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Localization of transcripts of the zebrafish morphogen Squint is dependent on egg activation and the microtubule cytoskeleton.

The generation of polarity and patterning in multicellular organisms depends in part on the asymmetric localization of molecules to specific subdomains within a cell. Localized transcripts for several molecules are known to be required for patterning oocytes and embryos in Drosophila as well as Caenorhabditis elegans. Here, we describe the localization of transcripts encoding the nodal-related morphogen, Squint (sqt), in zebrafish oocytes and early embryos, and the mechanisms by which sqt RNA is localized. sqt transcripts are uniformly distributed in oocytes through all stages of oogenesis. Upon egg activation, sqt RNA is localized to the blastoderm, and excluded from the yolk cell. The mechanism of sqt RNA transport was examined using cytoskeletal inhibitors. Disruption of actin microfilaments by treatment with latrunculin A does not alter the localization of sqt RNA to the blastoderm. However, disruption of the microtubule cytoskeleton by treatment with nocodazole affects sqt RNA localization. These results indicate that sqt transcripts are translocated by an RNA localization pathway which is initiated upon egg activation, and that sqt RNA localization through this pathway is mediated via the microtubule cytoskeleton.

Animals↗

Effects of active egg white product on neutrophil function in calves.

The effects of an active egg white product (AEWP) on neutrophil function in calves were investigated. Calves were administered AEWP orally at doses of 250 mg and 500 mg/kg either once or twice, with an interval of 5 days between doses in the latter case. The peripheral blood neutrophils of calves receiving a single 500 mg/kg dose displayed increased nitroblue tetrazolium reducing activity and increased intracellular killing of Staphylococcus aureus from 1 day after administration, with maximum levels being attained on the 3rd day. However, no such increase of these activities was observed after administration of 250 mg/kg. Calves receiving two doses of 500 mg/kg displayed the same changes as seen in the corresponding one-dose group, while the neutrophil activity of calves receiving 250 mg/kg also increased after the second dose. However, no increase in the peripheral blood neutrophil count was observed after AEWP administration. Thus, AEWP enhanced the nonspecific antibacterial activity of neutrophils when given to calves by the oral route.

Animals↗

Calcium signals for egg activation in mammals.

A dramatic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) occurs in eggs at fertilization common to all animal species examined to date, and this serves as a pivotal signal for egg activation characterized by resumption of meiotic cell division and formation of the pronuclei. In mammalian eggs, repetitive [Ca(2+)](i) rises (Ca(2+) oscillations) each of which accompanies a propagating wave across the egg occur due to release of Ca(2+) from the endoplasmic reticulum mainly through type 1 inositol 1,4,5-trisphosphate (IP(3)) receptor. Ca(2+) oscillations are induced by a cytosolic sperm factor driven into the egg cytoplasm upon sperm-egg fusion. A current strong candidate of the sperm factor is a novel sperm-specific isozyme of phospholipase C (IP(3)-producing enzyme), PLCzeta. Recent extensive research has reveled characteristics of PLCzeta such as the Ca(2+) oscillation-inducing activity after injection of PLCzeta-encoding RNA or recombinant PLCzeta into mouse eggs, extremely high Ca(2+)-sensitivity of the enzymatic activity in vitro, and nuclear translocation ability possibly related to cell-cycle-dependent regulation of Ca(2+) oscillations. [Ca(2+)](i) rises cause successive activation of calmodulin-dependent kinase II and E3 ubiquitin ligase, lead to proteolysis of ubiquitinated cyclin B1 and inactivation of metaphase-promoting factor (Cdk1/cyclin B1 complex), and result in the release of eggs from meiotic arrest.

Animals↗

MAP kinase activity is downregulated by phorbol ester during mouse oocyte maturation and egg activation in vitro.

