Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

In view of previous reports associating mitochondrial DNA deletions with male reproductive disorders, levels of the 'common' 4977 bp mitochondrial DNA deletion were evaluated semi-quantitatively in 64 men, without prior knowledge of the clinical diagnosis. Significant levels of deletions were detected in 34/64 men (53%) but 29 of these (45%) had a normal semen profile and were phenotypically normal. No deletions were detected in 30 men, of whom 21 were normospermic, six were oligozoospermic and three were azoospermic. It is concluded that although mitochondrial DNA deletions within the testis may be associated with primary testicular disease, no correlation with semen quality was evident in this study, thus limiting its potential use as a diagnostic test.

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase gamma ( POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)(10) allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee ( Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.

The "common" 4977 bp deletion in mitochondrial DNA (Delta4977) is commonly used as an indicator of tissue deterioration in ageing and bioenergetic diseases. Deletion levels are normally measured by a serial dilution polymerase chain reaction (PCR) approach, where test reactions are compared with dilutions of control amplifications of DNA from a similar sized stable region of the mitochondrial genome. The end-point of this assay is the dilution that can just detect any PCR product; however, this is an inherently unstable measure. We constructed a chimaeric DNA construct that binds to both control and deletion primers with similar annealing properties. This was used in a competitive PCR assay to quantify Delta4977 in human testicular tissues that had been well-characterized using the serial dilution approach. We found the competitive assay to be highly replicable as it compares the PCR product of the construct with that of test DNA samples during the linear growth phase of the PCR reaction. Moreover, the serial dilution assay was shown to significantly overestimate the amounts of deleted mitochondrial DNA present. The assay promises to throw new light on the role of mitochondrial DNA deletions in tissue dysfunction and ageing, as such deletions can now be determined with high accuracy and repeatability and is much cheaper to apply than real-time fluorescent quantitative PCR.