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 pronucleus development and DNA synthesis, as well as the ultrastructure of intranuclear bodies, in bovine zygotes produced in vitro. Bovine oocytes were matured and fertilized in vitro, and sperm penetration and pronucleus development were examined. DNA synthesis was investigated by sequential incubation with [3H]- and [14C]thymidine followed by autoradiography on semithin sections. Ultrathin sections for transmission electron microscopy were prepared from the same zygotes. Sperm penetration 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 developed than its paternal counterpart. DNA synthesis was initiated at 14-15 hr, and the S-phase lasted for 8-10 hr. The most prominent ultrastructural entities of the pronuclei were the nucleolus precursor bodies (NPBs). During the S- and G2-phases, the NPBs spatially associated with clusters of interchromatin-like granules. The two components were firmly attached to each other by an electron-dense reticulum. During the late G2-phase, the NPBs were apparently detached from the interchromatin-like granules and the electron-dense reticulum again. The interaction between the intranuclear bodies and granules appears to be comparable with the situation previously described for in vivo-produced bovine zygotes (J Laurincík et al., Mol Reprod Dev 43:62-69, 1996), except for the lack of vacuolization of the NPBs during the S-phase in vitro.