The effects of protein kinase C (PKC) stimulator, phorbol 12-myriatate 13-acetate (PMA), on meiotic cell cycle regulation and mitogen-activated protein (MAP) kinase changes have been studied in mouse oocytes and eggs. The results showed that MAP kinase activation itself was not necessary for germinal vesicle breakdown (GVBD), but the ability of the ooplasm to phosphorylate MAP kinase was a prerequisite for this event. At concentrations of 1.6 nM, PMA effectively inhibited GVBD and MAP kinase activation, suggesting that PMA inhibits GVBD by inhibiting molecule(s) upstream to MAP kinase. At concentrations of 16.2 nM, PMA induced metaphase-interphase transition more effectively in eggs collected 19 hr after human chorionic gonadotropin (hCG) administration than in those collected 15 hr after hCG administration. The degree of MAP kinase activity decrease was well correlated with the time course and proportion of pronuclear formation. On the other hand, when the effect of PMA on cell cycle progression was abolished by protein phosphatase inhibitor, okadaic acid, MAP kinase was superactivated. The biologically inactive 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) had no evident effects on either GVBD and interphase transition or on MAP kinase activity. Furthermore, the effects of PMA on oocyte GVBD, egg activation, and MAP kinase activity could be overcome by the specific PKC inhibitor, calphostin C, suggesting the possible involvement of this enzyme in the regulation of MAP kinase activity. The results suggest that activation of PKC by PMA entrains a cascade of events that ultimately inhibits MAP kinase activation and GVBD in mouse oocytes and induces MAP kinase inactivation and metaphase-interphase transition in mouse eggs.

Animals↗

Potential role of a sperm-derived phospholipase C in triggering the egg-activating Ca2+ signal at fertilization.

An increase in intracellular Ca2+ at fertilization is the trigger for egg activation in all species that have been studied. Exactly how sperm-egg interaction leads to this Ca2+ increase has not been established. There is increasing support for the hypothesis that the spermatozoon introduces a Ca2+-releasing protein into the egg cytoplasm after gamete membrane fusion. This review discusses the merits of this 'sperm factor' hypothesis and presents evidence indicating that the sperm factor, at least in mammals, consists of a phospholipase C with distinctive properties. This evidence leads us to propose that, after gamete fusion, a sperm-derived phospholipase C causes production of inositol 1,4,5- trisphosphate, which then generates Ca2+ waves from within the egg cytoplasm.

Animals↗

Effect of active egg white product on neutrophil function in weanling piglets.

The effect of oral administration of an active egg white product (AEWP) on neutrophil function in weanling piglets was investigated. The peripheral blood neutrophils of piglets receiving single doses of 20 mg/kg and 100 mg/kg displayed increased phagocytic activity for latex particles from 1 day after administration. This increased phagocytic activity persisted for 5 days in the 100 mg/kg group. Piglets receiving a dose of 30 mg/kg/day for a week displayed increased neutrophil phagocytic activity throughout the administration period. Moreover, when the administration of AEWP was resumed 10 days after withdrawal, neutrophil phagocytic activity rapidly increased again. Thus, AEWP enhanced the nonspecific phagocytic activity of neutrophils when given to piglets by the oral route.

Animals↗

Characterization of protein kinase C-delta in mouse oocytes throughout meiotic maturation and following egg activation.

Changes in protein kinase C (PKC) activity influence the progression of meiosis; however, the specific function of the various PKC isoforms in female gametes is not known. In the current study, the protein expression and subcellular distribution profile of PKC-delta (PKC-delta), a novel isoform of the PKC family, was determined in mouse oocytes undergoing meiotic maturation and following egg activation. The full-length protein was observed as a doublet (76 and 78 kDa) on Western blot analysis. A smaller (47 kDa) carboxyl-terminal fragment, presumably the truncated catalytic domain of PKC-delta, was also strongly expressed. Both the full-length protein and the catalytic fragment became phosphorylated coincident with the resumption of meiosis and remained phosphorylated throughout metaphase II (MII) arrest. Immunofluorescence staining showed PKC-delta distributed diffusely throughout the cytoplasm of oocytes during maturation and associated with the spindle apparatus during the first meiotic division. Discrete foci of the protein also localized with the chromosomes in some mature eggs. Following the completion of meiosis, PKC-delta became dephosphorylated within 2 h of in vitro fertilization or parthenogenetic activation. The protein also accumulated in the nuclei of early embryos and was phosphorylated during M-phase of the initial mitotic cleavage division. By the two-cell stage, expression of the truncated catalytic fragment was minimal. These data demonstrate that the subcellular distribution and posttranslational modification of PKC-delta is cell cycle dependent, suggesting that its activity and/or function likely vary with the progression of meiosis and egg activation.

