Hormonal control of meiosis reinitiation in starfish oocytes : New evidence for the absence of efficient intracellular receptors for 1-methyladenine recognition

Intracellular Ca²⁺ ions release and Na⁺ pump stimulation are triggered only 1 or 2 min after 1-methyladenine injection into starfish oocytes, whereas both events occur less than 2 sec after external application of the hormone. These data confirm and extend previous observations pointing to the existence of receptors localized on the external side of the plasma membrane. They make unlikely that the two elementary responses to the hormone here studied could be independently triggered through stimulation of eventual intracellular receptors, directly accessible from inside the oocyte.     

Hormonal control of meiosis reinitation in starfish oocytes. New evidence for the absence of efficient intracellular receptors for l-methyladenine recognition.

Nuclei isolated from testes of the house cricket were centrifuged in a gradient of colloidal silica with a density range of about 1.12 to 1.18 g/ml. Fractions were collected from the bottom to the top of the gradient, and the types of nuclei in them were classified by phase microscopy. The distribution of nuclear types in the gradient indicated relatively large increases in nuclear density during spermatogenesis, and that silica-gradient centrifugation can readily yield fractions enriched for nuclei of specific developmental stages needed to study basic protein changes during sperm development. Basic proteins could be extracted from nuclei spun through silica if they were washed with polyvinylpyrrolidone. The histones in different fractions of nuclei were analysed electrophoretically. Fractions of spermatocyte and early spermatid nuclei contained histones of the somatic types as their only basic proteins. Fractions with mixtures of mid-spermatid and earlier nuclei also yielded somatic histones primarily. Essentially pure samples of late spermatid nuclei were obtained. They lacked somatic histones. In one fraction of late nuclei, the spermatid-specific histones TH1 and TH2 were the major proteins present. In another, two additional histone-like components, not detected in previous studies, were also prominent.     

Spontaneous maturation of starfish oocytes: Role of follicle cells and calcium ions

Starfish oocytes released from the ovary into sea water during the breeding season underwent spontaneous meiotic maturation. In the absence of calcium ions, follicle cells surrounding the oocyte were dispersed and oocyte maturation did not occur. Immature oocytes in the absence of follicle cells did not mature even when calcium was available. Calcium ions stimulated the release of a meiosis-inducing substance from follicle cells which had been isolated in calcium-free sea water. Increasing numbers of follicle cells produced a parallel increase in the amount of meiosis inducing substance released. Spontaneous oocyte maturation appeared to be initiated and mediated in part by calcium sensitive processes in the follicle cells rather than the oocyte.     

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.     

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.     

Relationships between hormone-induced calcium release and 86rubidium uptake stimulation in starfish oocytes

86Rubidium+ uptake, but not 86Rubidium efflux, is strongly stimulated after addition of the meiosis inducing hormone 1-methyladenine (1-MeAde) to prophase blocked oocytes of the starfish Marthasterias glacialis. This stimulation is a transient process which does not require the continuous presence of 1-MeAde and is elicited within 1 minute of contact. 1-MeAde and its biologically active structural analogs fully stimulate Rb+ uptake at concentrations which are about two orders of magnitude lower than those required to trigger meiosis reinitiation but which already release underthreshold levels of Ca2+ from the inner part of the plasma membrane. External Ca2+ concentrations effective in triggering meiosis reinitiation also stimulate Rb+ influx, while drugs like D600, theophyllin and caffein which suppress the hormone induced Ca2+ release, simultaneously preclude the stimulation of Rb+ uptake. Dithiothreitol (DTT) which mimicks 1-MeAde action in releasing Ca2+ and inducing meiosis acts both on the efflux and on active and passive Rb+ influxes. Ouabain, the classical inhibitor of the Na+, K+ pump does not preclude meiosis reinitiation under the influence of 1-MeAde, its agonists of mimetics. It suppresses the active component of Rb+ uptake both in control or stimulate oocytes. When applied only in preincubation before starting the hormone treatment, it cannot however inhibit the stimulation of Rb+ uptake, while basal pump inhibition is preserved. These results demonstrate that stimulation of the active Rb+ or K+ transport is not indispensable to meiosis reinitiation. They suggest moreover that the hormone induced Ca2+ release from the plasma membrane may be responsible for unmasking new ouabain sensitive transport sites.     

