Our results demonstrate that in D. melanogaster
, the paternal inheritance of mtDNA is restricted by mechanisms that remove mtDNA from developing sperm. In addition to our findings, mtDNA appears to be removed from developing sperm in other species. For example, mtDNA copy number declines during human spermatogenesis (Larsson et al., 1997
), and the extremely low abundance of mtDNA (average of 1.4 copies per sperm) in highly purified sperm led to the suggestion that most human sperm lacked mtDNA (May-Panloup et al., 2003
). The sperm of mice and the medaka fish,Oryzias latipes
, also have reduced mtDNA levels (Nishimura et al., 2006
; Shitara et al., 2000
; Hecht et al., 1984
). While it is unclear if mtDNA is eliminated from all animal sperm as efficiently as it is eliminated in D. melanogaster
, it appears that other organisms also exhibit a developmental decline in mtDNA in spermatogenesis.
The molecular mechanisms that remove mtDNA from human, mouse, and medaka fish sperm are currently unknown. In C. reinhardtii
a nuclease has been proposed to digest mtDNA to enforce uniparental mtDNA inheritance, but it has yet to be identified (Aoyama et al., 2006
). In Arabidopsis thaliana
(which lacks an EndoG
homologue), a nuclease conserved in angiosperms eliminates mtDNA within mitochondria during pollen development (Matsushima et al., 2011
Our work implicates the known mitochondrial nuclease, EndoG, in the early elimination of mtDNA during D. melanogaster
spermatogenesis. Previous work in C. elegans
showed that EndoG contributed to destruction of DNA in apoptotic corpses, and the enzyme was thought to be stockpiled with other pro-apoptotic proteins in the mitochondrial intermembrane space (Li et al., 2001
). However, more recent studies questioned the role of EndoG in apoptosis, and argued that EndoG resides in the mitochondrial matrix, where it is proposed to participate in mtDNA replication, mtRNA processing, and mitochondrial biogenesis (David et al., 2006
; Côté and Ruiz-Carrillo, 1993
; McDermott-Roe et al., 2011
). Our findings suggest a function for EndoG in removing mtDNA from within mitochondria, though future work will be necessary to fully understand the generality and regulation of this function.
In addition to EndoG-dependent mtDNA elimination, we found that a cellular remodeling process that trims and shapes the D. melanogaster
sperm can remove residual mtDNA. In other species, the cytoplasm is similarly trimmed from developing spermatids. In mammals, this trimming discards at least some mitochondria from each spermatid into a residual body (Breucker et al., 1985
; Hecht et al., 1984
), suggesting that cellular remodeling contributes to low mtDNA levels in the sperm of other animals.
Most barriers to paternal mtDNA transmission have previously been proposed to act at, or following zygote formation. For example, simple dilution of the small allotment of sperm mtDNA by the large egg contribution is hypothesized to passively limit paternal mtDNA inheritance (Alberts et al., 1994
). Furthermore, selective destruction of paternal mitochondria occurs in the early zygotes of several animals (Sutovsky et al., 1999
; Sato and Sato, 2011
; Rawi et al., 2011
). Additionally, unusual cases of paternal mtDNA transmission in interspecies crosses suggest post-fertilization restriction of paternal mtDNA inheritance (Kaneda et al., 1995
; Kondo et al., 1990
). However, effective mtDNA elimination during D. melanogaster
spermatogenesis prevented us from evaluating the contribution of later zygotic processes to paternal mtDNA elimination. Additionally, we could not bypass the pre-zygotic mechanisms. since the individualization process, which removes mtDNA, is also required for sperm production. Importantly, whether or not other barriers to paternal transmission of mtDNA exist, the elimination of mtDNA from developing sperm removes the potential for paternal contribution to mitochondrial inheritance.
In concluding, we would like to consider why mechanisms might have evolved to eliminate mtDNA from sperm. Since biparental inheritance of mtDNA is rare, it presumably carries a disadvantage. Consequently, a sperm that transmits its mtDNA would put the resulting zygote at a disadvantage, while a sperm that eliminated its mtDNA would produce a more successful zygote. The advantage conferred to sperm without mtDNA might provide an evolutionary drive that would act widely to promote the evolution of mechanisms acting in spermatogenesis to limit paternal contributions of mtDNA to progeny.