Noninvasive recovery and detection of possum Trichosurus vulpecula DNA from bitten bait interference devices (WaxTags).

The brushtail possum is a major agricultural and ecological pest in New Zealand. A novel noninvasive DNA sampling tool for detecting its presence (WaxTags, or WT) was tested. DNA was recovered from saliva left on WT, and two lengths (407 bp and 648 bp) of the cytochrome c oxidase I (COI) barcoding region were amplified by polymerase chain reaction (PCR). PCR products were considered (+) when a DNA band was clearly visible by electrophoresis. Different factors that might affect PCR (+) were investigated with captive possums: (i) both extraction protocols of the QIAGEN DNeasy Blood and Tissue Kit, (ii) effect of an overnight or longer delay of up to 3 weeks before DNA extraction on both COI amplicons, and (iii) effect of the individual, order and magnitude of the bite. Extraction protocols were not significantly different. The effect of the overnight delay was not significant, and amplification of the short amplicon was significantly higher (100%) than for the long fragment (48%). After a two or 3-week delay, the short amplicon had 94% and 56% PCR (+), success rates, respectively. Individual, order and magnitude of a bite had no significant effect. The delay trial was repeated with WT from the wild, for which PCR (+) rate of the short amplicon was 63%, regardless of freshness. Four microsatellites were amplified from captive WT samples. We conclude that DNA from saliva traces can be recovered from WT, a potential new tool for noninvasive monitoring of possums and other wildlife.

Molecular analysis of polymerase gamma gene and mitochondrial polymorphism in fertile and subfertile men.

CAG trinucleotide repeat length in the nuclear polymerase gamma gene (POLgamma) has been shown to be associated with men with reduced fertility. The present study investigated the frequency of CAG repeat length genotypes and three exonuclease motifs of the POLgamma in relation to the frequency of mitochondrial nucleotide substitutions. DNA from semen samples of 93 normozoospermic men and 192 non-normozoospermic men was isolated and the specific regions of the genes were amplified by polymerase chain reactions (PCR) and sequenced to identify mutations. The genotypic frequencies of pooled POLgamma CAG repeat lengths, =10/ not equal 10 heterozygotes and not equal 10/ not equal 10 homozygotes, were significantly different between normozoospermic and non-normozoospermic men (p < 0.05), with non-normozoospermic men having a slightly higher frequency of the =10/=10 genotypes. The allelic frequency for =10 is 0.79 and not equal10 is 0.21 for normozoospermic men and 0.85 and 0.15, respectively, for non-normozoospermic men (p < 0.025). There was no mutation detected in the exonuclease motifs in all the samples tested. Eighty normozoospermic and 124 non-normozoospermic semen samples were analysed for nucleotide substitutions in mitochondrial genes by PCR and sequencing. Heteroplasmic mutations were found in one azoospermic man, four asthenozoospermic men and two normozoospermic men. Only one asthenozoospermic man was heterozygous for the POLgamma genotype. Of the 17 men with non-synonymous nucleotide substitutions, 14 were homozygous for the POLgamma genotype. Non-normozoospermic men had twice as many nucleotide substitutions than normozoospermic men. However, there were no significant differences in the frequencies of nucleotide substitution and POLgamma genotypes in the two groups of men.

High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men.

Single nucleotide polymorphisms (SNPs) in 7000 bp of the mitochondrial genome, encompassing 15 coding regions from COI to ND5, were characterized by single strand polymorphism analysis and confirmed by DNA sequencing. About 2.4% of normozoospermic men and 8.4% of men with poor semen quality had at least one nucleotide substitution. Most of the substitutions occurred in the third codon and did not change the amino acid. Hydrophobicity plots of the proteins with changes in an amino acid as a result of a nucleotide substitution suggested that they did not affect the function of the protein. The two most common substitutions at nucleotide (nt) 9055 and 11719 had significantly higher frequencies in men with reduced sperm motility. Eleven percent of the men with poor semen parameters and 1.3% of normozoospermic men had a 9055 substitution, 12% of the men with poor semen parameters had a substitution at nt 11719, but none of the normozoospermic men had this substitution. All the patients with these substitutions had reduced sperm motility and/or low sperm count. These SNPs in the mitochondrial genome were in a homoplasmic state. Thus, we propose that possessing these mitochondrial mutations compromises the semen quality of these men.

Association of a novel human mtDNA ATPase6 mutation with immature sperm cells.

This study reports the first clearly defined heteroplasmic mutation in immature human sperm cells. The human sperm mitochondrial genome from residue 8186-9341 was analysed with the aim of identifying point mutations which may be associated with human male infertility. The semen samples analysed were obtained from 88 fertile men, 19 with oligozoospermia, and 12 with severe oligozoospermia. Using single strand conformation polymorphism analysis a heteroplasmic T to C transition was detected in the ATPase6 gene, at nucleotide position 8821, in semen samples from one out of 12 (8%) severely oligozoospermic men, but not in oligozoospermic men or normospermic men. This mutation changed the amino acid serine to proline at residue 99 of the mitochondrial ATPase6 in a region which is highly conserved in other vertebrates including rat, bovine, chicken, salmonids and Xenopus. The mutation was detected in semen samples collected from the same man 9 months apart and in peripheral blood lymphocytes. Single sperm cell analyses did not find this mutation in the mature sperm, but the mutation was detected in 7% of immature spermatids. Our finding suggests that immature spermatids with this mutation fail to develop fully.