Sexual reproduction requires meiosis, a specialized cell division program that halves the chromosome number of germ cells in order to generate gametes. Chromosome number reduction is achieved by two successive rounds of cell divisions after a single round of DNA replication. In meiosis I, homologous chromosomes (homologs) recombine to produce at least one reciprocal DNA exchange, called crossover (CO), in each chromosome. COs ensure proper segregation, as the resulting tetrad chromosomes are bisected during the first division to form dyads. In meiosis II, dyads split allowing segregation of single chromatids, resembling mitosis. During prophase I, the earliest stage of meiosis I, each pair of sister chromatids is arranged in series of loops tethered longitudinally by a structure called the chromosome axis, which serves as a scaffold for the machinery that promotes the formation of programmed DNA double-strand breaks (DSBs). Recombination is initiated when the DSBs are resected to produce single-stranded DNA (ssDNA) overhangs that seek out their homologs, promoting pairing. Then the synaptonemal complex (SC) forms, physically linking homolog axes through transverse filaments. In the context of the SC, meiotic recombination repairs the DSBs and turns a small fraction of them into COs. DSB formation will continue on unsynapsed axes and it is only terminated by the complete synapsis of all homolog pairs in pachytene stage. Meiocytes that fail to complete recombination and/or synapsis are eliminated (spermatocytes by mid-pachytene; oocytes from late prophase I, but at or before follicle formation), as these defects can cause chromosomal abnormalities that will be passed down to the offspring, causing severe diseases or death. Our group and others suggest that there are two simultaneous pachytene checkpoints, one that is activated by persistent DSBs and the other by asynapsis. This issue has proven very difficult to approach, since defective DSB formation/repair will inevitably affect synapsis by impairing homology search and strand invasion. The meiosis-specific HORMAD2 protein binds preferentially to unsynapsed axes, where HORMAD2 promotes the recruitment of ATR kinase. The ATR-dependent accumulation of γH2AX (histone H2AX phosphorylated on Ser139) is thought to generate a checkpoint signal from unsynapsed chromosomal regions as part of the synapsis checkpoint. In the case of spermatocytes ATR activity is concentrated to the non-homologous regions of sex chromosomes, which remain unsynapsed. This leads to γH2AX accumulation and subsequent transcriptional silencing of the sex chromosomes. This mechanism is called meiotic sex chromosome inactivation (MSCI) and is essential for the survival of spermatocytes, since the expression of sex chromosome-linked genes is toxic to spermatocytes beyond mid pachytene. Despite the importance of HORMAD2, the mechanism for its interaction with the unsynapsed chromosomes was unknown. Furthermore, it remained untested if HORMAD2 binding to the axis is actually required for the synapsis checkpoint. To address whether HORMAD2 binding to the axis is required for its function, I generated a mouse strain that expresses an altered version of the constitutive chromosome axis component SYCP2. The mutant SYCP2Δex16 lacks a short peptide sequence, called closure motif, which is predicted to bind HORMAD2. Immunofluorescence (IF) in spermatocytes showed that the localization of HORMAD2 to the chromosome axis is lost in Sycp2Δex16/Δex16, while axis formation is not impaired. Consistently, immunoblotting showed that HORMAD2 was depleted from insoluble fractions (chromatin-rich) of Sycp2Δex16/Δex16 testis extracts, while present in the soluble and total fractions. These results confirmed that the closure motif of SYCP2 serves as anchor for HORMAD2 on meiotic chromosome axis. Sycp2Δex16/Δex16 males are infertile. IF staining of cryosections of testes showed a complete loss of spermatocytes beyond pachytene. The number of cells undergoing apoptosis increased relative to the wild type, most of them in stage IV of the epithelium cycle, which corresponds to mid-pachytene. Thus, the onset of arrest coincides with the activation of the checkpoint that prevents asynaptic/DSB repair defective meiocytes from progressing beyond mid-pachytene. Nonetheless, global SC formation, and patterns of DSB formation and recombination foci in Sycp2Δex16/Δex16 are similar to the wild type. By early pachytene, the last stage reached by Sycp2Δex16/Δex16 spermatocytes, most cells have completed synapsis and DSBs are repaired. Only a small percentage of pachytene cells show a degree of incomplete synapsis. Although this suggests a synapsis-enhancing role of HORMAD2, it is thought to be concomitant to the MSCI failure observed in this mutant. In Sycp2Δex16/Δex16, 83.7% of pachytene spermatocytes have an abnormal distribution of γH2AX on sex chromosomes, accompanied by impaired accumulation of ATR and BRCA1 (another MSCI-promoting protein). The phenotype of Sycp2Δex16/Δex16 suggests a misregulation of pachytene checkpoint functions due to loss of HORMAD2-dependent ATR signaling from unsynapsed axes, and I propose that this misregulation accounts for the meiotic arrest phenotype. Females do not require MSCI, as their XX chromosomes can synapse. Accordingly, Sycp2Δex16/Δex16 females are fertile. Nonetheless, previous reports indicate that