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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.
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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 oocytenucleus 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.
FIG. 1. Proapoptotic proteins
Bax and p53 are sequestered in
Xenopus oocytenucleus 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 oocytenucleus 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.
