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J Cell Sci
2022 Dec 01;1351:. doi: 10.1242/jcs.259394.
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Comparative analysis of vertebrates reveals that mouse primordial oocytes do not contain a Balbiani body.
Dhandapani L
,
Salzer MC
,
Duran JM
,
Zaffagnini G
,
De Guirior C
,
Martínez-Zamora MA
,
Böke E
.
Abstract
Oocytes spend the majority of their lifetime in a primordial state. The cellular and molecular biology of primordial oocytes is largely unexplored; yet, it is necessary to study them to understand the mechanisms through which oocytes maintain cellular fitness for decades, and why they eventually fail with age. Here, we develop enabling methods for live-imaging-based comparative characterization of Xenopus, mouse and human primordial oocytes. We show that primordial oocytes in all three vertebrate species contain active mitochondria, Golgi and lysosomes. We further demonstrate that human and Xenopus oocytes have a Balbiani body characterized by a dense accumulation of mitochondria in their cytoplasm. However, despite previous reports, we did not find a Balbiani body in mouse oocytes. Instead, we demonstrate that what was previously used as a marker for the Balbiani body in mouse primordial oocytes is in fact a ring-shaped Golgi that is not functionally associated with oocyte dormancy. This study provides the first insights into the organization of the cytoplasm in mammalian primordial oocytes, and clarifies the relative advantages and limitations of choosing different model organisms for studying oocyte dormancy.
Fig. 1. Live-imaging of vertebrate primordial follicles reveals active organelles. (A,C,G) Live imaging of Xenopus, mouse and human primordial follicles probed with LysoTracker Deep Red (A) to assess lysosomal activity, NBD C6-ceramide (C) to image the Golgi, and TMRE (G) to assess mitochondrial activity. All left panels show the central plane of the oocyte. Middle panels are maximum z-projections of peri-equatorial regions and right panels are differential interference contrast (DIC) images of the same oocyte. The nuclear envelope and the plasma membrane are marked with dashed circles. Insets in the Xenopus images are 4× magnification of the marked boxes. Three individuals were examined for each species; at least two oocytes were imaged for each human, and at least three oocytes for each mouse and frog. (B) Quantification of mean fluorescence intensity of LysoTracker puncta in primordial oocytes and somatic cells in the indicated species. Each dot represents a lysosome; blue and red dots represent lysosomes from oocytes and somatic cells, respectively. For each follicle, the mean fluorescence intensity of at least three puncta was measured on the same z-section of the oocyte and somatic cells within that follicle. Data are mean±s.e.m. A.U., arbitrary units. (D) Live imaging of the Golgi in mouse primordial oocytes untreated (DMSO) or treated with BFA, and labelled with NBD C6-ceramide to assess trafficking of the Golgi. Three biological replicates were performed. (E) Quantification of mouse oocytes containing a Golgi ring in untreated or BFA-treated oocytes from three biological replicates. At least 15 oocytes were counted per condition for each replicate. Data are mean±s.d. ***P=0.00034 was calculated with an unpaired two-tailed Student's t-test. (F) Live imaging of the Golgi after incubation with NBD C6-ceramide in Xenopus primordial oocytes untreated (DMSO) or treated with BFA to assess trafficking of the Golgi. Three biological replicates were performed; more than five oocytes were imaged for each replicate. Notice the Balbiani body (dashed circle) is not disassembled by BFA treatment. Insets are 4× magnification of marked boxes. (H) Cartoon representation of oocytes illustrating the cytoplasmic organization of organelles in Xenopus, human and mouse primordial oocytes. The nucleus (n) is depicted in blue, mitochondria in magenta, the Golgi in green and lysosomes in black. Scale bars: 10 µm (A,C,D,G); 50 µm (F).
Fig. 2. Mouse primordial oocytes do not contain a large proteinaceous matrix. (A-D) Formalin-fixed paraffin-embedded sections of ovaries from Xenopus (A), human (B), neonatal mouse (PND4) (C) and adult mouse (8 weeks old) (D) were deparaffinized and labelled using a Proteostat Aggresome Detection Kit to detect an amyloid-like protein matrix. Proteostat marked a structure reminiscent of the mitochondrial cluster in Xenopus and human oocytes but not in mouse. Nuclei were marked with DAPI (blue). Three individuals were examined for each species; at least three oocytes were imaged for each human, ten oocytes for each mouse and five oocytes for each frog. The bottom rows in A-D feature magnified images of the regions marked by boxes in the images in the upper rows.
