In situ hybridization using [3H]poly(U) was applied to developing oocytes of Xenopus laevis, which had been fixed in Bouin's solution. Tissue sections were pretreated with DNase I, annealed with [3H]poly(U) and post-treated with RNase A and TCA. After the autoradiographical processing, silver grains over the oocyte were counted. As a result of the control experiments which included RNase A, RNase T2, DNase I and Pronase E hydrolysis and Cordycepin (3'-deoxyadenosine) incubation before in situ hybridization, it was concluded that the poly(U)-binding activity detected upon the oocytes was due to the possible presence of poly(A)+RNAs. Spatial distribution of the poly(U)-binding sites changed during the development of the oocytes; in a small oocyte before the pachytene stage, silver grains developed over the nucleus, while in a larger oocyte after the diplotene the grains were concentrated over the cytoplasm. After yolk platelets were deposited in the cytoplasm, two types of poly(U)-binding activities were noted; a bound-type activity which was firmly associated with the cytoplasm, so that the positions of the silver grains were not influenced by fixation, and an unbound type which did not bind so firmly to the cytoplasm and was therefore easily influenced by inflow of fixative. The bound-type activity persisted in the cytoplasm throughout the oogenesis, but the unbound type appeared only after the vitellogenesis, especially in the yolky cytoplasm. The total poly(U)-binding activity per oocyte increased continuously with the growth of the oocyte.
Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis
