Three-Dimensional Ultrastructural Analysis of RNA Distribution within Germinal Granules of Xenopus

Abstract

The germ plasm is a specialized region of oocyte cytoplasm 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.

Mouse oocytes within germ cell cysts and primordial follicles contain a Balbiani body

“…The Balbiani body GFM morphologically resembles germinal granule precursors. A connection to germinal granules has been proven in Xenopus, where GFM has been shown to contain germinal granule proteins and RNAs that are incorporated into germ cells (15,16,24,25). Likewise in Drosophila, a key component of the germinal granules, oskar RNA, associates with the newly formed Balbiani body (26).…”

Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud

“…In Xenopus, the formation of the mitochondrial cloud and germinal granules is a lengthy and elaborate process that starts in oogonia and concludes in stage I oocytes (al-Mukhtar and Webb, 1971;Coggins, 1973;Billet and Adam, 1976;;Heasman et al, 1984;Wylie et al, 1985;Kloc et al 2002Kloc et al , 2004a. In stage I oocytes, the mitochondrial cloud is a spherical organelle (approximately 40 µm in diameter) that juxtaposes the nucleus and always faces the future vegetal pole of the oocyte.…”

“…The mitochondrial cloud present in previtellogenic stage I oocytes of Xenopus is composed of numerous mitochondria and germinal granules that are believed to be a determinant of germ cell fate (Eddy, 1975;Billet and Adam, 1976;Heasman et al, 1984). The cloud also contains various localized RNAs, some of which (e.g., Xcat2 mRNA) are present within the germinal granules and others (e.g., Xpat mRNA) are present at the periphery and outside the germinal granules (Mosquera et al, 1993;Kloc and Etkin, 1995a, b;Hudson and Woodland, 1998;Kloc et al, 2000Kloc et al, , 2002Kloc et al, , 2004b). …”

Polarisation des oeufs et des embryons : principes communs

“…Ce domaine cytoplasmique, appelé nuage mitochondrial, contient des ARNm spécifiques, dont le détermi-nant germinal Xcat2 [52][53][54]. En s'étalant contre le cortex végé-tatif, le nuage mitochondrial localise les granules germinaux (Figure 4, Fc) [63,64].…”