Fig. 3. The Golgi ring is not a marker for the mouse Balbiani body. (A) Simultaneous live imaging of mitochondria and the Golgi in mouse primordial oocytes revealed an MEZ close to the Golgi ring. The MEZ is indicated by white arrowheads. (B) Schematic illustration of the experimental rationale for analyzing mitochondrial localization after Golgi disassembly. Hypothesis 1: the MEZ is maintained by the proteinaceous matrix of a Balbiani body-like compartment. Hence, the MEZ will be maintained after Golgi ring disassembly by BFA. Hypothesis 2: the MEZ is maintained by the Golgi ring. Hence, Golgi ring disassembly would lead to the disappearance of the MEZ as mitochondria would redistribute in the cytoplasm and the proportion of cytoplasm occupied by mitochondria would increase. Mitochondria are shown in magenta, the Golgi ring in green and the proteinaceous matrix in dark grey. (C) Live imaging of mitochondria and Golgi in untreated or BFA-treated mouse primordial oocytes. The white arrowhead indicates MEZ. (D) Quantification of the area of oocytecytoplasm occupied by mitochondria in untreated and BFA-treated oocytes. Each dot represents an oocyte and each colour an experiment. ***P<0.0001 (unpaired two-tailed Student's t-test). Data are mean±s.d. For A, C and D, two biological replicates from a total of six animals are shown. (E) Mouse primordial oocytes were left untreated or treated with nocodazole to dissociate the Golgi ring, and were incubated with NBD C6-ceramide and MitoTracker Deep Red FM. The MEZ is depicted by a white arrowhead. (F) Quantification of the cytoplasmic area occupied by mitochondria in untreated and nocodazole-treated oocytes. Each dot represents an oocyte and each colour a replicate. For E and F, n=3 biological replicates. ***P=0.0009 (unpaired two-tailed Student's t-test). Scale bars: 10 µm.
Fig. 4. The Golgi ring does not associate with the RBPs RNGTT and RAP55. (A) Immunostaining of neonatal mouse ovary sections using antibodies against the RBP RNGTT (magenta) and the cis-Golgi marker GM130 (green). Nuclei are labelled with DAPI and are shown in blue. The white dashed boxes depict the area magnified in the inset. Representative images from three biological replicates are shown. (B) Quantification of oocytes with nuclear localization of RNGTT. At least 30 primordial oocytes were counted per replicate. Three biological replicates were performed. All oocytes displayed nuclear RNGTT. (C) Whole-mount immunostaining of embryonic (E14.5) and neonatal (P1 and P4) ovaries for RAP55 (magenta) and the cis-Golgi marker GM130 (green). The Golgi are indicated by yellow arrows. In P1 and P4 ovaries a Golgi ring can be seen. The RAP55 granules in P1 and P4 are indicated by white arrowheads. RAP55 is excluded from the area of the Golgi ring (indicated by a blue arrow). n=3 for each stage. In some batches of mouse, the monoclonal GM130 antibody stained the basal membrane in mouse ovary sections, as reported previously (Lei and Spradling, 2016). (D) Whole-mount immunostaining of neonatal mouse ovary using antibodies against DDX6 (green) and RAP55 (magenta). Insets are 2× magnification of the white dashed boxes. For C and D, three biological replicates were performed and representative images are shown. Scale bars: 10 µm.
Fig. 5. The Golgi ring is not functionally associated with oocyte dormancy. (A-C) Whole-mount immunostaining of neonatal mouse ovaries with FOXO3 (magenta) and GM130 (green) antibodies. (A) Representative image of an ovary fixed immediately after extraction. The dashed boxes indicate the areas magnified in the panels below: 1 – primordial oocytes, 2 – primary oocyte. (B) Representative images of ovaries that were either left untreated or treated with BFA for 1 h in vitro to observe the Golgi ring disassembly (left panels), and of ovaries that were left untreated for 5 h or treated with BFA for 1 h followed by 4 h of culture without BFA to observe the Golgi ring reformation (right panels). (C) Representative images of nuclear or cytoplasmic FOXO3 localization in oocytes with the Golgi ring (top and top middle panels, respectively), and without the Golgi ring (bottom middle and bottom panels, respectively). Nuclei were marked by DAPI. Maximum z projections of three sections taken 1 µm apart are shown. White dashed circles denote the oocyte membrane, and yellow dashed circles denote the nuclear membrane. (D-F) Quantification of cytoplasmic FOXO3 (D), the Golgi ring (E) and the Golgi ring in oocytes (F) with cytoplasmic FOXO3 from whole-mount images of ovaries taken at the indicated timepoints. Each biological replicate is represented by a different colour. Filled squares represent ovaries that were treated with BFA for 1 h and then fixed or followed by 4 h of culture in BFA-free medium (wash). Filled circles represent untreated ovaries at different timepoints. Within each of the three replicates, ovaries were taken from neonatal pups born in the same litter. Correlation analysis of FOXO3 nuclear localization and the presence of the Golgi ring between all conditions revealed no relation between the two (linear fit; R2=9E-05). For A-E, three biological replicates were performed. In D and E, at least 30 oocytes per condition and timepoint were counted. Scale bars: 10 µm.
