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Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV x A2) suppresses female pronuclear development. The cytoplasm of GV x A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger ([approximately]x1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV x A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged ([approximately]x1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV x A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2-3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach M-phase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic x A2). By contrast to the micropronuclei formed in GV x A2 heterokaryons, normal pronuclear development occurred in G2 mitotic x A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development.

Two experiments analyzing the impact of suppression of chromatid separation and protein synthesis on the metaphase to anaphase transition and maturation promoting factor (MPF) functions reveal that suppression of the former blocks the transition while that of the latter reduces MPF functions. Protein synthesis and chromatid separation suppression do not affect meiotic progression and MPF functions, respectively, indicating the specific effects of the two processes. The escape of the oocytes from metaphase arrest with cycloheximids and etoposide indicates the gradual decline in MPF functions to basal levels due to normal protein turnover.

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. [ABSTRACT FROM AUTHOR]

The presence of checkpoint mechanisms which are able to recognize damaged chromatin and thereafter to prevent exit from metaphase I has been investigated in giant mouse oocytes produced by fusion of a normal metaphase I oocyte with an equivalent oocyte with damaged chromatin. The presence of damaged chromatin did not prevent the onset of anaphase I in both sets of chromatin in the fused cells. Interestingly, fused or unfused cells containing only damaged chromatin failed to enter anaphase and persisted instead in a metaphase-like state. These results demonstrate the fragility of checkpoint controls in mammalian female germ cells. [ABSTRACT FROM AUTHOR]