Mature Starfish Oocytes Research Articles

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Ca2+-dependent phosphatidylserine synthesis in immature and mature starfish oocytes.

We found that in starfish oocytes two different enzymes, phosphatidylserine synthase-1 (PSS1) and -2 (PSS2), which synthesize phosphatidylserine by a base-exchange reaction, are present. We studied phosphatidylserine synthesis in immature oocytes which still contain the nucleus (germinal vesicles) and in mature cells, in which the re-initiation of the meiotic cycle induced by the hormone 1-methyladenine led to structural changes in the endoplasmic reticulum, to the disappearance of the nuclear envelope and to the intermixing of the nucleoplasm with the cytoplasm. It was found that the levels of PSS1 and PSS2 transcripts were higher in immature and mature oocytes, respectively. The level of the expressed PSS2 protein, higher than that of PSS1, was not influenced by the maturation process, whereas the level of PSS1 protein was higher in immature than in mature oocytes. Serine incorporation into phosphatidylserine was enhanced in immature oocytes. The depletion of calcium stores by thapsigargin resulted in 50% lowering of phosphatidylserine synthesis. We suggest that changes in phosphatidylserine synthesis may be affected by the release of calcium stored in the nuclear envelope and in the endoplasmic reticulum, the membranes that undergo disintegration and fragmentation during meiosis. The reason for the greater synthesis of PS may be the higher level of expression of PSS1 in immature oocytes.

Ca2+-dependent phosphatidylserine synthesis in immature and mature starfish oocytes.

We found that in starfish oocytes two different enzymes, phosphatidylserine synthase-1 (PSS1) and -2 (PSS2), which synthesize phosphatidylserine by a base-exchange reaction, are present. We studied phosphatidylserine synthesis in immature oocytes which still contain the nucleus (germinal vesicles) and in mature cells, in which the re-initiation of the meiotic cycle induced by the hormone 1-methyladenine led to structural changes in the endoplasmic reticulum, to the disappearance of the nuclear envelope and to the intermixing of the nucleoplasm with the cytoplasm. It was found that the levels of PSS1 and PSS2 transcripts were higher in immature and mature oocytes, respectively. The level of the expressed PSS2 protein, higher than that of PSS1, was not influenced by the maturation process, whereas the level of PSS1 protein was higher in immature than in mature oocytes. Serine incorporation into phosphatidylserine was enhanced in immature oocytes. The depletion of calcium stores by thapsigargin resulted in 50% lowering of phosphatidylserine synthesis. We suggest that changes in phosphatidylserine synthesis may be affected by the release of calcium stored in the nuclear envelope and in the endoplasmic reticulum, the membranes that undergo disintegration and fragmentation during meiosis. The reason for the greater synthesis of PS may be the higher level of expression of PSS1 in immature oocytes.

Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit

We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15 kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219–231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission.

Activation of oocytes by latrunculin A.

Actin depolymerization by latrunculin A (LAT-A) in mature starfish oocytes induces a massive calcium mobilization that results in the discharge of the cortical granules and in the elevation of the fertilization envelope. The Ca2+ liberation starts as a circumscribed subplasma membrane hotspot, which is followed by a flash of Ca2+ increase restricted to the cortical layer. Ca2+ propagates rapidly from these peripheral regions to the center of the oocyte, initiating calcium oscillations. Blockade of the inositol 1,4,5-trisphosphate receptors with heparin does not affect the liberation of Ca2+ at the initial hotspot or the cortical flash, but abolishes the centripetal spreading of the wave and the Ca2+ oscillations. In Ca2+-free medium, LAT-A also initiates Ca2+ release at a discrete cortical point, but then propagates throughout the cell without first forming the uniform cortical flash. The latter is thus linked to the influx of external Ca2+, somehow promoted by the depolymerization of cortical (microvillar) actin. The Ca2+ response to spermatozoa (i.e., peripheral hotspot, cortical flash, globalization of the signal) closely mimics that promoted by LAT-A. Thus, the initial cortical release of Ca2+ promoted by the sperm may be due to the depolymerization of actin.

Induction of germinal vesicle breakdown in a cell-free preparation from starfish oocytes.

Incubation of isolated germinal vesicles in the homogenate from maturing starfish oocytes resulted in synchronous germinal vesicle breakdown (GVBD), and chromosome condensation and gathering within 30 min. GVBD in this cell-free system required aerobic conditions. The endogenous ATP-generation system was preserved in the homogenate and effective under aerobic conditions, and thus exogenous ATP was not added to the homogenate. Injection of the homogenate into immature starfish oocytes induced meiotic maturation without 1-methyladenine, indicating high activity of maturation-promoting factor (MPF) in the homogenate. MPF activity in the homogenate was stable for 2 h at room temperature, while it disappeared within 1 h in the supernatant prepared by centrifugation of the homogenate. This disappearance of MPF activity is regulated by cyclin B destruction, similar to that seen in vivo.

