CYTOCHEMICAL AND IMMUNOCYTOCHEMICAL ULTRASTRUCTURAL-STUDY OF NUCLEOPROTEINS DURING NUCLEOLOGENESIS IN 8-CELL BOVINE EMBRYOS

The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, bas ic lysine-rich and histone proteins in 8-cell bovine embryos. Two silv er staining techniques, ethanolic phosphotungstic acid (PTA) and immun ocytochemical methods using specific antibodies, were applied at the u ltrastructural level. The silver-stained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus prec ursor bodies (NPBs). The amounts of these proteins increased during th e transformation of the NPBs into the fibrillogranular nucleolus. At t his stage the well-developed dense fibrillar components encircling fib rillar centres showed intense staining. PTA-positive (basic lysine-ric h) proteins were present within most nucleolar structures during nucle ologenesis as well as in the chromatin. Histones H2B, H3 and H4 were c oncentrated throughout the chromatin including the nucleolus-associate d chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multiva cuolated NPBs, both in the fibrous mass as well as inside the vacuoles . Nucleolar histones appeared with the massive penetration of DNA into the NPBs. We suggest that nucleologenesis may serve as a criterion of normal early embryonic development and that the proteins involved in the process of nucleologenesis and transcription could be used as chem ical markers of nucleolar function.