Kogo N et al. (2011), Germ-line mitochondria exhibit suppressed respi...

XB-ART-42395

Dev Biol 2011 Jan 15;3492:462-9. doi: 10.1016/j.ydbio.2010.11.021.

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Germ-line mitochondria exhibit suppressed respiratory activity to support their accurate transmission to the next generation.

Kogo N , Tazaki A , Kashino Y , Morichika K , Orii H , Mochii M , Watanabe K .

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Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3'-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.

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Species referenced: Xenopus laevis
Genes referenced: atp5f1b atp5pb cox4i1 got2 mdh2 mt-co1 mt-co3 pgc

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	Fig. 1. Comparison between mitochondria of stage VI oocytes and mitochondrial cloud of stage I oocytes. (A, B) Purity of stage VI and I samples. High purity of stage VI oocyte mitochondria was shown by differential absorption spectrum giving peaks at 552, 563, and 604 nm ascribed to cytochromes c (c1), b, and a, respectively (A). Routine electron microscopic picture of the mitochondrial cloud in stage I oocytes. The mitochondrial cloud includes a large number of mitochondria (arrowheads) and other organelles such as endoplasmic reticulum (arrows). Mitochondria occupied about 42% of whole area presented. Scale bar represents 200 nm (B). Protein profile in gradient SDS-PAGE (C). Red arrows show bands that indicated a lower protein level in stage I sample than stage VI sample. The 8, 21, and 23 bands were identified as F1-β, F0-b, and F1-δ, respectively. Black arrows show bands that were common to two samples. The 12, 14, and 18 bands were identified as GOT, MDH, and AK, respectively. Western blotting (D). F0-b and F1-β were lower in terms of protein level in stage I sample than in stage VI sample. GOT and MDH were detected at similar levels between stage VI and I samples.
	Fig. 2. Expression of F0-b in stage I and VI oocytes. Sections of ovary double-stained with antibodies against GOT (red) and F0-b (green). (A–D) A stage I oocyte in the center of (A) hasa mitochondrial cloud (arrowhead) under the nucleus (N). A stage IV oocyte is at the upper right. All mitochondria of the two cells were visualized by anti-GOT (B). All mitochondria in a stage I oocyte were F0-b-negative (C, D). Mitochondria in the animal hemisphere of stage IV oocyte were F0-b-positive. There was background fluorescence at the peripheralcytoplasm (C, D). (E–H) In a stage VI oocyte, arrows indicate boundary of animal hemisphere with heavy melanin granules and vegetal hemisphere (E). A stage VI oocyte has a largenumber of mitochondria labeled with anti-GOT in the animal hemisphere (F, upper box of E). Mitochondria were also labeled with anti-F0-b (G), overlaying GOT staining (H). (I–K) Magnified view of lower box of (E) shows accumulation of mitochondria at the vegetal cortex (arrowheads in I) and a few mitochondria in the inner part (arrows in I). The formerwas F0-b-negative, and the latter was F0-b-positive (J, K). Scale bars represent 100 μm in (A, B, E) and 50 μm in (F, I).
	Fig. 3. Expression of F0-b during oogenesis. (A–D) Stage I, II, and III oocytes are shown in order from right to left (A). The mitochondrial cloud of stage I oocyte (arrow) and fragmented clouds in stage II and III oocytes (brackets) were localized at the vegetal cortex from the nucleus (N) (B). All mitochondria of a stage I oocyte were F0-b-negative, and all mitochondria in stage II and III oocytes were F0-b-positive (C, D). (E–H) A stage IV oocyte (E) has a large number of mitochondria marked with anti-GOT in the animal hemisphere (F, upper box of E). Mitochondria were also labeled with anti F0-b (G), overlaying GOT staining (H). (I–K) Magnified view of lower box of (E) shows mitochondria at the vegetal cortex (arrowheads in I) and mitochondria in the inner part (I). The former became F0-b-negative and the latter remained F0-b-positive (J, K). Scale bars represent 100 μm in (A, B, E) and 50 μm in (F, I).
	Fig. 4. Ultrastructural analysis of oocytes on COX activity during oogenesis. (A′–J′) KCN inhibits COX activity in all stages of oocytes. Dense granules (arrowheads) are mitochondrial granules, not reaction products. (A, B) No mitochondria in stage I oocytes showed COX activity. (C, D) All mitochondria in early stage II oocytes showed high COX activity. (F, H, J) COX activity was heterogeneous in the population and also in areas of one mitochondrion at the vegetal hemisphere of stage III oocytes (F). Mitochondria at the vegetal cortex showed noCOX activity in stage IV (H) and VI oocytes (J). (E, G, I) Mitochondria in the animal hemisphere showed high COX activity. Scale bars represent 200 nm.
	Fig. S1. Expression of F0-b in primordial germ cells (PGCs) of stage 36 embryos. (A–D) Sections of stage 36 embryos injected with venus-DEAD-South-3′UTR mRNA. PGC was identified by VENUS protein translated from this mRNA. DEAD-South-3′UTR had an anchoring signal to the germ plasm and enabled visualization of PGCs with VENUS protein (Kataoka et al., 2006). PGC was double-stained with antibodies against VENUS (green) and GOT (red). A VENUS-positive PGC (arrows in B, box A) was GOT-positive in endodermal cell masses that were very weak in GOT immunoreactivity (arrows in D, box C). (E–H) Sections of stage 36 embryos double-stained with antibodies against GOT (red) and F0-b (green). GOT-positive cell (arrow in F, box E) near the mesoderm in endodermal cell mass might be PGC, and it was also F0-b-positive (arrow in G, box H). Scale bars represent 100 μm.
	Fig. S2. Expression of F0-b in PGC of stage 50 tadpoles. (A–D) Frontal sections of stage 50 tadpoles labeled with Hoechst (blue), anti-GOT (red), and anti-F0-b (green). A pair of gonads (g) included PGCs, and pronephric tubules (pt) were situated on the peritoneum with heavy melanin (A). PGCs had a large nucleus poorly stained with Hoechst (arrows in B). PGCs localized in gonads were GOT- and F0-b-positive (arrows in C and D). Scale bars represent 20 μm.