Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The germ plasm is a specialized region of oocytecytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.
FIG. 1. Diagram of the formation and elaboration of the germinal granules during Xenopus oogenesis and early embryogenesis. Nest stage
oogonium contains the mitochondrial aggregate that is a precursor of premitochondrial cloud. Mitochondrial aggregate is located in the
vicinity of the nucleus (blue) and is composed of mitochondria (green) and electron dense mitochondrial cement (pink) that is probably the
precursor of the granulo-fibrillar material (GFM). Prestage 1 oocyte contains premitochondrial cloud composed of numerous mitochondria,
the remnants of mitochondrial cement, GFM (red branching structures), and germinal granules (red spheres) that form from the coalescing
GFM, and elecrtron dense matrix. Stage 1 oocyte contains mitochondrial cloud that has two distinct regions. The part closer to the vegetal
pole of the oocyte contains accumulation of germinal granules. This region of the mitochondrial cloud is called METRO and contains
various localized RNAs. The part closer to the nucleus is devoid of germinal granules. Between stages 2 and 5 of oocyte growth, the
mitochondrial cloud breaks apart, and its fragments migrate into the vegetal cortex of the oocyte. In stage 6 oocyte, the germinal granules
are located at the apical region of vegetal cortex. During first cleavages, the germinal granules and mitochondria form the islands of the
germ plasm at the vegetal tips of vegetal blastomeres and the individual germinal granules coalesce and change the ultrastructure. In all
drawings, the animal pole is at the top, and vegetal pole at the bottom. Oocyte and embryonic nuclei are depicted as the blue spheres. The
size of the all structures shown is not to scale
FIG. 2. Electron microscopy analysis of various RNA distributions within the germ plasm of nest stage and stage 1 oocytes and within the germ plasm
island in the embryo. Nest stage oogonia, stage 1 oocytes, and eight-cell embryos were hybridized in situ with anti-digoxigenin-labeled antisense RNA
probes. Hybridization was detected with anti-digoxigenin antibody conjugated with nanogold and a silver enhancement (see Material and Methods). In
all pictures, white arrows point to the silver-enhanced gold particles, long black arrows indicate mitochondrial cement, arrow heads point to the GFM,
and thick black arrows to the germinal granules. The nest panels (AâC) show the fragments of mitochondrial aggregate with mitochondria and
mitochondrial cement. Xcat2 mRNA is present in the matrix of the aggregate but is absent from the cement (A). Xpat mRNA is present at the matrix
at very low level (B). Xlsirts RNA is located in the central part of the aggregate (C). Oocyte panels (DâF) show the fragments of mitochondrial cloud
containing mitochondria, GFM, and germinal granules. Xcat2 mRNA is present inside the germinal granules and in the GFM (D). Xpat mRNA is
present in the GFM and at the periphery of germinal granules (E). DEADSouth mRNA is localized on GFM and at a very low level at periphery of
germinal granules (F). Embryo panels (GâI) show the fragments of germ plasm islands containing mitochondria and aggregated germinal granules at the
tip of vegetal blastomeres in four- to eight-cell embryos. Xcat2 mRNA is present inside of the germinal granule (G). Xpat mRNA is present in several
spots at the periphery of the granule and on the matrix (H). Xdazl mRNA is present at low level on the germ plasm matrix (I). Bar in nest panels is equal
to 375 nm, in oocyte panels is equal to 675 nm, and in embryo panels is equal to 750 nm.
FIG. 3. Elaboration of germinal granules in cleaving embryo. Electron microscopy in situ hybridization with Xpat antisense RNA probe.
The ultrathin cross sections through the germ plasm islands of cleaving embryos show that, between fertilization and the eight-cell stage,
individual germinal granules (A, B) appear to aggregate into progressively more elaborate intermediary complexes (CâG). The cross section
through the ultimate germinal granule shows its extremely complex shape and ultrastructure (H). Xpat RNA (arrows) represented by
silver-enhanced gold particles is concentrated on the periphery of the aggregating granules and of the ultimate granule. (I) Section through
the germ plasm of neurula stage embryo. Germinal granules are small, spherical, and resemble the germinal granules before aggregation
(shown in A and B). The germinal granules are outlined in white. M, mitochondria. Bar is equal to 600 nm
FIG. 4. Xcat2 mRNA distribution in primordial germ cells in neurula stage of development. Electron microscopy in situ hybridization
with Xcat2 antisense RNA probe. (A) Two primordial germ cells in the endodermal mass of the embryo. Arrows point to the nuclei. (B)
Primordial germ cell in the metaphase of mitotic division. Black arrow points to the chromosomes in metaphase plate. Germ plasm
mitochondria (m) are concentrated at the poles of the spindle (stars). White arrow points to the germinal granule located between the
mitochondria. Germinal granule is unlabeled and the label is dispersed over the spindle and the cytoplasm. (C, D) Two different examples
of unlabeled germinal granules (arrows) in the germ plasm of primordial germ cells. Label is present on the germ plasm matrix (arrowheads).
m, mitochondria. Bar in (A) is equal to 10 microns, in (B) to 2 microns, in (C) and (D) to 500 nm.
