The role of cyclins in the maturation of Patella vulgata oocytes.

We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.     

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.     

Germinal vesicle nucleoplasm and intracellular pH requirements for cytoplasmic maturity in oocytes of the prosobranch mollusk Patella vulgata

The respective roles of germinal vesicle (GV) nucleoplasm dispersion and intracellular alkalinization in the acquisition of cytoplasmic maturity by oocytes of the prosobranch mollusk Patella vulgata have been investigated in experiments involving premature fertilization of prophase-blocked oocytes. These were then either enucleated and treated with 10 mM NH₄Cl, pH 8.5, or induced to break their germinal vesicle in the absence of any evoked intracellular pH change. Results indicate that male pronuclear decondensation, sperm aster differentiation and cleavage require both GV nucleoplasm dispersion and intracellular pH alkalinization. These data are discussed in relation to the respective roles of calcium, pH, and nucleoplasm during maturation and activation of invertebrate and vertebrate oocytes.     

Development of dorsoventral polarity and mesentoblast determination in Patella vulgata

In Patella vulgata the 32-cell stage represents a pause in the mitotic activity prior to the differentiation of the mesentoblast mother cell 3D. At the onset of this stage, the embryo is radially symmetrical. Nevertheless, the plane of bilateral symmetry is indicated as it passes through the macromeres forming the vegetal cross-furrow. From the early beginning of the 32-cell stage, all four macromeres intrude far into the interior and touch the centrally radiating cells of the first quartet of micromeres. The two cross-furrow forming macromeres (3B and 3D) intrude the farthest and come into contact with the greatest number of micromeres. Finally, the contacts are extended significantly and maintained with only one of these macromeres. From that moment, this cell can be called the macromere 3D and the dorsoventral axis is determined. The evolution of the internal cell contacts between the micromeres of the first quartet and the macromeres indicates an essential role of the former in the determination of one of the latter as the mesentoblast mother cell, and thus in the determination of dorsoventral polarity.     

In vitro maturation and artificial activation of donkey oocytes

Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.     

Communication compartments in the ectoderm of embryos of Patella vulgata

Summary Patterns of gap junctional communication in the ectoderm of embryos of Patella vulgata have been studied by intracellular injection of the fluorescent dye Lucifer Yellow, and by analysis of its subsequent spread to adjacent cells (dye-coupling). We found that dye-coupling became progressively restricted to different domains of the ectoderm, forming communication compartments. These communication compartments are characterized by their high coupling abilities within the compartment, and reduction of coupling across their boundaries. During development, the pretrochal (anterior) ectoderm becomes subdivided into two communication compartments, the apical organ and the anlage of the head ectoderm. The posttrochal (posterior) ectoderm becomes subdivided into different communication compartments in two successive phases. Firstly, in the 15-h embryo the dorsal and ventral domains of the ectoderm form separate communication compartments. A dorso-ventral communication boundary restricts the passage of dye between the two domains. Secondly, in the 24-h embryo dye-coupling becomes further compartmentalized in both the dorsal and ventral domains. These compartments correspond to the anlagen of different ectodermal structures. In order to study whether any level of coupling persists between the ectodermal compartments we injected currents through a microelectrode inserted into one cell of one compartment and monitored its spread by means of a second microelectrode inserted into one cell of another compartment (electrical coupling). Despite the absence of dye-coupling, electrical coupling between the ectodermal dye-coupling compartments was detected, which suggests that some level of communication is maintained between compartments. Our results demonstrate that within the ectoderm layer of Patella vulgata the transfer of dyes becomes progressively restricted to communication compartments and, concomitantly with the specification of the different ectodermal anlagen, these compartments become subdivided into smaller communication compartments.     

Measurement of the arylsulphatase of Patella vulgata with 4-methylumbelliferone sulphate

1. The preparation, purification, chemical and spectral properties of potassium 4-methylumbelliferone sulphate are described. 2. The use of 4-methylumbelliferone sulphate as a substrate for the arylsulphatase of Patella vulgata is presented with specific reference to the fluorimetric assay procedure used with this substrate. 3. 4-Methylumbelliferone sulphate is compared with the previously used synthetic sulphatase substrates nitrocatechol sulphate and p-nitrophenyl sulphate with respect to K_m, V_max. and sensitivity in the assay of arylsulphatase. 4. 4-Methylumbelliferone sulphate was strongly inhibited by phosphate. Sulphate, a less potent inhibitor, appeared to be of the competitive type with some anomalous characteristics.     

Inhibition of maturation and metabolism in rat oocytes by cyclic amp.

It is known that dibutyryl cyclic AMP (dbcAMP) and theophylline inhibit the spontaneous maturation of isolated mouse oocytes. The present study demonstrates that dbcAMP (0.01-1.0 mM) as well as cyclic AMP (cAMP, 10 mM) and a phosphodiesterase inhibitor (IBMX, 0.01-1.0 mM) prevent spontaneous maturation of isolated rat oocytes. As reported earlier an increase in oxygen consumption by the oocyte was found following maturation. When the oocytes were cultured in the presence of dbcAMP or cAMP no change in respiration occurred during culture. These results argue against the theory that cAMP acts as a direct mediator of the action of luteinizing hormone (LH) on oocyte maturation. Furthermore they suggest that changes in oocyte energy metabolism are closely related to the maturation process.     

