Mouse early oocytes are transiently polar: three-dimensional and ultrastructural analysis

Abstract

The oocytes of many invertebrate and non-mammalian vertebrate species are not only asymmetrical but also polar in the distribution of organelles, localized RNAs and proteins, and the oocyte polarity dictates the patterning of the future embryo. Polarily located within the oocytes of many species is the Balbiani body (Bb), which in Xenopus is known to be associated with the germinal granules responsible for the determination of germ cell fate. In contrast, in mammals, it is widely believed that the patterning of the embryo does not occur before implantation, and that oocytes are non-polar and symmetrical. Although the oocytes of many mammals, including mice and humans, contain Bbs, it remains unknown how and if the presence of Bbs relates to mouse oocyte and egg polarity. Using three-dimensional reconstruction of mouse neonatal oocytes, we showed that mouse early oocytes are both asymmetrical and transiently polar. In addition, the specifics of polarity in mouse oocytes are highly reminiscent of those in Xenopus early oocytes. Based on these findings, we conclude that the polarity of early oocytes imposed by the position of the centrioles at the cytoplasmic bridges is a fundamental and ancestral feature across the animal kingdom.

Fig. 1. Asymmetric distribution of organelles in mouse early oocytes

Electron microscopy images of P0 (A) and P4 (B) oocytes. A. An asymmetrically positioned Balbiani body (Bb) is composed of multiple Golgi stacks (arrows) arranged around the centriole (arrowhead). Mitochondria (m) and cisternae of the rough endoplasmic reticulum (RER) are distributed at the Bb periphery; an oocyte nucleus (n) is also marked. The oocyte is surrounded by somatic cells (sc). Somatic cell nucleus (ns), nucleolus (nu). In P4 oocytes, the Bb disperses (dashed line marks the remnants of the Bb). The nucleus (n), m – mitochondria (m), and somatic cell (sc) are also marked. Scale bars are equal to 2 μm.

Fig. 2. Semithin sections of oocyte cluster

Examples of some of the semithin serial sections of the oocyte cluster (2 oocytes are visible) within a mouse ovary (stage P0) used for three-dimesional reconstruction shown in Figs. 3 and 6. The oocyte nucleus is encircled, the Bb (Golgi stacks) is indicated by arrows; and mitochondria are labeled with the letter “m.” Sections were stained with Methylene blue. The scale bar is equal to10 μm.

Fig. 3. Three-dimensional reconstruction of the distribution of organelles in mouse early oocytes

Sixteen semithin sections of a P0 oocyte were used for the reconstruction (see Fig. 2 and Materials and methods for details). A-C. Three different views of a single P0 oocyte showing the asymmetrical distribution of the Bb (yellow), nucleus (red), and mitochondria (black). A. For clarity, this view shows the nucleus and Bb only. B, C. These views show the nucleus, Bb, and mitochondria (which partially mask the visibility of Bb).

Fig. 4. Three-dimensional reconstruction of the ultrastructure of Balbiani body in mouse early oocytes

Eighteen ultrathin sections of a P0 oocyte were used for the reconstruction (see Materials and methods for details). A-D. These views represent different fragments of the oocyte. The Bb is located to one side of the nucleus (a fragment of the nucleus is colored in red) and contains an elaborate network of Golgi stacks (brown). Golgi and mitochondria (blue) are arranged around the centriole (green; only one centriole of the pair is visible).

Fig. 5. Polarity of mouse early oocytes

Electron microscopy images of ultrastructure (A, B) and immunogold staining (C) of P0 oocytes. A. An intercellular bridge (circled) connects a P0 oocyte to another oocyte, which is not visitble because it is located on a different plane. Note the characteristic distribution of Bb components: a centriole (arrowhead) at the intercellular bridge is surrounded by Golgi stacks (G); mitochondria (m) are more externally located and are interspersed with the cisternae of the rough endoplasmic reticulum (RER). The synaptonemal complex (open arrow) is visible in the oocyte nucleus (n). The somatic cell (sc) is also labeled. B. In this view, using a higher magnification of the Bb in a P0 oocyte, the centriole (arrowhead), with the adjacent pericentriolar material (PCM; arrows), is surrounded by the Golgi stacks (G). Mitochondrion (m), nucleus (n), and cisternae of the rough endoplasmic reticulum (RER) also also labeled. C. Immunogold labeling with PCM-1 antibody: nanogold particles marking the location of PCM-1 protein are visible within the aggregates of pericentriolar material (arrows) located in the vicinity of the centriole (arrowhead). Scale bar is equal to 1 μm in A, 500 nm in B, and 200 nm in C.

Fig. 6. Polarity of mouse early oocyte; three-dimensional reconstruction of two P0 oocytes connected by intercellular bridge

A-C. Three different views of two oocytes connected by an intercellular bridge. A, B. For clarity, these views show the nucleus (red) and Bb (yellow) only. Panel B shows the cytoplasmic bridge (black) connecting two oocytes. In panel A, the same bridge, although present, is not shown. C. This view shows the position of the Bb, nucleus (red), mitochondria (gray), and the cytoplasmic bridge (black).

Fig. 7. Polar formation of multivesicular aggregates (MVAs)

Electron microscopy images of a P0 oocyte showing the location of MVAs, which form by budding off from the Golgi stacks. A. Located at one pole of the nucleus (n) is the Bb, with a pair of centrioles (double arrowhead and arrowhead). The centrioles are surrounded by electron-dense pericentriolar material (arrows) and more peripherally by hemispherically distributed Golgi stacks (G). The asymmetry in the distribution of organelles is further shown by the localization of MVAs (v) at one pole of the oocyte, close to the oocyte membrane. The vesicles are filled with electron-dense material. Mitochondria (m), nuclear envelope (ne), and somatic cell (sc) are also labeled. B. The perinuclear cytoplasm bordering the Bb is occupied by mitochondria interspersed with cisternae of the rough endoplasmic reticulum (RER). The nuclear envelope (ne) and nucleus (n) are also labeled. Scale bars are equal to1 μm.

Fig. 8. The ancestral polarity of animal oocyte

We believe that it is the positioning of the centriole at the cytoplasmic bridge that imposes the axial polarity on the oocyte, and that the cytocentric role of the centrioles in the aggregation of various cytoplasmic organelles are the features characteristic of developing oocytes across the animal kingdom, i.e., in hypothetical ancestor species. The cytocentric role of the centrioles results in the formation of Bbs that are composed of numerous mitochondria and some Golgi stacks in Xenopus and of numerous Golgi stacks and some mitochondria in mouse early oocytes. In Xenopus, the aggregation of mitochondria around the centrioles probably occurs via mitochondrial movement on the microtubules radiating from the cytocenter (Kloc et al., 2004). Although, at present, it remains unknown if the microtubules participate in the aggregation of the organelles around the centrioles in mouse oocytes, the presence of pericentriolar material (PCM) that is known to be involved in microtubule polymerization strongly suggests such a possibility.