Testis

Estimating Mitochondrial DNA Content of Chinook Salmon Spermatozoa Using Quantitative Real-Time Polymerase Chain Reaction

Abstract
Animal mitochondrial DNA (mtDNA) is inherited predominantly maternally. Various mechanisms to avoid the transmission of paternal mtDNA to offspring have been proposed including the dilution of paternal mtDNA by maternal mtDNA in the zygote. The effectiveness of dilution as a barrier will be determined by the number of mtDNA molecules contributed by each parental gamete and is expected to be highly variable among different taxa due to interspecific differences in mating systems and gamete investment. Estimates of this ratio are currently limited to few mammalian species and data from other taxa are therefore needed to understand better the mechanisms of mitochondrial inheritance. The present study estimates mtDNA content in salmon sperm, the first non-mammalian vertebrate to be examined. Although highly divergent, it appears that the mitochondrial DNA content may be conserved within vertebrate taxa, indicating the reduction of mtDNA being a key factor of spermatogenesis to ensure mitochondrial functionality on the one hand and to avoid paternal leakage at a significant or detectable level on the other hand. We employ quantitative real-time PCR (Q-PCR) and demonstrate the accuracy and high reproducibility of our experiments. Furthermore, we compare and evaluate two standard approaches used for the quantification of genes, Q-PCR and blotting methods, in regard to their utility to accurately quantify mitochondrial genes.

Key words: Testis • Sperm • mtDNA content