Over the past few years, we have witnessed a burgeoning series of papers addressing the role of calcium signalling in cell cycle control. In this review I will attempt to bring together all the diverse threads and discuss new concepts that have arisen from the most recent data. Because the major part of the data concerns mitosis/meiosis entry and exit, I have focused on these areas. I will jointly refer to meiotic and mitotic phases of the cell cycle as M-phase because these phases are highly comparable. Studies of the cell cycle involve a huge range of species, from plants to humans. I will, however, restrict this review to the work performed in early embryos. I apologise in advance to contributors to this field whose names I do not mention because they do not work on embryos.

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethyl-aminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.

Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.

Microsomal fractions of Xenopus oocytes release preloaded 45Ca2+ when treated with inositol triphosphate (InsP3). The effective concentration of InsP3 required for half-maximal release (EC50) is 59 nM and maximal release occurs at ∼ 2 μM InsP3. Uptake and release of 45Ca2+ are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50 for InsP3-induced Ca2+ release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+ release causes the tyrosine phorphorylation of a 42000 (Mr 42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+ release, thereby inhibiting phosphorylation of MAP kinase.

It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By peforming immunofluorescence labelling and Western blots with antibodies directed against some of the α and β subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, α2 and α5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that α2 and α5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich three cytoskeletal proteins known to be linked to the β chain of integrins: talin, vinculin and α-actinin. Moreover, we found that talin and α-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.

Following fertilisation, the sperm triggers a series of intracellular changes which initiate oocyte activation and pronuclear formation. Oocyte activation can also be induced artificially by several chemicals, such as the calcium ionophore A23187. The sperm nucleus is transformed into the male pronucleus through the interaction of oocyte cytoplasmic factors. The profile of protein synthesis is different in bovine oocytes following fertilisation and parthenogenetic activation. The formation of male and female pronuclei was not blocked by the presence of the protein synthesis inhibitor cycloheximide. These results suggest that bovine oocyte activation by sperm and parthenogenetic activation induce different cytoplasmic responses for protein synthesis and that new protein synthesis is not required for male pronuclear formation in bovine zygotes.

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Previous studies of tetraploid↔diploid mouse chimaeras and mosaics have revealed that tetraploid cells do not contribute equally to all tissues of the conceptus. In this study we have shown that, within 30 h of aggregating cleavage stage embryos, tetraploid cells were non-randomly distributed among different tissues of the early blastocyst. They were preferentially allocated to the mural trophectoderm regardless of cell size at the time of aggregation. This early effect may underlie the restricted distribution of tetraploid cells at later stages. We have demonstrated for the first time that ploidy can influence the relative position of blastomeres in the preimplantation embryo.

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes during in vitro oocyte maturation and fertilisation. Binding was quantified by the intensity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at the periphery of oocytes following incubation with biotinylated sperm plasma membrane proteins and subsequent incubation with TRITC-avidin. Bovine oocytes were matured in vitro. Sample groups were removed after 0,6 and 22 h, or inseminated and further cultured for 24 or 48 h. Oocytes were denuded of cumulus cells and zona pellucida and co-incubated with 56 μg biotinylated bovine sperm plasma membrane protein for 45 min in 150 μl drops of saline-BSA. Controls were incubated for the same time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixed and transferred to mounting medium. Oocytes were scanned with a confocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intensity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ellipse. Binding of sperm plasma membrane continued to increase throughout in vitro oocyte maturation and fertilisation (9051, 24318 and 49953 for 0 and 22 h in vitro matured oocytes and fertilised oocytes, respectively; p = 0.0001). A dramatic decrease in sperm plasma membrane binding to the oocyte plasma membrane was observed in 2-cell embryos (mean intensity = 24477, p = 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocytes incubated with TRITC- avidin only were barely visible (integrated fluorescence intensity values ranged from 8 to 3757).

Nuclei of diplotene (dictyate) primordial oocytes (PO) were transferred to metaphase II oocytes and to activated mouse oocytes using cell fusion techniques. In a metaphase II oocyte, the PO nucleus condenses within 2–3 h to bivalents which become arranged on the first meiotic spindle. After oocyte activation, homologous chromosomes segregate between the oocyte and the first polar body, and a diploid pronucleus-like nucleus reforms from the one set of dyads. This nucleus condenses in the first embryonic mitosis into 40 ‘somatic’ chromosomes which coexist in the common metaphase plate with 20 somatic chromosomes originating from the female pronucleus. Shortening of the time between fusion and activation to about 1 h prevents bivalent differentiation. The PO nucleus condenses only partially and reforms, after oocyte activation, a pronucleus-like nucleus. This nucleus gives rise at the first embryonic mitosis to 20 bivalents which coexist with 20 somatic chromosomes originating from the female pronucleus. A PO nucleus introduced into an activated egg completes the first cell cycle as an intact interphase nucleus. It never condenses in the first embryonic mitosis into bivalents, and undergoes only initial condensation (preceding bivalent differentiation). These results indicate that: (1) condensation into bivalents, meiotic spindle formation and first meiotic division can be greatly accelerated by the introduction of an early diplotene (dictyate) oocyte nucleus into a metaphase II oocyte, and (2) depending on whether the diplotene nucleus enters the first embryonic (mitotic) cell cycle after just initiating or after completing the first meiosis, it gives rise at the first cleavage division to meiotic (bivalents) or ‘somatic’ chromosomes respectively.