Changes in the pattern of protein phosphorylation during meiosis reinitiation in starfish oocytes

Endogenous protein phosphorylation in oocytes of Marthasterias glacialis was examined by incubating living oocytes with [³²P]phosphate and cortical and endoplasmic fractions with [γ-³²P]ATP. Individual phosphorylated proteins were detected by autoradiography after bidimensional and monodimensional electrophoresis, using SDS-polyacrylamide gradient gel slabs. An increased phosphorylation of several protein species was observed as early as 5 min following in vivo hormonal stimulation by 1-methyladenine. No dephosphorylation nor any change in protein staining of the gels was observed. In vitro phosphorylation patterns were consistent with those observed in vivo. They did not change upon in vitro 1-methyladenine addition and remained unaffected when the incubations were carried out in the presence of cAMP or beef heart protein kinase inhibitor. Cortical phosphorylation was inhibited by calcium ions. The results suggest that the hormone promotes alterations in the availability of phosphorylation sites in some proteins already present in the control oocytes which, as well as the corresponding activated cAMP-independent protein kinases, may play a significant role during formation of the maturation promoting factor.     

Rapid increase in plasma membrane chloride permeability during wound resealing in starfish oocytes

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation-induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca(2+). Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca(2+) to reseal. Within a few seconds the membrane resistance is approximately 100-fold lower, while the membrane potential has depolarized from -80 to -30 mV and is now sensitive to the Cl(-) concentration but not to that of Na(+), K(+), or H(+). We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.     

The effects of quercetin on meiosis initiation in clam and starfish oocytes

We have treated Spisula and Asterias oocytes with quercetin to determine the effects of this drug on germinal vesicle breakdown (GVBD). Quercetin (100-200 microM) reversibly inhibited GVBD when induced by excess KCl or ionophore A23187. Kinetic studies showed that quercetin blocked an early event in GVBD. Lower concentrations of quercetin (10-20 microM) blocked fertilization. However, quercetin sensitized the oocytes to initiation of GVBD by excess ions which do not normally trigger GVBD. Quercetin (100-200 microM) also blocked 1-methyladenine-induced GVBD in the starfish. In subthreshold concentrations of the hormone or in its absence, lower concentrations (20-40 microM) stimulated GVBD. The results support the hypothesis that quercetin exerts its effects on meiosis initiation through its effects on calcium sequestration.     

Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes

Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization.     

Irreversible effects of calcium ions on the plasma membrane calcium pump

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mm Ca²⁺, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca²⁺ affinity at 37°C and is unaffected by serine-protease inhibitors. The activation caused by 1 mm Ca²⁺ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 m Ca²⁺ is not inhibited. Preincubations at 0°C also lead to activation, to a level up to half that seen at 37°C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca²⁺-containing solutions at 37°C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 m Ca²⁺. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca²⁺ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.We thank The Wellcome Trust for a research grant, the Medical Research Council for an equipment grant and the Regional Transfusion Service (Sheffield) for bank blood supplies.     

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.     

Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca²⁺ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.     

Effect of calcium ions on plasma membrane structure

The structure of thymocyte's plasma membranes was studied as affected by calcium ions (0-1.126 mM). The fluorescence intensity of 1-anilinonaphthalene-8-sulphonate, the epimerization degree of pyrene and fluorescence anisotropy of membrane proteins were studied. The change of electrochemical properties of membranes, conformation of membrane proteins and lipid fluidity has been shown.     

