Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish.

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.

The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio.

Meiosis is a cellular program that generates haploid gametes for sexual reproduction. While chromosome events that contribute to reducing ploidy (homologous chromosome pairing, synapsis, and recombination) are well conserved, their execution varies across species and even between sexes of the same species. The telomere bouquet is a conserved feature of meiosis that was first described nearly a century ago, yet its role is still debated. Here we took advantage of the prominent telomere bouquet in zebrafish, Danio rerio, and super-resolution microscopy to show that axis morphogenesis, synapsis, and the formation of double-strand breaks (DSBs) all take place within the immediate vicinity of telomeres. We established a coherent timeline of events and tested the dependence of each event on the formation of Spo11-induced DSBs. First, we found that the axis protein Sycp3 loads adjacent to telomeres and extends inward, suggesting a specific feature common to all telomeres seeds the development of the axis. Second, we found that newly formed axes near telomeres engage in presynaptic co-alignment by a mechanism that depends on DSBs, even when stable juxtaposition of homologous chromosomes at interstitial regions is not yet evident. Third, we were surprised to discover that ~30% of telomeres in early prophase I engage in associations between two or more chromosome ends and these interactions decrease in later stages. Finally, while pairing and synapsis were disrupted in both spo11 males and females, their reproductive phenotypes were starkly different; spo11 mutant males failed to produce sperm while females produced offspring with severe developmental defects. Our results support zebrafish as an important vertebrate model for meiosis with implications for differences in fertility and genetically derived birth defects in males and females.

Moving fast and breaking things: Incidence and repair of DNA damage within ribosomal DNA repeats.

The genes that code for ribosomal RNA are present in hundreds of tandemly arrayed copies in the human genome. Ribosomal DNA repeats transcribe vast amounts of ribosomal RNA in order to meet the cell's relentless demand for ribosome production. Intrinsic features of ribosomal DNA repeats render them uniquely vulnerable to DNA damage. Sensing and repairing damage to ribosomal DNA involves dramatic spatial reorganization of the nucleolus, the phase-separated nuclear subdomain where ribosomes are made. We highlight recent advances in detecting the incidence of DNA damage and defining the mechanisms of DNA repair on these essential genes.

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.