Distinct regulators for Plk1 activation in starfish meiotic and early embryonic cycles

The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. Here we have studied the interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, were unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 was required for cyclin B-Cdc2 activation, the action of Plk1 was mediated primarily through suppression of Myt1 rather than through activation of Cdc25. We propose that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1.     

Effects of egg quality on normal fertilization and early development of the cod, Gadus morhua L

Normal cod eggs respond to insemination by a rapid cortical reaction followed by an increase in total osmolarity and a small increase in egg diameter. The chorion becomes harder, but this is a slower process reaching its maximum strength after c . 24 h. Bad eggs are characterized by a slower or incomplete cortical reaction, resulting in a slower rise in osmolarity and a softer chorion. Bad eggs rapidly lose their capacity for fertilization. In unfertilized eggs in sea water, no cortical reaction is observed. There is, however, a rise in total osmolarity and a hardening of the chorion.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

Heat production during early development of frog egg

The production of heat in the fertilized eggs, during the early development of the Japanese pond frog, Rana brevipoda porosa, was measured using a pyroelectric detector constructed with a polyvinylidene fluoride film. One and 2 μW of heat production was detected during the cleavage periods in the embryogenesis of the 2-cell embryo and the 4-cell embryo, respectively. The heat production increased stepwise with the cleavage in the embryogenesis of each stage. In comparison with the heat production during the inter-cleavage periods, a larger amount was noted at the cleavage periods in the two embryonic stages.     

Calcium transients during early development in single starfish (Asterias forbesi) oocytes

Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. We have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. We used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approximately 1 microM at the point of insemination and the region of elevated free calcium expands across the oocyte in approximately 20 s (17-19 degrees C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.     

Irregularities in the early development of the Drosophila Melanogaster egg

Summary Polyspermy is normally present in the Drosophila egg.Extreme polyspermy may lead to disturbances which may prevent the egg from further development.Polar body formation may be disorganized by supernumerary sperms.Supernumerary sperms may form unipolar, bipolar and multipolar spindles.In rarer cases sperms will enter into normal spindles and form multipolar spindles. In some cases such interference seems to adjust itself to the original bipolar condition and division figures with the polyploid number of chromosomes will result. Either by the conjugation of two sperms, or by dimegaly, or by the doubling of chromosomes of a single sperm, supernumerary nuclei may develop. The origin of certain mosaics and gynandromorphs may be explained on this basis.     

Anuran and pig egg zona pellucida glycoproteins in fertilization and early development

The envelope surrounding the eggs of all animals has many biological functions in fertilization and development. This review focuses on the anuran egg envelope in terms of its biochemistry, biophysics, structural biology and function in sperm-egg interactions and early development (embryo hatching). Egg envelopes from Xenopus laevis are among the most studied of the anurans, and are the central theme of this review. Comparisons of Xenopus laevis envelopes with those of other anurans and with pig egg envelopes are also included. This article presents historical as well as contemporary comparative perspectives on molecular and cellular mechanisms of sperm-egg envelope binding, block to polyspermy, envelope hardening, and hatching.     

Quantitative measurement of caspase-3 activity in a living starfish egg

If not fertilized, synchronous apoptosis is induced in starfish eggs at approximately 11h after stimulation with the hormone, 1-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected approximately 30min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3h after 1-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at approximately 10h after 1-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis.     

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 Ca²⁺ at that time, although it is shown that Ca²⁺ 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.     

Effects of ionomycin and thapsigargin on ion currents in oocytes of Bufo arenarum

In this study, two electrode voltage clamp technique was used to assess the ionic current of oocytes of the South American toad Bufo arenarum and to study the dependence of these currents on the extracellular and intracellular Ca2+ concentrations. Ca2+ chelators, ionomycin -a calcium ionophore- and thapsigargin, a blocker of the Ca2+ pump of the sarcoplasmic reticulum, were used. The main results were the following: Most oocytes showed a voltage activated rectifying conductance. Ionomycin (1 microM) increased inward and outward currents in control solution. The effect of ionomycin was blocked partially at negative potentials and was blocked completely at positive potentials in absence of extracellular Ca2+. When the oocytes were treated with thapsigargin (2 microM) or BAPTA-am, a membrane-permeant intracellular chelator in control solution (10 microM), ionomycin did not increased either inward nor outward currents. The conclusion of our experiments is that there are two sources of Ca2+ for activation of the current induced by ionomycin, the cytoplasmic stores and the extracellular space. We believe ionomycin directly translocates Ca2+ from the SER into the cytoplasm but not from the extracellular medium. Ca2+ entry probably occurs through store-operated-Ca-channels.     

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKC alpha isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKC alpha isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.     

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.     

Effects of the egg incubation environment on turtle carapace development

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26–30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.     

G-proteins and egg activation

G-proteins are present in eggs, and experiments in which GTP-γ-S, GDP-β-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.