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The capacity of oocytes to fully support meiotic maturation develops gradually during oocyte growth. Growing oocytes accumulate proteins and mRNAs required for this process. However, little is known about the identity of these factors. We performed a differential proteomic screen comparing the proteomes of growing stage-IV oocytes, which do not undergo meiotic maturation in response to progesterone, with fully grown stage-VI ones, which do. In 2D gels of stage-VI oocytes, we identified a group of four protein spots as EP45 (estrogen-regulated protein 45 kDa), which belongs to the family of serine protease inhibitors and is also known as Seryp or pNiXa. Western blot analysis after mono- and bi-dimensional electrophoreses confirmed the accumulation of certain forms of this protein in oocytes between stages IV and VI. EP45 mRNA was not detectable in oocytes or ovaries, but was expressed in the liver. A low-mobility isoform of EP45 was detected in liver and blood, whereas two (occasionally three or four) higher-mobility isoforms were found exclusively in oocytes, suggesting that liver-synthesized protein is taken up by oocytes from the blood and rapidly modified. Alone, overexpression of RNA encoding either full-length or N-terminally truncated protein had no effect on meiotic resumption in stage-IV or -VI oocytes. However, in oocytes moderately reacting to low doses of progesterone, it significantly enhanced germinal-vesicle breakdown, showing a novel and unsuspected activity of this protein. Thus, EP45 accumulates in growing oocytes through uptake from the blood and has the capacity to act as an 'oocyte-maturation enhancer' ('Omen').

Deadenylation is an intimate part of the post-transcriptional regulation of maternal mRNAs in embryos. EDEN-BP is so far the only known member of a complex regulating the deadenylation of maternal mRNA in Xenopus laevis embryos in a manner that is dependent on the 3'-untranslated region called EDEN (embryo deadenylation element). In this report, we show that calcium activation of cell-free extracts triggers EDEN binding protein (EDEN-BP) dephosphorylation and concomitant deadenylation of a chimeric RNA bearing Aurora A/Eg2 EDEN sequence. Deadenylation of mRNA deprived of EDEN sequence (default deadenylation) does not change with egg activation. Kinase and phosphatase inhibitors downregulate EDEN-dependent deadenylation but they do not substantially influence default deadenylation. Using indestructible Delta90 cyclin B to revert interphase extracts to the M-phase, we show that modulation of EDEN-dependent deadenylation is independent of M-phase promoting factor (MPF) activity. These results suggest that the increase in EDEN-dependent deadenylation following egg activation is achieved, at least partially, via dephosphorylation and/or phosphorylation of regulatory proteins, including EDEN-BP dephosphorylation. This regulation proceeds in a manner independent from MPF inactivation.

Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

Fusion of large and small karyoplasts produced from metaphase II mouse oocytes with interphase blastomeres from 2-cell and 8-cell embryos (volume ratio of partners, 1:1) results in premature chromosome condensation (PCC) of the interphase nucleus in the majority of the fusion products (hybrids). Fused under the same experimental protocol, oocyte-derived cytoplasts also induce PCC of the blastomere nucleus in the fusion products (cybrids) provided they originate from recently ovulated oocytes (141/2-15 h after injection of human chorionic gonadotrophin (HCG)). In cytoplasts derived from older oocytes (16-20 h post-HCG) chromosome condensation activity gradually decreases with time as can be inferred from the increasing proportion of cybrids retaining interphase blastomere nuclei. However, even the oldest cytoplasts (19-20 h post-HCG) can induce PCC if the cytoplast volume significantly exceeds the volume of the interphase partner (7:1). We postulate that the condensation activity is predominantly bound to the nuclear apparatus (most probably to the chromosomes), and that in the cytoplasm of metaphase II mouse oocyte it decreases with post-ovulatory age.