Animals↗

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus).

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

Animals↗

Differential contribution of cytoplasmic Ca2+ and Ca2+ influx to gamete fusion and egg activation in maize.

In multicellular organisms, gamete fusion triggers a set of events, collectively known as egg activation, that leads to the development of a new individual. Every species that has been studied shows at least one rise in cytoplasmic Ca2+ concentration ([Ca2+]Cyt) after gamete fusion which is believed to be involved in activation. Yet the source and regulation of this Ca2+ signal and the way it is transduced inside the zygote are controversial. In higher plants, in vitro fertilization (IVF) has enabled the description of a rise in [Ca2+]Cyt (ref. 4) that is sufficient for activation, and of a Ca2+ influx that spreads as a wavefront from the fusion site The relationship between these two responses is unknown. Using a new combination of methods that simultaneously monitor the extracellular flux with a Ca2+-vibrating probe, and [Ca2+]Cyt by widefield imaging, we directly determined that the Ca2+ influx precedes the [Ca2+]Cyt elevation by 40-120 s. In addition, results from experiments using the Ca2+-channel inhibitor gadolinium (Gd3+) suggest that the Ca2+ influx may be necessary for sperm incorporation. We also present evidence for a putative sperm-dependent Gd3+-insensitive localized Ca2+ influx confined to the fusion point.

Calcium Channel Blockers↗

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

Anaphase↗

The absence of a Ca(2+) signal during mouse egg activation can affect parthenogenetic preimplantation development, gene expression patterns, and blastocyst quality.

A series of Ca(2+) oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca(2+) signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca(2+) increase, with ethanol that causes a single large Ca(2+) increase, or with Sr(2+) that causes Ca(2+) oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stagecompared tothose activated by Sr(2+) orethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr(2+)-activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca(2+) increase because they developed to blastocyst stages at high rates when co-treated with Sr(2+) or ethanol. Embryos activated by either Sr(2+) or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr(2+) leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca(2+) signal does not affect initial parthenogenetic events, but can influence later gene expression and development.

Animals↗

Setting the stage for development: mRNA translation and stability during oocyte maturation and egg activation in Drosophila.

Early animal development is controlled by maternally encoded RNAs and proteins, which are loaded into the egg during oogenesis. Oocyte maturation and egg activation trigger changes in the translational status and the stability of specific maternal mRNAs. Whereas both maturation and activation have been studied in depth in amphibians and echinoderms, only recently have these processes begun to be dissected using the powerful genetic and molecular tools available in Drosophila. This review focuses on the mechanisms and functions of regulated maternal mRNA translation and stability in Drosophila--and compares these mechanisms with those elucidated in other animal models, particularly Xenopus--beginning late in oogenesis and continuing to the mid-blastula transition, when developmental control is transferred to zygotically synthesized transcripts.

Animals↗

Calcium oscillations and mammalian egg activation.

Fertilization in all species studied to date induces an increase in the intracellular concentration of free calcium ions ([Ca2+]i) within the egg. In mammals, this [Ca2+]i signal is delivered in the form of long-lasting [Ca2+]i oscillations that begin shortly after fusion of the gametes and persist beyond the time of completion of meiosis. While not fully elucidated, recent evidence supports the notion that the sperm delivers into the ooplasm a trigger of oscillations, the so-called sperm factor (SF). The recent discovery that mammalian sperm harbor a specific phospholipase C (PLC), PLCzeta has consolidated this view. The fertilizing sperm, and presumably PLCzeta promote Ca2+ release in eggs via the production of inositol 1,4,5-trisphosphate (IP3), which binds and gates its receptor, the type-1 IP3 receptor, located on the endoplasmic reticulum, the Ca2+ store of the cell. Repetitive Ca2+ release in this manner results in a positive cumulative effect on downstream signaling molecules that are responsible for the completion of all the events comprising egg activation. This review will discuss recent advances in our understanding of how [Ca2+]i oscillations are initiated and regulated in mammals, highlight areas of discrepancies, and emphasize the need to better characterize the downstream molecular cascades that are dependent on [Ca2+]i oscillations and that may impact embryo development.

Animals↗

Substrate-specific regulation of RNA deadenylation in Xenopus embryo and activated egg extracts.

