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Title: Dissecting the meiotic defects of Tex19.1-/- mouse spermatocytes
Author: Crichton, James Hugh
ISNI:       0000 0004 6062 7753
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity.
Supervisor: Adams, Ian Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: meiosis ; mouse ; recombination ; retrotransposon ; chromosome synapsis ; spermatocyte ; fertility