Separate Activation of the Cytoplasmic and Nuclear Calcium Pools in Maturing Starfish Oocytes

The dynamics of the cytoplasmic and nuclear Ca2+pools in starfish oocytes arrested at the prophase of the first meiotic division or after induction of meiosis by 1-methyladenine (1-MA) have been studied by confocal microscopy. A 70 kDa fluorescent Ca2+indicator has been injected in either the cytoplasm or the nucleus, and shown to remain restricted to the compartment of injection. 1-MA induced a first Ca2+transient in the cytosol, followed by a nuclear transient, and eventually by a second cytosolic transient. The latter failed to occur if the nuclear peak was suppressed. This required the nuclear injection of antagonists of the inositol 1,4,5-trisphosphate (InsP3) and cyclic-ADPribose (cADPr) Ca2+channels, showing that both channel types were active in the inner envelope membrane. The nuclear injection of the Ca2+channel antagonists affected the process of meiosis reinitiation: in about one third of the injected oocytes no breakdown of the nuclear envelope (GVBD) was observed. In the others, even if GVBD eventually occurred, the intermixing of the nucleoplasm and cytoplasm was inhibited.

MAP kinase inactivation is required only for G2-M phase transition in early embryogenesis cell cycles of the starfishes Marthasterias glacialis and Astropecten aranciacus.

Downregulation of MAP kinase is a universal consequence of fertilization in the animal kingdom. Here we show that oocytes of the starfishes Astropecten aranciacus and Marthasterias glacialis complete meiotic maturation and form a pronucleus when treated with 1-methyladenine and then complete DNA replication and arrest at G2 if not fertilized. Release of G2 by fertilization or a variety of parthenogenetic treatments is associated with inactivation of MAP kinase. Prevention of MAP kinase inactivation by microinjection of Ste11-DeltaN, a constitutively active budding yeast MAP kinase kinase kinase, arrests fertilized eggs at G2 in either the first or the second mitotic cell cycle, in a dose-dependent manner. G1 arrest is never observed. Conversely, inactivation of MAP kinase by microinjection of the MAP kinase-specific phosphatase Pyst-1 releases mature starfish oocytes from G2 arrest. The role of MAP kinase in arresting cell cycle at various stages in oocytes of different animal species is discussed.

The cell cycle can occur in starfish oocytes and embryos without the production of transferable MPF (maturation-promoting factor)

All cells undergoing the transition from interphase to metaphase have been postulated to contain a “maturation-promoting factor” (MPF) capable of causing meiotic maturation when injected into immature oocytes. We have shown in an accompanying paper ( A. Picard, M. C. Harricane, J. C. Labbe, and M. Doreé, 1988, Dev. Biol. 128, 121–128) that the basic oscillator driving the cell cycle still operates in maturing starfish oocytes and fertilized eggs in the absence of germinal vesicle (GV) material. Under such conditions of enucleation, we now show, however, that MPF activity cannot be detected after hormonal stimulation of prophase-arrested oocytes in Astropecten or after the normal time of second meiotic cleavage in Marthasterias. In contrast, cell cycles occur with the production of transferable MPF activity in embryos from which both pronuclei have been removed after fertilization. Reinjection of the entire contents of a GV after the normal time of second meiotic cleavage restores the ability of cytoplasm to induce meiotic maturation in immature recipient oocytes after transfer. Transduction of the hormonal stimulus at the level of the plasma membrane, stimulation of the phosphorylation of cytoplasmic proteins, and activation of a cycling Ca 2+- and cyclic nucleotide-independent histone kinase still occur in the absence of GV material. Since previous studies have demonstrated that the presence of GV material in the recipient oocytes is absolutely required in starfish for the amplification of microinjected MPF ( Kishimoto et al., 1981 ; Picard and Doree, 1984), we propose that some unidentified component of the GV is required, at least after the normal time of second meiotic cleavage in donor oocytes and at any time in recipient oocytes, for the successful transfer of MPF activity in starfish.

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes.

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-β-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobie chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 × 10 3 D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin.

Physico-chemical properties and interaction with actin of an actin-depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.

Hormone-induced parthenogenetic activation of mature starfish oocytes

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca 2+ at that time, although it is shown that Ca 2+ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation. It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.

Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187.

The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.

Changes in adenine nucleotide levels and respiration during 1-methyladenine induced maturation of starfish oocytes

The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to 1 1 2 h after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for 2 1 2 h after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations.