Aspects of mitochondrial DNA mutations in relation to human male fertilising potential
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Fertilisation is a chance event dependent on, amongst other things, the number, motility, and physical characteristics of sperm. There is a strong link: between ATP production and sperm motility. The mitochondrially encoded OXPHOS ATP production pathway provides ATP for sperm motility. Thirteen polypeptides involved in OXPHOS are encoded in mtDNA. Therefore, the aim of this study was to assess the influence of point mutations and small deletions in mtDNA on human male fertilising potential. Two hundred and fifty two semen samples were screened for mutations in the ATPase 6 and 8, and COII genes using polymerase chain reaction (PCR), followed by single-strand conformation polymorphism (SSCP) analysis of restriction digests of two overlapping amplicons. ATPase 6 and 8 gene analysis of 210 semen samples identified three singly occurring known homoplasmic polymorphisms (G → A 8860, G → A 8856, and G → A 8839); one polymorphism found in 13 samples that created a known HaeII RFLP; and a novel heteroplasmic point mutation (T → C 8821) found in the immature sperm from two semen samples from an oligo-astheno-teratozoospermic individual. Based on amino acid homologies and protein structure this new heteroplasmic mutation is predicted to be potentially pathogenic. COII gene analysis of 223 semen samples identified two previously described, singly occurring homoplasmic polymorphisms (C → T 7476, and G → A 7853); three novel singly occurring homoplasmic polymorphisms (T → C 8077, G → A 7789, and G → A 7754); two known homoplasmic polymorphisms found in multiple samples (9 bp deletion, 7 samples; G → A 8251, 11 samples) and one novel heteroplasmic homopolymeric tract insertion in the COII/tRNALys intergenic spacer. Of the multiply occurring variants found in these genes only a 9 bp deletion from the COII/RNALys intergenic spacer of 7 semen samples could be associated with asthenozoospermia. This association was negated when disease and cultural factors were considered. In addition to the characterisation of true heteroplasmy, one case of pseudogenecreated heteroplasmy was characterised in the COII amplicon from one semen sample. During the analysis of this known pseudogene-created heteroplasmy, two other novel pseudogenes paralogous to the mitochondrial ND4/ND5 genes and the HV1 region of the D-loop were isolated. All three pseudogenes were characterised and the times of pseudogene insertion were calculated. The COII and ND4/ND5 pseudogenes are predicted to have inserted into nuclear DNA since the time of the last common ancestor of humans and chimpanzees, whilst the HV1 pseudogene is predicted to have an ancient origin. These pseudogenes may act as useful markers for molecular evolution.