Kinetic analysis of cell population growth during cultivation in vitro.

XL-2 cells (Xenopus laevis) were used for kinetic analysis of cell population growth. The dependence of the time of cell duplication on the percentage of cells in the G0 phase of the cell cycle was studied and described by a mathematical expression. Possible causes of the changes in the ratio between the percentage of cells in the cell cycle and that in the G0 phase were analyzed. These are the decrease in the percentage of cells in the G0 phase due to the increase in the number of dividing cells, their position in the cell islets, the number of nuclei, the relative position of cells in the G0 phase. It was shown that the loss of the free edge by cells during their transition to the second layer of the cell islets without any changes in spreading led to a significant increase in the percentage of cells in the G0 phase. The percentage of cells in the G0 phase increased about five times for multinuclear cells. Analysis of the position of cells in the G0 phase showed that these cells were mostly in groups of two, three or four. Studies of a real cell culture in the logarithmic phase of growth (48-120 h of cultivation) showed that the percentage of cells in the G0 phase did not virtually change and all processes were equalized by one another. We propose a new method to determine the cell cycle duration under conditions from the time of cell culture duplication and the data on the percentage of cells in the G0 phase. This method can be used when traditional approaches using BrdU or [3H]]thymidine are difficult to implement or are unacceptable.

pEg7, a new Xenopus protein required for mitotic chromosome condensation in egg extracts.

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

Cell cycle analysis and synchronization of the Xenopus cell line XL2.

We have determined the length of the cell cycle and its different phases in a permanent Xenopus tadpole cell line, XL2. Following BrdU labeling, the total length of the cell cycle was estimated as 28 h. The different phases of the cell cycle, G1, S, G2, and M were, respectively, 14 h, 10 h 45 min, 2 h 30 min, and 54 min. Knowing these parameters, we were able to develop methods that selectively enrich cells in different phases of the cycle. Treatment with aphidicolin resulted in a S phase block in which more than 85% of the cells showed S phase chromosomes. Almost 60% of the cells were arrested in mitosis after a double block with aphidicolin/nocodazole or aphidicolin/ALLN (acetyl-leucyl-leucyl-norleucinal) treatment. This synchronization protocol will greatly facilitate studies of biochemical events associated with specific gene regulation through the cell cycle. Our synchronization protocol does not disturb cell metabolism as the expression of cyclin B2 during the cell cycle is in agreement with the results obtained with mammalian cells.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly.

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Cyclin D2 arrests Xenopus early embryonic cell cycles.

Xenopus cyclin D2 mRNA is a member of the class of maternal RNAs. It is rare and stable during early embryonic development. To investigate the potential role of cyclin D2 during early embryonic cell cycles, cyclin D2 was injected into one blastomere of a two-cell embryo. This injection induced a cell cycle arrest in the injected blastomere. To analyze more precisely the mechanism of this arrest, we took advantage of cycling egg extracts that recapitulate major events of the cell cycle when supplemented with demembranated sperm heads. When Xenopus cyclin D2 is added to egg extracts, the first round of DNA replication occurs as in control extracts. However, Xenopus cyclin D2 blocks subsequent rounds of DNA replication and the oscillations of histone H1 kinase activity associated with cdc2 kinase, indicating that the cell cycle is arrested after the first S-phase. The block induced by Xenopus cyclin D2 is not due to a lack of the mitotic cyclin B2 that accumulates normally. Radiolabeled Xenopus cyclin D2 enters nuclei after completion of the first S-phase and remains stable over the entire period of the arrest. These features suggest that Xenopus cyclin D2 could play an original role during early development, controlling the G2-phase and/or the G2/M transition.

Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication.

The capacity to initiate DNA replication appears during oocyte maturation in Xenopus. Initiation of S phase is driven by several components which include active cyclin/cdk complexes. We have identified three Xenopus cyclin E clones showing 59% amino acid identity with human cyclin E. The recruitment of cyclin E mRNA, like cdk2 mRNA, into the polysomal fraction during oocyte maturation, results in the accumulation of the corresponding proteins in unfertilized eggs. Cyclin E mRNA remains polyadenylated during cleavage and anti-cyclin E antibodies detect Xlcyclin E in embryonic nuclei at this time. Cdk2 protein is necessary for the phosphorylation of radiolabelled cyclin E added to egg extracts. Radiolabelled Xlcyclin E enters interphase nuclei and, though stable through interphase and mitosis, is not associated with condensed mitotic chromatin. In egg extracts, endogenous Xlcyclin E rapidly associates with nuclei before S phase and remains nuclear throughout interphase, becoming nucleoplasmic in G2/prophase. Under conditions where initiation of replication is limiting in extracts, Xlcyclin E associates only with those nuclei that undergo S phase. These features are entirely consistent with the view that Xlcyclin E is required for initiation of S phase.

Overview of the regulation of S-phase in Xenopus egg extracts.

