Female and male gamete mitochondria are distinct and complementary in transcription, structure, and genome function

Abstract

Respiratory electron transport in mitochondria is coupled to ATP synthesis while generating mutagenic oxygen free radicals. Mitochondrial DNA mutation then accumulates with age, and may set a limit to the lifespan of individual, multicellular organisms. Why is this mutation not inherited? Here we demonstrate that female gametes-oocytes-have unusually small and simple mitochondria that are suppressed for DNA transcription, electron transport, and free radical production. By contrast, male gametes-sperm-and somatic cells of both sexes transcribe mitochondrial genes for respiratory electron carriers and produce oxygen free radicals. This germ-line division between mitochondria of sperm and egg is observed in both the vinegar fruitfly and the zebrafish-species spanning a major evolutionary divide within the animal kingdom. We interpret these findings as an evidence that oocyte mitochondria serve primarily as genetic templates, giving rise, irreversibly and in each new generation, to the familiar energy-transducing mitochondria of somatic cells and male gametes. Suppressed mitochondrial metabolism in the female germ line may therefore constitute a mechanism for increasing the fidelity of mitochondrial DNA inheritance.

Keywords: Danio rerio; Drosophila melanogaster; aging; maternal inheritance; mitochondrial DNA; reactive oxygen species.

Fig. 1.—

Relative quantities of mitochondrial mRNA measured for three key protein subunits of the mitochondrial respiratory chain. (a) A schematic representation of the respiratory electron transport chain of the mitochondrial inner membrane. The protein subunits highlighted in blue, green, and yellow represent the products of the mitochondrial genes nad1, cob, and cox1, respectively. Structures are surface models drawn using PyMol (Schrodinger 2010) from Protein Data Bank (PDB) atomic coordinate files with the following accession numbers: respiratory complex I (NADH-ubiquinone oxidoreductase), 3M9S; respiratory complex II (succinate dehydrogenase), 1ZOY; respiratory complex III (the cytochrome b-c₁ complex), 1QCR; complex IV (cytochrome c oxidase), 1V54. (b) Respiratory electron transport gene expression profile. Quantities are shown for mitochondrial mRNA expressed in different tissues of male and female individuals of D. melanogaster and D. rerio. The color coding is the same as that used in (a): blue, nad1; green, cob; yellow, coxI. Error bars indicate standard error of the mean (SEM). P ≤ 0.05. See also supplementary tables S1 and S2, Supplementary Material online.

Fig. 2.—

Mitochondrial inner membrane electrical potential visualized in ovary and sperm cells. Mitochondrial membrane potential in Drosophila ovary (a), zebrafish ovary (b), Drosophila sperm (c), and zebrafish sperm (d). The bright field micrograph shows the corresponding scale bars. Mitochondrial YFP (Drosophila) and Mitotracker Green FM (zebrafish) report the presence of intact mitochondria in the green channel. Mitotracker Red FM reports the relative membrane potential in those mitochondria in the red channel. Overlay of both channels highlights two different populations of mitochondria seen in (a and b) ovary. White arrows point to inactive female gamete mitochondria. Yellow arrows indicate somatic, active mitochondria, which accumulate the red dye indicating presence of membrane potential. In (c) and (d), blue arrows indicate active male gamete (sperm) mitochondria as a control. See also supplementary figure S1 and movie S1, Supplementary Material online.

Fig. 3.—

Mitochondrial production of ROS visualized in ovary and in sperm cells. ROS accumulation in Drosophila ovary (a), zebrafish ovary (b), Drosophila sperm (c), and zebrafish sperm (d). The bright field micrograph shows the corresponding scale bars. DAPI indicates nuclear DNA in the blue channel. Oxidized H₂DCF-DA is seen in the green channel and reports the relative amount of ROS in different tissues. Merged overlay of both channels highlights the abundance of ROS in diploid follicle cells compared with the female germ line cells in images (a) and (b), suggesting a reduced rate of electron transfer to oxygen in this cell type. Yellow arrows point to sperm mitochondria, which accumulate ROS as shown in images (c) and (d). See also supplementary figure S2 and movie S2, Supplementary Material online.

Fig. 4.—

Mitochondrial ultrastructure in somatic cells and in male and female gametes. Transmission electron micrographs of D. melanogaster (a) flight muscle, (b) sperm, and (c) oocyte; and D. rerio (e) cardiac muscle, (f) sperm, and (g) oocyte. Letter (m) indicates mitochondria, (n) a haploid nucleus, and (f) a flagellum. Oocyte mitochondria are seen as simpler structures, ranging from 200 to 500 nm, lacking cristae development and matrix electron density (c and g). Muscle (a and e) and sperm (b and f) mitochondria were used as a somatic and male gametic tissue control samples for normal development, respectively. Images were taken using 9,300× magnification. The scale bar corresponds to 500 nm. Stereological analysis of the morphological variations among the three samples: (d) Drosophila; (h) Danio. V(c,m) is the ratio of crista volume to mitochondrion volume. Error bars are SEM, P ≤ 0.01.

Fig. 5.—

A model for maintenance of mtDNA by maternal inheritance of template mitochondria transmitted in the cytoplasm. An oocyte (egg cell) contains a nucleus with a haploid chromosome number (n) and a cytoplasm with multiple template mitochondria. A sperm cell, also with a haploid nucleus (n), is motile, and its motility requires ATP from active mitochondria performing oxidative phosphorylation (OXPHOS). Following fertilization, active sperm mitochondria are rapidly degraded, leaving only the maternal, template mitochondria in the cytoplasm of the diploid (2 n) zygote (or fertilized egg). Successive cell divisions in embryogenesis involve mitosis and differentiation—and division—of most template mitochondria into active OXPHOS mitochondria, which eventually dominate and populate not only somatic tissues but also the male germ line in which sperm are generated by meiosis in males for the next generation. However, some cells are sequestered and continue to carry only quiescent, template mitochondria, through meiosis and oogenesis to give the oocytes of females in the next generation. These cells comprise the female germ line. Female germ cells are never supplied with ATP by oxidative phosphorylation in their own mitochondria, but depend for their maintenance, at low metabolic rate, on ATP supplied, directly or indirectly, by neighboring somatic cells (follicle cells or nurse cells) that are specially adapted for this role. This hypothesis, after Allen (1996), predicts that the female germ line forms an indefinitely replicating vehicle for accurate transmission of mtDNA between generations. See also supplementary movie S3, Supplementary Material online.