Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs.

The effects of the new cyclin-dependent kinase inhibitors, roscovitine and olomoucine, on oocytes and eggs of Xenopus laevis were investigated and compared with those of 6-dimethylamino purine (6-DMAP). The inhibitory properties of 6-DMAP, olomoucine and roscovitine towards p34cdc2-cyclin B isolated from Xenopus eggs revealed K-IC50 values of 300, 40 and 10 microM respectively. The three compounds inhibited progesterone-induced maturation with M-IC50 values of 200, 100 and 20 microM. These values were consistent with the K-IC50 values but the ratio M-IC50/K-IC50 was higher for roscovitine and olomoucine than for 6-DMAP. The disappearance of spindle and condensed chromosomes without pronucleus formation was observed when 1 mM 6-DMAP was applied for 4 h at germinal vesicle breakdown or at metaphase II, whereas no effect was observed using 1 mM olomoucine or 50 microM roscovitine. Changes in the electrophoretic mobility of p34cdc2 and erk2 were observed only in homogenates of matured oocytes or eggs exposed for 4 h to 1 mM 6-DMAP. When the drugs were microinjected into matured oocytes, olomoucine (100 microM) and roscovitine (50 microM) induced pronucleus formation more efficiently than did 6-DMAP (100 microM). Taken together, these results demonstrate that Xenopus oocytes possess a lower permeability to olomoucine and roscovitine and that these new compounds are suitable for in vivo studies after germinal vesicle breakdown provided they are microinjected.

Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis.

In Xenopus eggs, metaphase II arrest is due to the cytostatic factor that maintains a high level of MPF activity. Kinases are important in this phenomenon since p39(mos) and MAPK play a part in the cytostatic activity whereas p34(cdc2) is the catalytic subunit of MPF. Fertilization induces a rise in intracellular calcium leading to egg activation that can be mimicked by calcium-increasing agents such as calcium ionophore. We have performed on Xenopus eggs a biochemical comparison of the effects of the kinase inhibitor 6-DMAP and the calcium ionophore. Both drugs were able to induce pronucleus formation but the underlying molecular events were different. The inactivation of MAPK occurred earlier in eggs exposed to 6-DMAP. Cyclins B1 and B2 were stable and p39(mos) was proteolysed in 6-DMAP-treated eggs while the three proteins underwent degradation in A23187-treated ones. These results suggest a differential regulation of ubiquitin-dependent proteolysis of cyclin B and p39(mos).

Inhibition of protein tyrosine phosphatases blocks calcium-induced activation of metaphase II-arrested oocytes of Xenopus laevis.

We have studied the effect of a protein tyrosine phosphatases (PTP) inhibitor on calcium-induced activation of Xenopus laevis oocytes arrested at metaphase II. Ammonium molybdate microinjection blocked pronucleus formation following A23187 treatment while cortical granules still underwent exocytosis. Pronuclei still occurred in ammonium molybdate-injected oocytes following 6-DMAP addition. Changes that usually occurred following A23187 exposure were inhibited in the presence of ammonium molybdate in the oocyte: MAPK dephosphorylation, p34(cdc2) rephosphorylation and cyclin B2 and p39(mos) proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin-dependent degradation machinery.

Rat yolk sac explants as a system for studying the regulation of endodermal genes: down-regulation of the alpha-fetoprotein gene by dexamethasone and phorbol ester.

The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis.

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca2+ in follicle cells of amphibians. The possible involvements of these Ca2+ stores in mediating the hormonal control of meiotic maturation are discussed.

Procaine-induced maturation of Xenopus oocytes is mediated by a transient activation of M-phase promoting factor.

We have recently shown that the incubation of Xenopus laevis oocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2 kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16 h, but it was not detected in those exposed for 24 h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16 h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.

Xenopus oocyte maturation: cytoplasm alkalization is involved in germinal vesicle migration.

In Xenopus laevis oocytes a transient increase in intracellular pH has been reported to occur during progesterone-induced maturation. Using a cytological approach, we have systematically analyzed germinal vesicle breakdown and meiotic spindle formation in various experimental conditions either preventing or promoting pHi changes. Injection of a neutral buffer (MOPS pH 6.9) induced a cytosolic acidification of 0.3 pH unit and inhibited by 30% the formation of the maturation white spot after progesterone exposure; in oocytes displaying a white spot, only half showed a spindle, often located far from the plasma membrane. Similar results were observed with a Na-free medium which prevents oocyte alkalization. Injection of an alkaline buffer (Tris pH 9) was able to induce migration of the germinal vesicle in 25% of the oocytes in the absence of progesterone, but failed to induce GVBD. Taken together, these results suggest that the increase in pHi observed during maturation may be involved in the migration of the germinal vesicle towards the plasma membrane. We also incubated oocytes in the presence of procaine, a weak base often used to artificially alkalize the oocyte cytoplasm. The changes induced by exposure to procaine were different from those resulting from alkaline buffer injection. Indeed procaine promoted GVBD, as well as spindle formation and chromosome condensation. However these events appeared without migration of the germinal vesicle, suggesting that the expected alkalization did not occur.

Intracellular calcium and breast-cancer cell-growth and differentiation.