FIG. 6. Diagramatic summary of RNA distribution in the GFM and in the germinal granules in oocytes and embryos. Xcat2 (red dots), Xpat
(blue dots), Xdazl (stars), DEADSouth (squares), and C10 (triangles) mRNAs are all present on branching GFM in prestage 1 and stage 1
oocytes. Mature germinal granules that form from the aggregating GFM contain Xcat2, Xpat, and low level of DEADSouth mRNAs. The
germinal granules in the embryo that form from the coalescing oocyte granules contain Xcat2 and Xpat RNAs. Xcat2 is the only RNA that
is consistently sequestered inside of the GFM and inside of the germinal granules in oocytes and embryos
FIG. 7. Three-dimensional ultrastructural reconstruction of mitochondrial cloud and germinal garnules in stage 1 oocyte. (A) Mitochondrial
cloud was reconstructed from 21 serial EM sections. The cloud is a sphere composed of thousands of mitochondria (green speckles)
and germinal granules (red spheres). Germinal granules are concentrated in the form of a ring in the METRO region that is the part of the
cloud facing vegetal pole and are excluded from the center of the cloud. Oocyte nucleus, not visible in the picture, is above, and the vegetal
pole of the oocyte is below the plane of the picture. (B, D) The half sections of three germinal granules from a mitochondrial cloud similar
to one shown in (A). The images were reconstructed from four serial sections of oocytes hybridized in situ with Xpat (B, D) and Xcat2 (C,
D) antisense RNA probes. The Xpat RNA (green dots) is predominantly on the granule periphery with a small portion localized internally
while the majority of Xcat2 (black dots) is sequestered internally in the granule. For better clarity of the image, the original silver-enhanced
gold label was replaced (using proper logarithm and computational programs; see Materials and Methods) with uniform sized dots. Bar is
equal to 4.5 mm in (A), and 250 nm in (BâD).
FIG. 8. Three-dimentional ultrastructural reconstruction of aggregating germinal granule in germ plasm of eight-cell-stage embryo.
Germinal granule in intermediary stage of aggregation was reconstructed from 10 serial EM sections hybridized with Xpat antisense RNA.
One of the serial sections labeled with silver enhanced gold particles of Xpat RNA (black speckles) is shown in (A). The same section on
the cut surface of reconstructed granule is shown in (B and C). Arrow in (AâC) indicates the orientation of the section. (B, C) The same cut
surface of the granule viewed from different angle. (D) A different cut surface of the same granule viewed from the opposite side. Xpat RNA
is represented as green dots. Bar is equal to 300 nm.
FIG. 9. Three-dimensional ultrastructural reconstruction of germinal granule in germ plasm of eight-cell-stage embryo. Germinal
granules were reconstructed from 10 serial EM sections hybridized with Xpat and Xcat2 antisense RNA probes. The example of one of the
serial sections labeled with silver-enhanced gold particles of Xcat2 mRNA (black speckles) is shown in (A). The same section on the surface
of reconstructed granule is shown in (B), arrow indicates the orientation of the section. Nontransparent (C) and semitransparent (D) images
of half section of the granule show the distribution of Xcat2 (black dots) and Xpat (green dots) RNA. The Xpat RNA distribution is limited
to the granule periphery and the majority of Xcat2 RNA is sequestered inside the granule. Nontransparent (E) and semitransparent (F)
images of whole granule show the distribution of Xcat2 and Xpat RNAs. For better clarity, original silver-enhanced gold label shown in (A)
was replaced (using proper logarithm and computational program) with uniform sized dots (CâF). Bar is equal to 270 nm in (A) and 200 nm
in (BâF).
FIG. 5. (A, B) Studentâs t test statistical analysis of the label distribution in electron microscopy in situ hybridization samples. Graphs
depict the distribution of the grains in three different compartments of the germ plasm (GFM, germinal granules, matrix) for nine different
RNA probes in oocytes (A) and the distribution of the grains in granules and matrix for two different RNAs in embryos (B). Each bar
represents the average (with the standard deviation) number of grains counted in several independent samples in 10â14 squares of 1.4 mm2
.
Numbers below the graph represent the P values. All the P , 0.05 are statistically significant. NS, statistically not significant.
