High-resolution autoradiographic localization of DNA-containing sites and RNA synthesis in developing nucleoli of human preimplantation embryos: a new concept of embryonic nucleologenesis

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Abstract
Human embryos from the 2-cell to the morula stage, obtained by in vitro fertilization, were incubated with [3H]thymidine or [3H]uridine so as to achieve labelling of all replicating nuclear DNA and the newly synthesized RNA, respectively. The label was localized in different structural components of developing nucleoli using electron microscopic autoradiography. Careful study of the relationship between the structural pattern and nucleic acid distribution made it possible to define four stages of embryonic nucleologenesis. Homogeneous nucleolar precursors (i) consist of nucleolar matrix elements appearing as filaments of 3 nm thickness, (ii) do not contain recently replicated DNA and (iii) lack RNA synthetic activity. Penetration of DNA into these bodies is a key event leading to their transformation into heterogeneous nucleolar precursors. In addition to the 3nm matrix filaments, two types of 5 nm fibrillar components can be recognized in them. The denser type contains DNA and is the site of nucleolar RNA synthesis, while the more loosely arranged 5 nm fibrils are not labelled with [3H]thymidine and apparently represent the newly produced pre-rRNA detached from the transcribing rDNA filament. Compact fibrillogranular nucleoli are characterized by the first appearance of the granular component and reduction of the nontranscribing part of the fibrillar component, both indicating the activation of the machinery for rRNA processing. Finally, the granular component is most evident in reticulated nucleoli, occupying mostly the inner parts of their nucleolonema, while the transcription sites tend to be located at the nucleolar periphery. Our findings advocate a unique concept of embryonic nucleologenesis, different from any other nucleolar event during the cell cycle of differentiated cells. This developmental pattern is characterized by a gradual activation of rRNA synthesis and processing, mediated by progressive association of rDNA and, later on, the newly formed pre-rRNA with pre-existing nucleolar matrix elements that are originally topically separated from nucleolar organizer regions. This model may have a general validity in early animal embryos despite some interspecies variability in the timing of individual steps and resulting structural peculiarities.