The origins and consequences of DNA damage in the male germ line
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Infertility affects ~20% of couples in Europe and in 50% of cases the problem lies with the male. The development of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intra-cytoplasmic spermatozoa injection (ICSI) has allowed some couples to overcome male-factor infertility. However concerns remain over the increasing use of ART as elevated levels of DNA damage in sperm from infertile men have been reported and a link between DNA damage in sperm and early embryonic failure has been demonstrated. DNA damage in sperm, caused by oxidative stress may also be passed on from father to child resulting in an increased incidence of childhood cancer. This has led to fears that the use of damaged sperm in ART could contribute to early embryonic failure and/or birth defects. The studies described in this thesis used mouse models to investigate the relationship between DNA integrity in male germ cells and male fertility. This was achieved by studying both the effects of targeted ablation of genes involved in DNA repair and the impact of scrotal heat stress on testicular function and sperm DNA integrity. Three lines of transgenic mice with deletions in genes involved in genomic integrity (Ercc1, Msh2 and p53) were studied. All three genes are expressed in the testis. These studies confirmed and extended studies on Ercc1 knockout (-/-) mice showing reduced germ cell complement, increased apopotosis, an increased percentage of damaged sperm and demonstrated for the first time that depletion of Ercc1 results in an increased incidence of unrepaired double strand DNA breaks (DSB) in pachytene spermatocytes. The persistence of DSBs in spermatocytes and abnormal sperm chromatin structure confirmed that the repair functions of Ercc1 are essential for normal germ cell maturation. In the p53-/- mice these studies showed for the first time that there was an increase in DSBs in spermatocytes and an increase in numbers of sperm with damaged DNA. The level of apoptosis was also increased in the testes suggesting that caspase-3 mediated apoptosis is not entirely p53 dependent as been previously suggested. These studies demonstrated for the first time that targeted ablation of Msh2 compromises germ cell complement and as in the Ercc1-/- this resulted in gaps in the seminiferous epithelium consistent with clonal loss of germ cells. Consistent with a role for MSH2 in mismatch repair no DSBs were detected in spermatocytes from Msh2-/-. Testicular function is temperature dependant and due to their location in the scrotum testes are normally kept between 2ºC and 8ºC below core body temperature. In mice transient scrotal heat stress (30 minutes at 38°C, 40°C and 42°C) disrupted testicular function. Analysis of sperm and testis parameters revealed that stress at 38°C was sufficient to have subtle effects on epididymal function but the higher temperatures had additional consequences for testicular function which resulted in DNA damage in spermatocytes, germ cells loss and increased apoptosis. Further studies into the pathways of apoptosis demonstrated that the mitochondrial/intrinsic pathway plays a role in heat stress response. The fertility of males was altered in those heated to 42°C resulting in reduced pregnancy rate and litter size. Given that the paternal genome is reported to be required for the development of extraembryonic tissues and this will influence growth of the embryo, it was interesting to note an increase in resorption sites in pregnancies using 40°C males. IVF was used to demonstrate that embryos formed using sperm from males stressed at 42°C were compromised between the 4-cell and blastocyst stage suggesting that though sperm with DNA damage are still capable of fertilisation, the paternal DNA was introducing genomic instability to the embryo and having fatal effects on development. These studies have also shown that one possible underlying cause of the disturbance in testicular function is hypoxia, as a marked increase in Hif1 alpha (a marker of hypoxia) mRNA and relocalisation of the protein was observed in the testis. In conclusion, DNA damage in the male germ line caused either by induced stress, or by targeted ablation of DNA repair genes, can disrupt testicular architecture, function and therefore the fertility of mice. These data have demonstrated that deletion of Ercc1, Msh2 and p53 can have differential but overlapping affects on germ cell function and sperm production and that increased scrotal temperature can cause subfertility in male mice. This study has provided further confirmation of possible male-mediated effects on embryo survival and these findings should be taken into consideration when using sperm from infertile men in IVF/ICSI treatments where the normal quality control processes involved in fertilisation are bypassed.