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.
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/14386 |
| Date | January 2015 |
| Creators | Habas, Khaled S.A. |
| Contributors | Brinkworth, Martin H., Anderson, Diana |
| Publisher | University of Bradford, Faculty of Life Sciences |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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