The growth of normal human breast epithelial cells in vitro, as well as those of other cell types is strongly influenced by the concentration of calcium in the culture medium [Ca++]e. The aim of this study was to ascertain if calcium also affects breast tumor cell growth in vitro. To address this question, the metastatic breast cancer cells MCF-7 were grown at low (0.04 mM, L-Ca) and high (2.5 mM, H-Ca) [Ca++]e. In each culture condition, we estimated intracellular calcium levels (Ca++]i from Indo-1 fluorescence by the ratio method. We showed that [Ca++]i increased with [Ca++]e, the [Ca++]i values ranging from approximately 50 to 250 nM. Changes of [Ca++]i ware accompanied by changes of cell shape and cell kinetic parameters. In H-Ca, cells were flat and 3 times larger than in L-Ca and the percentage of cells in the S+G2+M phases as well as the percentage of Ki-67 positive cells rapidly dropped on days 3-4 of culture in contrast to cells grown in L-Ca. In H-Ca, the cell growth arrest corresponded to maximal [Ca++]i which was stable during the stationary phase; at that time, a switch from H-Ca to L-Ca resulted in a drop of [Ca++]i and a resumption of cell growth.. In H-Ca, modifications in cell differentiation parameters such as diminution of ER expression and increases of lipid content and EMA expression were observed as compared to cells grown m L-Ca. Our results suggest that MCF-7 cells have retained some calcium dependency and that agents that can increase [Ca++]i in breast tumor cells may limit their proliferation and trigger at least a partial differentiation.

Syntheses of nucleic acids during spermatogenesis in Nereis diversicolor (annelida polychaeta): a quantitative autoradiographic study.

The development of DNA and RNA synthesis in the germ cell population was studied after a 3H-thymidine or 3H-uridine pulse at each stage of spermatogenesis. The autoradiographic results show that the first sign (after 3 days in vitro) of cellular changes is an increase in RNA synthesis which reaches a maximum at day 5. DNA replication (premeiotic S phase) occurred at day 7, then cells entered meiotic prophase (day 9). Meiotic divisions and spermiogenesis occurred after 11 days. Silver grain counts permit the conclusion that RNA synthesis is clearly higher during premeiotic interphase (days 3-7) than during spermatogonial proliferation (day 0). It appears therefore that male meiotic differentiation in Nereidae is accompanied by increased RNA synthesis.

Endocrine regulation of spermatogenesis in Nereis diversicolor (annelida polychaeta): experimental study of the control of meiotic differentiation.

In Nereidae, spermatogenesis is regulated by a hormone secreted by the supraesophageal ganglion (brain hormone). The hormonal concentration decreases with worm age. Spermatogonial proliferation in young worms proceeds under a high hormonal level whereas differentiation of spermatozoa in aged worms occurs in the absence of brain hormone. Spermatogonia were removed from the endocrine influence of the brain in vitro by the organ culture method. Isolated parapodia produced spermatozoa after 13 days. Control cultures were obtained by associating a parapodium with the prostomium of a young worm. It seems that the brain hormone inhibits the spermatocyte differentiation since removal of the brain results in increased RNA synthesis, followed by DNA replication, meiosis, and spermiogenesis (Bertout, '83). Modalities of the hormonal control of meiosis, especially at the level of the RNA syntheses related to meiotic differentiation, were investigated. Effects of reintroducing the hormonal influence during meiotic differentiation were studied through parapodium-prostomium associations at various time after the brain inhibition has been lifted. Effects or RNA inhibitors (actinomycin D, alpha-amanitin, cordycepin) were also tested. The results lead us to propose a hypothesis according to which brain hormone would interfere with molecular events related to the transition from the stage of spermatogonial proliferation (mitotic behavior) to the stage of meiotic differentiation (meiotic behavior).

[Relations between the evolution of the nucleolus and the brain hormone activity during oogenesis inNereis diversicolor O.F. Müller (Annelida, Polychaeta) under natural and experimental conditions]. / Relations entre l'évolution du nucléole et l'activité endocrine cérébrale au cours de l'ovogenèse deNereis diversicolor O. F. Müller (annélide, polychète), dans les conditions naturelles et expérimentales.

Each stage of oogenesis: previtellogenesis, proteic yolk elaboration, carbohydrates synthesis, maturity, take place under a given hormonal rate. During the previtellogenesis and protein yolk elaboration stages (high hormonal rate), nucleolus is composed of three concentric zones: a fibrillar center, a fibrillar intermediate belt and a granular cortex. During the carbohydrates synthesis stage, which occurs after a rapid decrease of the hormonal rate, a new nucleolar organization appears. Nucleolus is then composed of fibrillar places scattered among a fibrillo-granular component. During the maturity stage (very low hormonal rate), the nucleolar material segregates into two hemispheres, then breaks up.The result of removal of cerebral activity was considered. In oocytes undergoing protein yolk elaboration, the absence of hormone rapidly produces the development of a large nucleolar vacuole. Then, vacuole decreases and nucleolus shows again a compact shape. The end of the experimental evolution is marked by the segregation of the nucleolar fibrillar and fibrillogranular components.Chronological study of the modifications occuring at the different cellular regions shows that the consequences of removal of hormonal activity are seen at the nucleolar level in advance of being detectable in cytoplasm. These results are discussed accordingly present notions about hormonal regulation of RNA synthesis.