Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus

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.