Preimplantation development of giant triploid zygotes in the mouse.

Giant oocytes or two-cell embryos have been reported in various mammalian species. They may arise during multiplication of oogonia, after fusion of two oogonia or, more probably, when nuclear division is not accompanied by cytoplasmic division. The ultimate fate of these giant embryos is not well known. In our laboratory, giant two-cell mouse embryos have been occasionally observed. Recently, we observed two giant one-cell zygotes in the same species. Both showed two female pronuclei and one male pronucleus, as well as two second polar bodies localized at opposite poles of the embryo. These two giant zygotes showed normal viability and developmental capacity. Their triploid nature was confirmed by cytogenetic analysis. In order to study this interesting phenomenon in more detail, we produced giant oocytes containing two germinal vesicles by cell fusion and cultured them in vitro. About one third of them extruded two first polar bodies; in the second group only one polar body was observed, whilst the last group was without polar bodies. When parthenogenetically activated, the consistent answer analogical to that observed in "in vivo" oocytes was only observed when oocytes with two polar bodies were activated. The implication for IVF technologies is discussed.

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

The objective of the study was to characterize the ultrastructure changes and biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural studies demonstrated that the morphology of cytoplasts produced by etoposide-cycloheximide treatment were indistinguishable from intact metaphase I and II oocytes. Moreover, polar bodies formed by chemical enucleation were in almost all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and because 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 chromosomes during chemical enucleation. After etoposide-cycloheximide treatment, histone H1 kinase activity remains high for the ensuing 6-8 h before declining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity reached basal levels. The activity of this kinase rose sharply to reach maximal levels within 4 h when the enucleated oocytes were removed from the inhibitor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphologically normal, thus suggesting that these enucleated cells are suitable for 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.

Mouse oocyte maturation: the effect of modified nucleocytoplasmic ratio.

Fully grown mouse oocytes isolated from large antral follicles and cultured in vitro complete their maturation up to the second metaphase with extrusion of the first polar body (1PB) with a 40/50 proportion (80%). When their cytoplasmic volume is, however, reduced before the onset of culture, the frequency of oocytes completing maturation gradually decreases. In the half oocytes, 66% (33/50) extruded 1PB, while in third oocytes the proportion was 57% (28/49) and in quarter oocytes no polar bodies were extruded. The time course of germinal vesicle breakdown was also delayed in comparison to the decreased cytoplasmic volume. Moreover, the isolated germinal vesicles surrounded with a thin cytoplasmic rim only remained intact after a prolonged culture. The full competence of complete maturation can be restored by fusion of an additional cytoplast to the manipulated nucleate parts. We postulate that a critical nucleocytoplasmic volume ratio is absolutely necessary for normal maturation in mammalian oocytes.