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    In vitro studies on genotoxicity and gene expression in spermatogenic cells: mechanisms and assay development

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    PhD Thesis (8.210Mb)
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    Publication date
    2015
    Author
    Habas, Khaled S.A.
    Supervisor
    Brinkworth, Martin H.
    Anderson, Diana
    Keyword
    Spermatogenesis; In vitro; Doxorubicin; Hydrogen peroxide; Rodent; Staput; TUNEL assay; Comet assay; Cloning; Gene expression
    Rights
    Creative Commons License
    The University of Bradford theses are licenced under a Creative Commons Licence.
    Institution
    University of Bradford
    Department
    Faculty of Life Sciences
    Awarded
    2015
    
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    Abstract
    Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.
    URI
    http://hdl.handle.net/10454/14386
    Type
    Thesis
    Qualification name
    PhD
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