Inositol 1,4,5-trisphosphate-induced calcium release from platelet plasma membrane vesicles

A platelet membrane preparation, enriched in plasma membrane markers, took up 45Ca2+ in exchange for intravesicular Na+ and released it after the addition of inositol 1,4,5-trisphosphate (IP3). The possibility that contaminating dense tubular membrane (DTS) vesicles contributed the Ca2+ released by IP3 was eliminated by the addition of vanadate to inhibit Ca+-ATPase-mediated DTS Ca2+ sequestration and by the finding that only plasma membrane vesicles exhibit Na+-dependent Ca2+ uptake. Ca2+ released by IP3 was dependent on low extravesicular Ca2+ concentrations. IP3-induced Ca2+ release was additive to that released by Na+ addition while GTP or polyethylene glycol (PEG) had no effect. These results strongly suggest that IP3 facilitates extracellular Ca2+ influx in addition to release from DTS membranes.     

Calcium regulation and calcium function in the nucleus of starfish oocytes

Summary

The nucleus (germinal vesicle) of starfish oocytes can be injected in vivo to introduce into it calcium indicators and various effectors or inhibitors of calcium signalling pathways. This is advantageous to the study of the debated problem of nuclear calcium homeostasis, which is related to that of the function of calcium in the nucleus. The work described here has shown that, at variance with other cell types, the nuclear envelope of starfish oocytes is relatively impermeable to calcium and to calcium sensitive dyes. It has also shown that a rise in free nuclear calcium is required for the reinitiation of meiosis induced by 1-methyladenine. Bom inositol 1, 4, 5-trisphosphate (InsP₃) and cyclic ADP-ribose (cADPr) receptors are present and functional in the membrane enveloping the nucleus. The chief processor of the calcium signal, caknodulin, interacts in the nucleus with the heterogeneous ribonucleoprotein particles and could thus play an important role in the processing of pre-mRNA.     

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

Ca(2+) is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca(2+) to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca(2+) signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca(2+)-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca(2+) signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca(2+)-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.     

Hormone-induced loss of surface membrane during maturation of starfish oocytes: differential effects on potassium and calcium channels

Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered by the hormone 1-methyladenine (1-MA). Maturation involves a variety of complex biochemical, morphological, and electrical changes, many of which are similar to those caused by progesterone in vertebrates. Using voltage-clamp and ultrastructural techniques to study maturation in starfish, we have discovered a novel process by which 1-MA alters the electrical properties of the oocyte. The surface area of the oocyte decreases by more than 50% during the first hour of maturation, due to the elimination of microvilli, but the calcium and potassium currents present are affected differently by the loss of membrane. The amplitudes of both the transient K current ("A-current") and the inwardly rectifying K current decrease, following the time course of the decrease in surface area, while the Ca current amplitude remains virtually unaffected, and may even increase in some oocytes. The kinetics of the currents do not change. This selective removal of K channels results in a larger and more rapidly rising action potential in the mature egg, which may aid in the fast block to polyspermy. The differential accessibility of various ion channels to mechanisms of membrane removal and insertion may play an important role in the development of excitable cells.     

Cultivation of mouse oocytes in vitro: the ability to resume meiosis

It is well established that fully grown oocytes from sexually mature mice are capable of resuming meiosis when cultured in vitro. However it has not been established at what stage during the growth the oocyte acquires the ability to mature. The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in ICR strain mice. Oocytes of various sizes were isolated from the ovary by mechanical dissection. This method yielded about 30% growing and fully-grown oocytes from the ovary. Cumulus-free oocytes dissociated from the ovaries of 5-week-old mice were cultured in vitro in a chemically defined medium for 3 hr. None of the 61- to 65-microns-diameter oocytes resumed meiosis; however 81.8% of the 66- to 70-microns-diameter oocytes were able to resume meiosis. Fifty- to 60-microns-diameter oocytes recovered by enzymatic dispersal of the basement membrane of isolated growing follicles were unable to resume meiosis. Follicle-enclosed 50- to 60-microns-diameter oocytes did not resume meiosis even when cultured in a medium containing Ca-ionophore A 23187 or 8-bromoadenosine 3', 5'-cyclic monophosphate which are known to induce resumption of meiosis of oocytes in Graafian follicles. These results indicate that the ability to resume meiosis is acquired dramatically at a specific stage of oocyte growth.