Kloc M et al. (2007), Differential subcellular sequestration of proap...

XB-ART-36301

Genesis 2007 Aug 01;458:523-31. doi: 10.1002/dvg.20322.

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Differential subcellular sequestration of proapoptotic and antiapoptotic proteins and colocalization of Bcl-x(L) with the germ plasm, in Xenopus laevis oocytes.

Kloc M , Shirato Y , Bilinski S , Browder LW , Johnston J .

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Apoptosis is an important element of normal embryonic development and gametogenesis in invertebrate and vertebrate species. Although the components of apoptotic machinery are present in Xenopus laevis fully grown stage VI oocytes and eggs, apoptosis in the developing Xenopus ovary is limited to the somatic cells with no indication of apoptosis in the germ cells. Considering the possibility that Xenopus previtellogenic oocytes might lack the components of the apoptotic pathway, we analyzed Xenopus Stage I oocytes for the presence of the proapoptotic factors Bax and tumor suppressor p53, and antiapoptotic factors Bcl-x(L) and mitochondrial heat shock protein 60 (Hsp60). We found that pro- and antiapoptotic proteins are present in Xenopus oocytes but, surprisingly, they are located in distinct subcellular compartments with proapoptotic proteins Bax and p53 being sequestered in the oocyte nucleus and antiapoptotic protein Bcl-x(L) sequestered in the cytoplasm and highly enriched in the METRO region of the mitochondrial cloud, where it colocalized with the germ plasm, and Hsp60 colocalizing with all mitochondria. The absence of apoptosis in Xenopus early oogenesis is maybe due to differential sequestration of pro- and antiapoptotic molecules.

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Species referenced: Xenopus laevis
Genes referenced: bax bcl2l1 cat2 hspd1 nanos1 tp53
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	FIG. 1. Proapoptotic proteins Bax and p53 are sequestered in Xenopus oocyte nucleus and excluded from the mitochondrial cloud. (A, B, D, F, G) Whole-mount and (C, E, H) section immunostaining of Stage I–II (A, F) albino, and (B, C and G, H) and Stage IV (D, E) wild type oocytes with Bax antibody. In (A–C) control and (F–H) heat-shocked Stage I–II oocytes, Bax protein is concentrated in the nucleus and excluded from the mitochondrial cloud (arrows in B, C, H). Arrow in F points to the heavily damaged oocyte with cytoplasmic blebbing. In Stage IV oocytes Bax is exluded from the nucleus and concentrated (arrow) in perinuclear region (D, E). Whole-mount (I) control and (J) heat-shocked oocytes immunostained with p53 antibody showing the protein concentrated in the nucleus and excluded from the mitochondrial cloud (arrows). The level of p53 expression seems to be similar in control and heat-shocked oocytes, the oocyte in panel J showing lower than control expression of p53 was chosen just to clearly visualize the mitochondrial cloud. N, nucleus. Scale bars represent 50 lm in A–C and F–J, 200 lm in D and 75 lm in E.
	FIG. 2. Antiapoptotic protein Bcl-xL is sequestered in the mitochondrial cloud, and Hsp60 is localized both in the mitochondrial cloud and in the cytoplasmic mitochondria. (A, B, E, F) whole mount and (C, D) section immunostaining of wild type Stage I to early Stage II oocytes with (A–D) Bcl-xL and (E, F) Hsp60 antibodies. In (A–C) control and (D) heat-shocked oocytes, the Bcl-xL protein is excluded from the oocyte nucleus and concentrated in the METRO (germ plasm) region of the mitochondrial cloud (arrows in A, B, C, D). In (E) control and (F) heatshocked oocytes, the Hsp60 protein colocalizes with the mitochondrial cloud (long arrows) and the network of cytoplasmic mitochondria (short arrows). N, nucleus. Scale bars represent 50 lm in panels A, B, E, and F, and 40 lm in panels C and D.
	FIG. 3. Western blots of Bcl-xL and Bax proteins in mitochondrial fractions of Xenopus oocytes. (A) Bcl-xL is present in the mitochondrial cloud fraction of control (C) and heat shocked (HS) Stage I–II oocytes. (B, Lanes 1, 2) Bax is present in the lysate of control and heatshocked whole Stage I–II oocytes and absent from the (Lanes 3, 4) cytoplasmic mitochondria fraction and the (Lanes 5, 6) mitochondrial cloud fraction. Mitochondrial protein porin was used as a loading control. We always observed porin as a doublet, which may represent the phosphorylated and unphosphorylated forms.
	FIG. 4. Bcl-xL protein is concentrated in the METRO (germ plasm) region of the mitochondrial cloud of Xenopus oocytes. (A) Whole mount and (B) section of oocyte hybridized in situ with digoxigenin labeled antisense Xcat2 RNA probe. Xcat2 mRNA is localized in the germinal granules, which are restricted to the METRO region of the mitochondrial cloud (arrows). (A) Top view of the oocyte showing the ring of labeled germinal granules at the apex of the mitochondrial cloud (arrow). The center of the mitochondrial cloud is devoid of germinal granules and remains unlabeled. (C) Bcl-xL protein is, as is Xcat2 mRNA, distributed in the mitochondrial cloud in the ringlike pattern (arrow). (D) Electron microscopy image of two independent fragments of the mitochondrial cloud with the METRO region; i.e., the peripheral ring of germinal granules (marked with red stars) between mitochondria. (E, F) Electron microscopy image of the fragment of the METRO region of the mitochondrial cloud with the germinal granules (short arrows) visible between the mitochondria (long arrows) from (E) control and (F) heat-shocked Stage I oocytes. Mitochondria and germinal granules in the heat-shocked oocyte show abnormal ultrastructure. Mitochondria are swollen and devoid of cristae, and the germinal granules are swollen and less homogeneous than those in controls. N, nucleus. Scale bars represent 50 lm in panels A–C and 4 lm in D.
	FIG. 5. Ultrastructure of mitochondrial cloud and cytoplasmic mitochondria in control and heatshocked oocytes. In control oocytes, the (A) mitochondrial cloud and (B) cytoplasmic mitochondria show prominent cristae and intra-mitochondrial granules (arrows), which are more abundant in mitochondrial cloud mitochondria. In (C, D) heat-shocked oocytes, mitochondria located in the (C) mitochondrial cloud and in the (D) cytoplasm are visibly swollen and have less prominent cristae than those in (A, B) control oocytes. In addition, the intra-mitochondrial granules are much more abundant in the (C, arrow) mitochondrial cloud mitochondria than in the (D) cytoplasmic mitochondria.