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Title: The origins of DNA damage in mammalian spermatazoa
Author: King, Sasha Ann
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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The development of in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) techniques has revolutionised treatment for couples with so-called male-factor infertility. However concern over the integrity of the DNA in spermatozoa used for ICSI has been expressed. Poor semen quality and morphologically abnormal spermatozoa are associated with poor embryonic development following IVF. Furthermore, studies have demonstrated that the transmission of defective DNA (e.g. adducts, gene deletions) from the spermatozoa to the developing embryo can occur and that this may lead to developmental failure of the embryo or future health risks for the child. The aims of this project were; a) to develop a Comet assay for the study of DNA integrity of murine spermatozoa, b) to use this assay to assess DNA integrity in spermatozoa from mice with known sub/infertility and to examine the susceptibility of these spermatozoa to heat -induced DNA damage, c) to identify the stages of spermatogenesis in wild type mice which are susceptible to heat-induced DNA damage, and d) to determine whether this damage is present in the mature spermatozoa developed from heat -treated germ cells. The single -cell gel- electrophoresis (Comet) assay was originally developed to study DNA damage in somatic cells and has been modified to study both endogenous and induced DNA damage in human ejaculated spermatozoa. DNA packaging and condensation in mouse spermatozoa is not identical to that in the human. The human spermatozoa Comet assay was therefore not suitable for use on murine spermatozoa. A series of modifications were made to the human spermatozoa Comet assay and a usable assay based on a commercially- available Comet Assay Kit (Trevigen) was developed for the study of murine spermatozoa. The motility, morphology and DNA integrity of motile spermatozoa recovered from the epidiymes of four transgenic lines of mice, all of which suffered from male infertility /subfertility, were examined. Deleted in azoospermia -like autosomal (Dazla)-deficient mice (-/-) are infertile, and males fail to produce any mature spermatozoa; heterozygous (+/-) males are fertile but exhibit reduced numbers of spermatozoa with high incidence of morphological abnormality. Reduced numbers of motile spermatozoa and increased incidence of morphological abnormalities in motile spermatozoa from +l- Dazla mice was observed. Compared to wild type (+/+) mice, the level of DNA damage in the spermatozoa of +/- mice was significantly higher. Levels of DNA damage in both +1+ and +/- were increased following in vitro heat treatment. Similar findings were also observed in mice with excision repair cross - complementation gene 1 (ercc -1) genotypes (+/- and -/-). ERCC1 is involved in the nucleotide excision repair (NER) pathway and mitotic recombination process, and is highly expressed in the testis. Deletion of the Prion protein (PrP) or the PrP- related gene Doppel (PrnD) also resulted in lower numbers of motile spermatozoa with higher levels of DNA damage. However, following in vitro heating, levels of DNA damage in the motile spermatozoa from these mice did not increase. To investigate the effects of heat stress on DNA integrity, wild type mice (Dazia +/+) underwent scrotal heating (42 °C, 30 min) and were then sacrificed at various time points; 1, 2, 4, 6 or 24 hours (h) or 7, 14, 21, 24, 28, and 32 days (d) after heat stress. Testes and epididymides were removed and fixed for histological analysis, and motile epididymal spermatozoa were retrieved. Altered expression of Cirp (a heat -response protein), heat shock protein 105 (HSP105) and Bax (a pro - apoptotic protein), together with increased numbers of TUNEL- positive cells were detected in testes. Expression of Cirp, Bax and the macrophage marker CD68 were also altered in the epididymis. Levels of DNA damage in motile spermatozoa increased significantly within lh of heating, reaching a peak at 4h and then recovering to control levels at 7 and 14d. At 21d after heating, DNA damage increased again reaching a second peak at 28d and failing to recover by 32d. These results indicate that motile spermatozoa in the epididymis are susceptible to heat -induced DNA damage. Furthermore, while DNA integrity of spermatozoa derived from spermatids subjected to heat stress is normal, loss of DNA integrity in pre-meiotic germ cells caused by heat stress is not repaired as these cells mature, resulting in motile spermatozoa with impaired DNA integrity. These findings suggest that it may be necessary for additional criteria to be taken into consideration when selecting spermatozoa for ICSI/IVF treatment.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available