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.

Animals↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Oocyte maturation and embryonic failure.

Embryonic development is readily compromised by imperfections introduced during the process of oocyte maturation. We discuss the nature and causes of these imperfections, particularly in oocytes exposed to inappropriate hormonal regimes in vivo or to culture systems designed to induce the maturation of oocytes in vitro. The acquisition of developmental competence involves the synthesis and storage of a wide range of molecules during oocyte growth followed by the reprogramming and ordered utilization of these stored products during maturation, fertilization and early embryogenesis. The regulatory signals for these molecular changes are produced by the follicle cells in response to circulating levels of gonadotrophins; we report that some ovarian stimulation protocols distort these signals thereby disrupting molecular reprogramming of the oocyte and reducing subsequent developmental competence. The aspiration of immature oocytes from antral follicles followed by their maturation in vitro is a potential alternative to hormonal stimulation of patients in IVF treatment. Although relatively successful in a variety of animals, the production of fully viable human embryos by in-vitro maturation is still unsatisfactory despite the use of a wide variety of culture protocols. Our data suggests that the key to maturation and embryo viability in vitro resides in the follicle cell compartment rather than the oocyte. Because of rapid luteinization changes, follicle cells in culture probably fail to provide the maturing oocyte with the necessary ordered set of instructive signals and nutrients needed for the acquisition of developmental competence. Although much remains to be discovered about the nature, concentration and transmission of signals, nevertheless it is already clear that different steroids, matrix metalloproteinases and growth factors are involved in conferring viability on the maturing oocyte. Major improvements in the yield of viable embryos from in-vitro matured oocytes can be anticipated from a systematic analysis of somatic signals from the pre-ovulatory follicle.

Animals↗