HORMAD2 has a role in sensing asynapsis, but not persistent DSB, as part of a checkpoint for quality control in oocytes. As expected, Sycp2Δex16/Δex16 cannot rescue oocytes in a DSB repair and synapsis defective background. Sycp2Δex16/Δex16 females have higher oocyte numbers than wild type. This could suggest that oocytes with defective synapsis that are usually culled in wild type ovaries, are not eliminated in Sycp2Δex16/Δex16, supporting HORMAD2 role in signaling asynapsis. Further testing in a synapsis defective background is underway. Sycp2Δex16/Δex16 seems to phenocopy Hormad2-/-, supporting a model where HORMAD2 binding to the unsynapsed axis is required for enabling its checkpoint-activating functions.:List of figures I List of tables II List of abbreviations III Acknowledgments V 1. Introduction 1 1.1. Meiosis and gametogenesis in mouse 1 1.2. Prophase of the first meiotic division 3 1.2.1. Meiotic recombination 4 1.2.2. The chromosome axis and the synaptonemal complex 5 1.2.3. The interplay between DSBs and synaptonemal complex formation 8 1.3. Surveillance mechanisms in prophase I 9 1.3.1. HORMAD1 and HORMAD2 in the male pachytene checkpoint 11 1.3.2. HORMAD1 and HORMAD2 in the female prophase checkpoint 12 1.4. HORMAD1/2 interaction with the chromosome axis 14 1.5. Aim of the project 15 2. Materials and methods 16 2.1. Generation of a mouse model lacking the closure motif 16 2.1.1. Generation of a mutant using CRISPR-Cas9 system 16 2.1.2. Identification of mutant candidates by PCR and HMA 17 2.1.3. Genomic DNA sequencing for the selection of founders 19 2.1.4. cDNA sequencing 20 2.2. Mice 21 2.2.1 Mice from a homogenized genetic background 21 2.3. Methods for the fixation of tissues 22 2.3.1. Nuclear surface spread spermatocytes 22 2.3.2. No-spin sucrose spreads 23 2.3.3. Testis fixation and cryosectioning 23 2.3.4. Ovary fixation and cryosectioning 23 2.3.4. Fixed Wild Type Cells (FWTC) 24 2.4 Staging 24 2.4.1. Staging of spermatocytes from nuclear surface spreads 24 2.4.2. Staging of epithelial cycles in cross-sections of seminiferous tubules 25 2.4.3. Staging of oocytes 26 2.5. Protein extraction 27 2.5.1. Total protein extraction and western blotting 27 2.5.2. Fractionation assay and western blotting 27 2.6. Immunofluorescence microscopy 29 2.6.1. Staining conditions 29 2.6.2. Imaging 30 3. Results 31 3.1. HORMAD2 recruitment to the axis requires the closure motif of SYCP2 31 3.1.1. Generation of a mouse mutant lacking the closure motif 31 3.1.2. Axial HORMAD2 localization is lost in Sycp2Δex16/Δex16 spermatocytes 34 3.1.3. HORMAD2 is present in Sycp2Δex16/Δex16 spermatocytes but greatly decreased from the chromatin-rich fraction 36 3.2. HORMAD1 localization to the axis seems to be independent of the closure motif of SYCP2 37 3.3. Loss of HORMAD2 localization to the axis causes infertility in males 38 3.3.1. Low levels of H1t signal indicate that Sycp2Δex16/Δex16 spermatocytes do not progress beyond mid-pachytene 39 3.3.2. Defect in spermatogenesis of Sycp2Δex16/Δex16 is caused by mid-pachytene arrest 41 3.4. Axis formation is not impaired in Sycp2Δex16/Δex16 spermatocytes 44 3.5. HORMAD2 localization to the axis plays a minor role in SC formation 45 3.5.1. In Sycp2Δex16/Δex16 spermatocytes, autosomal SC formation does not seem impaired, but abnormal XY synapsis is observed 45 3.5.2. IHO1 is properly removed from axes in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 48 3.5.3. Synapsis at early pachytene is mildly defective in Sycp2Δex16/Δex16 and Hormad2-/- spermatocytes 50 3.6. HORMAD2 localization to the axis is not essential for DSB repair 52 3.7. Loss of HORMAD2-dependent ATR signaling from unsynapsed axes causes a misregulation of pachytene checkpoint 55 3.7.1. Abnormal sex body formation in Sycp2Δex16/Δex16 is an indicator of defective ATR signaling on unsynapsed sex chromosomes 55 3.7.2. ATR localization is affected by the loss of HORMAD2 localization to the axes 58 3.8. HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 60 3.8.1. HORMAD2 is not essential for fertility in females 60 3.8.3. HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 60 4. Discussion 63 4.1. The closure motif in SYCP2 is the binding site of HORMAD2 to the unsynapsed axes 63 4.1.1 The role of HORMAD1 in the recruitment of HORMAD2 to the chromosome axis 64 4.2. Loss of HORMAD2 localization to the axes causes mid-pachytene arrest 65 4.2.1 Persistent DSBs and/or asynapsis are unlikely to trigger the mid-pachytene arrest in Sycp2Δex16/Δex16 spermatocytes 66 4.3. Loss of HORMAD2 from the unsynapsed axes impairs the recruitment of ATR activity on sex chromosomes, triggering the elimination by the mid-pachytene checkpoint 67 4.3.1 Increased asynapsis of sex chromosomes is most likely an effect of defective ATR signaling 68 4.3.2 ATR-dependent silencing of sex chromosomes is required for survival of spermatocytes beyond pachytene 69 4.4. HORMAD2 localization to the axis has a limited role in the prophase checkpoint in females 69 4.4.1 HORMAD2 seems to be required for a checkpoint mechanism in females that is activated by persistent asynapsis 70 4.4.2 HORMAD2 localization to the axis is not required for the elimination of oocytes with persistent DSB 70 4.4.3 HORMAD2 might play a role in the checkpoint that monitors unrepaired DSBs during female prophase 71 4.5. Final remarks 72 5. Summary 73 5. Zusammenfassung 75 References 78