1 Mammalian TRIP13 .............................................................................................................. 31 1.4 DNA damage response in somatic cells .................................................................................. 33 1.4.2 P53 family members ............................................................................................................. 36 2. OBJECTIVES ................................................................................................................................................ 47 3. MATERIALS AND METHODS .............................................................................................................. 53 3.1 BIOLOGICAL MATERIAL ............................................................................................................... 53 3.1.1 Experimental mutant mice ................................................................................................ 53 3.2 MOLECULAR BIOLOGY .................................................................................................................. 53 3.2.1 Genomic DNA extraction ..................................................................................................... 53 3.2.2 Genotyping................................................................................................................................ 54 1 3.2.3 RNA extraction ........................................................................................................................ 59 3.2.4 RNA sequencing ...................................................................................................................... 60 3.2.5 Real-time PCR .......................................................................................................................... 60 3.2.6 Protein extraction and quantification ........................................................................... 62 3.2.7 Western Blot ............................................................................................................................ 63 3.3 HISTOLOGY TECHNIQUES ........................................................................................................... 65 3.3.1 Fixation, embedding and sectioning .............................................................................. 65 3.3.2 PAS-Hematoxylin staining .................................................................................................. 67 3.3.3 TUNEL test: In situ cell death detection over tissue sections .............................. 67 3.4 CELL BIOLOGY AND CYTOLOGY TECHNIQUES .................................................................. 68 3.4.1 Spermatocyte nuclei spreads from fresh testis ......................................................... 68 3.4.2 Spermatocytes nuclei spreads from frozen testis .................................................... 69 3.4.3 Spermatocytes squashed preparation .......................................................................... 70 3.4.4 Immunofluorescence ............................................................................................................ 70 3.4.5 TUNEL staining over IF-stained slides .......................................................................... 72 3.4.6 RNA FISH and Immunofluorescence on spermatocytes ........................................ 73 3.4.7 EU Imaging on spermatocytes .......................................................................................... 78 3.4.8 Cell Sorting................................................................................................................................ 81 4. RESULTS ....................................................................................................................................................... 89 4.1 Study the pachytene arrest in male mammalian meiosis ............................................... 89 4.1.1 Determine other members of the signaling pathway that activates the pachytene arrest in mouse spermatocytes ..................................................................................... 89 4.1.2 Evaluate the possibility of separating prophase stages through cell sorting technique ...................................................................................................................................................... 91 2 4.1.3 Analysis of p53 role in an unperturbed mouse meiosis and possible implications in the pachytene arrest ................................................................................................ 93 4.2 Study p53 family participation in the pachytene recombination-dependent arrest mechanism ..................................................................................................................................................... 100 4.2.1 Evaluate p53 family members role in the activation of the Recombination- dependent arrest in mouse spermatocytes ................................................................................. 100 4.2.2 Trip13 double mutants still present stage IV arrest ............................................. 104 4.2.3 TRIP13 involvement in the formation of the sex body ....................................... 107 4.2.4 p53 and TAp63 do not activate the sex body-deficient arrest ......................... 112 4.3 Analysis of TRIP13 role in meiotic transcription ............................................................ 114 4.3.1 Transcription levels in Trip13 mutants ..................................................................... 114 4.3.2 Specific RNA expression analysis in Trip13 mutants: RNA sequencing ....... 119 5. DISCUSSION ............................................................................................................................................. 131 5.1 Study the components that activate the pachytene arrest occurring in mammalian spermatocytes ..................................................................................................................... 131 5.2 P53 role in meiosis ....................................................................................................................... 132 5.3 P53 family members p53 and TAp63 participate in the recombination-dependent arrest …………………………………………………………………………………………………………………135 5.4 TRIP13’s new role in meiotic silencing ............................................................................... 141 5.4.1 TRIP13 is required to implement the meiotic sex chromosomes inactivation ……………………………………………………………………………………………………………141 5.4.2 TRIP13 mediates transcriptional silencing of the unsynapsed regions at early meiotic stages ............................................................................................................................... 142 6. CONCLUSIONS .......................................................................................................................................