In vitro studies on genotoxicity and gene expression in spermatogenic cells: mechanisms and assay development
Year: 2015
Type: dissertation
Abstract: Spermatogenesis is a complex process of male germ cell development from \ndiploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital \nrole in limiting cell numbers and eliminating defective germ cells. This requires \nnovel gene products, and precise and well-coordinated programmes of gene \nexpression. It is therefore possible that a disruption of transcription factor \nfunction would significantly impact germ cell development. \n \nThe present work was undertaken to use Staput separation followed by culture \nof purified germ cells of rodent testis since mammalian spermatogenesis cannot \nyet be recreated in vitro. Specificity of separation was assessed using \nimmunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The \ngenotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, \n6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate \nand ethyl methanesulphonate were investigated. \n \nCells were cultured and treated with different concentrations for each agent. \nDNA damage and apoptosis were measured by Comet and TUNEL assay \nrespectively. Up-regulation of expression of the transcription factors Tbpl1, \nFHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- \nPCR and qPCR. Protein production was evaluated using Western blotting. \n \nTbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression \nvector pET/100-TOPO. The recombinant clones were induced and successful \nexpression of the proteins in E. coli was confirmed by SDS-PAGE and Western \nblotting. The recombinant clones obtained were used to demonstrate genotoxin \ninduced impairment of gene expression. \n \nThus, Staput-isolated rodent testicular germ cells seem to be a suitable model \nto study genotoxicity in vitro yielding result comparable to those reported in vivo. \nFurthermore, the work shows that genotoxins can impair gene expression.
Author: Khaled Habas
Language: English
Subfield: Reproductive Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2782043545