The mineralization and hardness of the radular teeth of the limpet Patella vulgata L.

Summary A study of the Patella vulgata radula has been made using: the scanning electron microscope in its normal and compositional contrast modes of operation, the electron microprobe analyser, ion etching with argon ions and microhardness testing.Only iron, silicon and small amounts of sulphur were detected in the radula. The teeth can be subdivided into a cusp, a junctional area where the cusp is joined to the base, and the base which is embedded in the radular membrane. From a study of longitudinal vertical and transverse sections of the mature teeth it was found that the cusp could be subdivided into a posterior iron-rich area (44–51% Fe, 1–6% Si) and an anterior silicon-rich area (22–30% Fe, 27–32% Si). The junctional zone consisted of a poorly mineralised layer at its border with the cusp and an iron-rich layer where it joined the base. The upper part of the base (5% Fe, 16% Si) could be clearly differentiated from the silicon-rich anterior and lower parts of the base (3–4% Fe, 28–35% Si). No minerals were detected in the membrane. The changes in the mineral content of the teeth cusps along the length of the radula were studied. Iron appeared in the cusps at the 25th row and the concentration increased to 28% at the 50th row. The iron was here evenly distributed throughout the cusp. Silicon appeared in the anterior part of the cusp at the 50th row and as it increased in concentration so the iron was displaced, and at the same time the concentration of iron increased in the posterior part of the cusp. Mineralization appeared to be complete by the 150th row.The teeth cusps appear to consist of 800 Å fibres grouped into 1 thick bundles and the tooth appears to be covered by a thin enamel-like layer. It is suggested that the fibres contain the silicon-rich phase and the matrix the iron-rich phase.The significance of the arrangement of the fibres and the distribution of the minerals are discussed with relation to the function of the teeth.We wish to thank Mr. A. Rees and Mr. A. Davies for their technical assistance; Prof. Lewis and Dr. James for the use of the Electron Microprobe; and the S.R.C. for their financial support.     

The expression of a caudal homologue in a mollusc,Patella vulgata

We cloned and analyzed the expression of a caudal homologue (PvuCdx) during the early development of the marine gastropod, Patella vulgata. PvuCdx is expressed at the onset of gastrulation in the ectodermal cells that constitute the posterior edge of the blastopore, as well as in the paired mesentoblasts, the stem cells that generate the posterior mesoderm of the trochophore larva. During larval stages, PvuCdx is expressed in the posterior neurectoderm of the larva, as well as in part of the mesoderm. This is the first report of the expression of a caudal gene in a lophotrochozoan species. The striking similarities with the expression of caudal in other organisms, such as chordates, suggest that a posterior expression of caudal is ancestral to Bilateria.     

Activation of MPF at meiosis reinitiation in starfish oocytes.

Although maturation or M-phase-promoting factor (MPF) was originally identified as a cytoplasmic activity responsible for induction of maturation or meiosis reinitiation in oocytes, MPF is now thought to be the universal trigger of G2/M-phase transition in all eukaryotic cells, and its activity is ascribed to cyclin B. Cdc2 kinase. Here, the activation process of cyclin B. Cdc2 at meiosis reinitiation in starfish oocytes is compared with that at G2/M-phase transition in mitotic somatic cells. Based on this comparison, the role of cyclin B. Cdc2 in the original cytoplasmic MPF activity is reexamined.     

Conditions for in vitro maturation and artificial activation of ferret oocytes

The ferret represents an attractive species for animal modeling of lung diseases because of the similarity between ferret and human lung biology and its relatively small size and short gestation time. In an effort to establish experimental protocols necessary for cloning ferrets, optimized conditions for in vitro maturation and artificial activation of ferret oocytes were examined. Cumulus-oocyte complexes were harvested from ovaries of superovulated ferrets, and in vitro maturation was evaluated in three different culture media: medium 1 (TCM-199 + 10% FBS), medium 2 (TCM-199 + 10% FBS with eCG [10 IU/ml] and hCG [5 IU/ml]), or medium 3 (TCM-199 + 10% FBS with eCG, hCG, and 17beta-estradiol [2 microg/ml]). After 24 h of maturation in vitro, the maturation rate of oocytes cultured in medium 2 (70%, n = 79) was significantly greater (P < 0.01) than those of oocytes cultured in the other two media (27%-36%, n = 67-73). At 48 h, similar maturation rates (56%-69%, n = 76-87) were observed for all three types of media. For activation experiments, oocytes cultured in medium 2 were stimulated with electrical and chemical stimuli either individually or in combination. Treatment with cycloheximide and 6-dimethylaminopurine (6-DMAP) following electrical stimulation resulted in 43% (n = 58) of the oocytes developing to the blastocyst stage. Such an activation rate represented a significant improvement over those obtainable under other tested conditions, including individual treatment with electrical pulses (10%, n = 41), cycloheximide (3%, n = 58), or 6-DMAP (5%, n = 59). Blastocysts derived from in vitro activation appeared to be normal morphologically and were composed of an appropriate number of both inner cell mass (mean +/- SEM, 10.3 +/- 1.1; n = 11) and trophectoderm (60.8 +/- 2.9, n = 11) cells. These results have begun to elucidate parameters important for animal modeling and cloning with ferrets.