Jean-Pierre Vilain

Free calcium changes associated with hormone action in amphibian oocytes

Abstract Ca2+-sensitive electrodes and the photoproteins obelin and aequorin were used with the oocytes of the anuran Xenopus laevis and the urodeles Ambystoma mexicanum and Pleurodeles waltlii in order to detect any changes in internal free Ca2+ which might result from progesterone or agonist stimulation. A dramatic Ca2+ surge was recorded: from 0.7 × 10−6 M in the unstimulated oocyte to 7 × 10−6 M after stimulation but before germinal vesicle breakdown (GVBD). Ca2+ efflux was also measured, but it accounted for less than 0.2% of the internal Ca2+ transient; this efflux did not take place in the absence of external calcium. The Ca2+ surge and maturation in response to progesterone, p-hydroxymethylenesulfonate (PHMPS), or Mn2+ occurred normally even when divalent cations were absent from the external medium. In contrast, external divalent cations were necessary for the induction of meiosis and a Ca2+ transient by the K+ ionophore valinomycin. HCO3− also triggers meiosis and causes Ca2+ release, but the release occurs with quite different kinetics. Incompletely grown or seasonally dormant oocytes as well as 10 mM theophilline- or procaine-treated oocytes neither release Ca2+ nor respond to the hormone. We conclude that intracellular released Ca2+ is likely to be the major “second messenger” following hormone stimulation in amphibian oocytes as in starfish.

Fertilization of amphibian eggs: a comparison of electrical responses between anurans and urodeles.

In Pleurodeles waltl and Ambystoma mexicanum, which exhibit physiological polyspermy, the membrane potential in most eggs did not change in any consistent pattern during 45 min after fertilization; in some cases, a slow hyperpolarization began 5 to 15 min after insemination and continued for 10-15 min. These eggs then slowly depolarized, reaching a stable value of -10 to +10 mV, about 45 min after fertilization. Membranes of eggs activated by A23187 or by electrical stimulus showed a similar behavior. The diversity of responses does not correlate with the number of sperm fusing with the egg. Holding the membrane potential at a constant value between -40 and +40 mV during insemination did not prevent fertilization nor delay sperm-egg interactions. The fertilization or activation potential of Rana temporaria eggs consists of a rapid (1 sec) depolarization accompanied by a sudden decrease in membrane resistance. The activation potential can be triggered by A23187 and by calcium iontophoresis; its amplitude depends on the (Cl-)0 and to a lesser extent on the (Na+)0. Fertilization was prevented when the membrane potential was clamped above +15 mV. However, slowing the rise time (5 to 8 sec instead of 1 sec) and reducing the amplitude (10-20 mV instead of 40-60 mV) of the fertilization potential, both by injecting negative current, never induced polyspermy.

UNCOUPLING OF OOCYTE‐FOLLICLE CELLS TRIGGERS REINITIATION OF MEIOSIS IN AMPHIBIAN OOCYTES

Reinitiation of meiosis has been triggered in vitro in oocytes of the anouran Xenopus laevis and the urodeles Pleurodeles waltlii and Ambystoma mexicanum by enzymatic, mechanical or manual defolliculation, without addition of hormone. By measuring changes in the membrane resistance and time constants, we also demonstrate the existence of an electrical polarized coupling between the oocyte and its follicle and show that progesterone breaks it definitively within the first two hours. These results are discussed in relation to mammalian maturation.

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.

The Alphavirus 6K Protein Activates Endogenous Ionic Conductances when Expressed in Xenopus Oocytes

The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (Iin) and a calcium-dependent chloride current (ICl). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (ICl upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.

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, p34cdc2 rephosphorylation and cyclin B2 and p39mos proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin‐dependent degradation machinery.

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI‐induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI‐induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1–2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50–105 sec) than in monospermic eggs (10–40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.

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.

MPF and procaine-induced maturation of Xenopus oocyte

BODART Jean-Francois’, FLAMENT..&?phanc’. BROWAEYS Edith’. BERTOUT ~arcl, ROUSSEAU Arlettel, GANNON Julian2 et VILAIN Jean-Pierrel. 1 Centre de Biologie Cellulaire, Unit6 de Dynamique des cellules embryonnaires et cancereuses, Laboratoire de Biologie du Developpcment. EA DRED 1033, UniversitC de Lille 1, SN3, F-59655 Villeneuve d’Ascq cedex, France. 2 Imperial Cancer Research Fund Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD. U.K.