Chemically enucleated mouse oocytes: ultrastructure and kinetics of histone H1 kinase activity

The objective of the study was to characterize the ultrastructure changes a nd biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural stu dies demonstrated that the morphology of cytoplasts produced by etoposide-c ycloheximide treatment were indistinguishable from intact metaphase I and I I oocytes. Moreover, polar bodies formed by chemical enucleation were in al most all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and bec ause they contained a slightly higher number of cytoplasmic organelles. The mode of polar body formation, however, in normal and chemically enucleated oocytes differs substantially: spindle involvement is important for normal polar body extrusion but plays no part in the protracted expulsion of chro mosomes during chemical enucleation. After etoposide-cycloheximide treatmen t, histone H1 kinase activity remains high for the ensuing 6-8 h before dec lining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity rea ched basal levels. The activity of this kinase rose sharply to reach maxima l levels within 4 h when the enucleated oocytes were removed from the inhib itor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphol ogically normal, thus suggesting that these enucleated cells are suitable f or cloning studies. Although effective in mouse oocytes, we postulate that certain modifications to the enucleation technology are necessary before a reliable non-invasive protocol for ungulate oocytes will be available. (C) Inra/Elsevier, Paris.