A DETAILED ANALYSIS OF PRONUCLEUS DEVELOPMENT IN BOVINE ZYGOTES IN-VITRO - CELL-CYCLE CHRONOLOGY AND ULTRASTRUCTURE

The aim of the present experiment was to analyze the chronology of pro nucleus development and DNA synthesis, as well as the ultrastructure o f intranuclear bodies, in bovine zygotes produced in vitro. Bovine ooc ytes were matured and fertilized in vitro, and sperm penetration and p ronucleus development were examined. DNA synthesis was investigated by sequential incubation with [H-3]. and [C-14]thymidine followed by aut oradiography on semithin sections. Ultrathin sections for transmission electron microscopy were prepared from the same zygotes. Sperm penetr ation was noted for the first time at 4 hr after in vitro insemination and reached a maximum at 6 hr. Pronucleus formation was initiated at 4 hr, and up to at least 11 hr the maternal pronucleus was more develo ped than its paternal counterpart. DNA synthesis was initiated at 14-1 5 hr, and the S-phase lasted for 8-10 hr. The most prominent ultrastru ctural entities of the pronuclei were the nucleolus precursor bodies ( NPBs). During the S- and G(2)-phases, the NPBs spatially associated wi th clusters of interchromatin-like granules. The two components were f irmly attached to each other by an electron-dense reticulum. During th e late G(2)-phase, the NPBs were apparently detached from the interchr omatin-like granules and the electron-dense reticulum again. The inter action between the intranuclear bodies and granules appears to be comp arable with the situation previously described for in vivo-produced bo vine zygotes (J Laurincik et at., Mot Reprod Dev 43:62-69, 1996), exce pt for the lack of vacuolization of the NPBs during the S-phase in vit ro. (C) 1998 Wiley-Liss, Inc.