Recently, several components required for nuclear DNA replication and involved in the regulation of S-phase within the cell cycle have been identified. Though detailed models of these events are not available, we focus on recent developments arisen from analysis in Xenopus egg extracts.

Production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae.

Bovine inner cell masses (ICMs) were isolated by immunosurgery at day 8 or 10, or by dissection at day 14, and combined with day-5.5 morulae. Aggregation was obtained between 89%, 62%, and 0% of the day-5:day-8, day-5:day-10, day-5:day-14 composites, respectively. Chromosome analysis of composites, respectively. Chromosome analysis of composites potentially carrying the 1/29 translocation as a chromosome marker and temporarily transferred to the bovine uterus for 8 days showed that chimaeric day-14 embryos can be obtained from day-5:day-8 aggregation. The definitive transfer of eight day-5:day-8 and 11 day-5:day-10 composites resulted in the birth of six and four calves, respectively; five of the six, but none of the four, were chimaeric. The five chimaeras showed mostly the ICM phenotype. The morphological differences between ICMs at different stages of development were examined by electron microscopy and related to the success of the aggregation technique. It is concluded that bovine embryonic cells can regulate for at least 3 days difference in development but not 5 days even though aggregation is still possible.

La récolte et le transfert d'embryons chez la chèvre Angora.

The objective of this study was to analyze the efficiency of embryo collection and transfer in Angora goats during the breeding season. Synchronization was obtained by placing an intravaginal sponge for either a short period of time (11 days + PGF(2alpha)) or a long period (17 to 21 days). Thirty-three goats were superovulated, embryo collection was not attempted on seven goats (four did not respond to the superovulation treatment, three did not show estrus), and four gave oocytes only. The embryos of one goat are still frozen. The remaining 21 goats produced 285 embryos of which 187 (66%) were transferred (fresh or thawed) to 66 recipients; 36 (55%) recipients remained pregnant and gave birth to 63 kids (34% of the transferred embryos). The superovulation and transfer techniques used in this study provided a large number of transferable embryos. However, the percentage of embryos that reach term could be improved by more strict selection of the recipients and of the embryos to be transferred, and by improving the embryo freezing technique.

Nucleolus organizer regions and nucleoli in preattachment bovine embryos.

Embryos (1-cell to elongated blastocyst stage) were recovered from superovulated heifers at surgery (Days 2-4; oestrus = Day 0), after slaughter (Day 4), or by transcervical flushing (Days 6, 7 and 14). The 175 embryos were cultured for 4, 8, 24 or 48 h, fixed on slides and sequentially stained with Giemsa and silver nitrate. Twenty-three 2-cell to blastocyst-stage embryos were fixed, embedded and examined by transmission electron microscopy. Argentophilic nucleolus organizer regions (Ag-NORs), indicative of transcriptionally active rRNA genes, were observed in embryos in which short- or long-term culture began at or after the late 8-cell stage. The nucleoli of embryonic cells also showed increased affinity for silver from the 8-cell stage onward. Differences in the number of Ag-NORs observed after the 8-cell stage reached statistical significance only when Day-5 and Day-7 embryos cultured for 4 h were compared. Ultrastructurally, the nucleoli were seen to develop from small, dense, fibrillar masses at the 2-cell stage, to ring-shaped structures (signifying a low level of activity) at the 8-cell stage. At the 16-cell stage the nucleoli became reticulated, suggesting an increase in activity, and by the morula and blastocyst stages they were characteristic of fully active nucleoli. It is concluded that a significant transcriptional activity of the rRNA genes in the embryos of cattle begins around the 8-cell stage.

Development of the nucleolus in early goat embryos.

In vivo nucleologenesis was studied in goat embryos from the pronuclear stage to the blastocyst stage by light and electron microscopy. Ultrastructural changes of the nucleoli were characterized by the following progression: homogeneous electron-dense fibrillar primary nucleoli in the pronucleus; small, dense fibrillar masses dispersed in clusters of chromatin at the two-cell stage; ring-shaped nucleoli made up of a fibrillar center surrounded by a layer of dense fibrillar components at the four-cell stage; reticulated nucleoli composed of a three-dimensional network of fibrillar components surrounded by small amounts of granular components at the eight-cell stage; fully developed compact-type nucleoli consisting of several fibrillar centers each surrounded by a layer of fibrillar components and abundant granular components in morulae and blastocysts. Moreover, it was concluded that activation of rRNA transcription, as evidenced by specific silver nitrate staining of the nucleolus organizer regions of metaphase chromosomes, occurs at the two- to four-cell stage and that the morphological changes accompanied a substantial increase in nucleolar transcriptional activity up to the blastocyst stage. This study provides evidence that a structure-function relationship exists during nucleologenesis in goat embryos.

Effect of serum on organogenesis of the rat testis in vitro.

It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the alpha globulin group. Among a series of purified serum proteins tested, alpha 2-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.