Estimating Mitochondrial DNA Content of Chinook Salmon Spermatozoa Using Quantitative Real-Time Polymerase Chain Reaction1

Abstract

Animal mitochondrial DNA (mtDNA) is predominantly inherited 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 better understand the mechanisms of mitochondrial inheritance. The present study estimates mtDNA content in salmon sperm, the first nonmammalian vertebrate to be examined. Although highly divergent, it appears that the mtDNA content may be conserved within vertebrate taxa, indicating that the reduction of mtDNA is 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 in the accurate quantification of mitochondrial genes.

Lost in the zygote: the dilution of paternal mtDNA upon fertilization

“…However, the key focus of our work was to describe the dilution effect of paternal mtDNA in the fertilized zygote. Recently, we determined the mtDNA content of chinook salmon sperm to be 5.73±2.28 per gamete (Wolff and Gemmell, 2008), and here, we estimate the average mtDNA content of salmon oocytes to be 3.2 Â 10 9 ± 1.0 Â 10 9 . If these estimates are indicative and if paternal mtDNA enters the egg upon fertilization without subsequent degradation, the ratio of paternal-tomaternal mtDNA is then 1:5.5 Â 10 8 (quotient of mean mtDNA content per sperm and mean mtDNA content of oocytes).…”

“…Finally, considering a comparable mtDNA content for mammalian and chinook salmon sperm (Wolff and Gemmell, 2008), it appears that the varying strength of the dilution effect between the two systems is mainly determined by the mtDNA content of the female gametes. If this relation finds general application across taxa and if the mtDNA content of oocytes is linked to oocyte size, as indicated by this work, the probability of paternal leakage occurring, based on the dilution effect only, might then be linked to oocyte size and gamete investment in general.…”

“…Here, we determine the mtDNA content of chinook salmon oocytes and build on earlier work examining the mtDNA content of sperm (Wolff and Gemmell, 2008), which enables us to estimate the mtDNA ratio of paternalto-maternal DNA in zygotes. The determination of this dilution effect is the first outside the mammalian system.…”

Morphological and molecular clues for recording the first appearance of Artemia franciscana ( ) in Egypt

“…DNA was extracted from entire bodies using 5 % Chelex ® 100 sodium form resin (Sigma-Aldrich, Madrid, Spain) in TE buffer (pH 8) according to the protocol described by Wolff and Gemmel (2008). 2.4 U of Proteinase K (ThermoFisher) was added to each tube.…”

The Trojan female technique: a novel, effective and humane approach for pest population control

“…Most tissues can function well when mitochondrial capacity is reduced by as much as 80% [32,33,44,45]. However, the dependence of sperm vigour on mitochondrial capacity, together with the relatively small numbers of mtDNA in a sperm [46,47], means that even a modest reduction in mitochondrial capacity can impact male fertility substantially [35,38,41,43,44,48]. Owing to the asymmetry between egg and sperm dependence of mtDNA function, mtDNA mutations can potentially cause large reductions in male fitness while having little or no effect on female fitness; a view supported by empirical data from mice [38,41] and more recently flies [34,35,37,43].…”