The poly(A) tail of mRNAs plays an important role in translational control. In Xenopus laevis matured oocytes, maternal mRNAs that contain a cytoplasmic polyadenylation element (CPE) are polyadenylated, whereas CPE deficient mRNAs are deadenylated by a default process. Eg mRNAs are maternal transcripts that are poly(A)+ in matured oocytes and rapidly deadenylated after fertilization. This post-fertilization deadenylation of Eg mRNAs requires specific sequence information. Such a deadenylation element has been identified previously in the 3'UTR of Eg2 mRNA. In this study, we show that cell-free extracts made from embryos or activated eggs contain two kinetically distinct deadenylation activities, with different substrate specificities. One, responsible for the slow deadenylation of RNAs that are devoid of a functional CPE, has the characteristics of a default PAN activity. The other effectuates the rapid deadenylation of RNAs containing a deadenylation element. The in vitro system described here will allow the characterization of factors controlling the deadenylation of Eg mRNAs in embryos.

Animals↗

Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

Animals↗

The analysis of differentially expressed novel transcripts in diapausing and diapause-activated eggs of Bombyx mori.

As an initial step to define the molecular mechanism of initiation and termination of diapause during the embryogenesis of silkworms, Bombyx mori, mRNA transcripts from maintained and activated diapause eggs were compared with differential expression using cDNA array. Twenty-four individual cDNA transcripts were expressed differentially in a total of 1,468 different cDNAs. Among those clones, mRNA transcript from cytochrome oxidase subunit I (COI), which was detected to be 2-kb transcripts, gradually increased in diapause-activated eggs during early embryogenesis. Further analysis revealed that mRNA transcripts from silkworm COI were highly expressed in testis, fat body, and midgut during the larval stage. These results may indicate that the expression of silkworm COI mRNA is regulated developmentally as well as tissue-specifically.

Animals↗

Propagated and nonpropagated calcium transients during egg activation in the annelid, Chaetopterus.

Transient waves of Ca2+ release cross-fertilizing deuterostome eggs from the point of sperm entry to its antipode and provide much of the activating stimulus for the egg. Based on several indirect lines of experimental evidence, it was proposed that protostome eggs are activated by a prolonged uptake of Ca2+ from the medium due to sperm-induced membrane depolarization and that this uptake then starts an activation wave similar to those in deuterostomes, except that it moves inward from the whole surface rather than through the egg from pole to pole. To test these hypotheses, we microinjected oocytes of the polychaete annelid, Chaetopterus pergamentaceus, with semisynthetic recombinant aequorins and measured light emission in response to both fertilization and artificial activation by excess K+. Both fertilization and K(+)-activation induced multiple, brief Ca2+ transients in the eggs. The first transient did not propagate, but it was followed by a series of globally propagated Ca2+ waves interspersed with additional nonpropagated pulses. The waves traversed the egg at about 30 micrometer/sec. Sequential propagated waves and nonpropagated pulses generally originated at different regions of the egg surface, except the last few, which originated in the same "pacemaker" region. These new data are consistent with the hypothesis that the activation of protostome eggs is initiated by Ca2+ waves. However, the fact that these waves propagated from pole to pole like those in deuterostome eggs refutes the notion that Ca2+ waves in activating protostome eggs move inward from the whole surface.

Animals↗

Egg activation: upstream of the fertilization calcium signal.

Interaction of sperm and egg at fertilization induces well-coordinated molecular events including specific recognition between species, adhesion and fusion, that lead to the formation of a zygote, a totipotent cell that develops into a new individual. A calcium signal, common to a great number of species, from marine invertebrates to mammals, is essential to activate the metabolism of the unfertilized oocyte. However, how fertilization triggers this calcium signal and initiates development of the early embryo is far from understood. The signaling pathways activated in eggs may be similar to those described in somatic cells, since changes in intracellular free calcium and in mitosis activating protein (MAP) kinase activity occur in both systems after activation. Several hypotheses are currently proposed, implying a spermatic ligand binding to a specific receptor expressed at the egg surface, or where the fused sperm either allows the transit of external calcium into the egg or injects one (or several) activating factor(s). It is still not known which of these ideas is true. We concentrate in this review on the possible signaling pathways involving IP3 (inositol trisphosphate), since its production is involved in most species to generate the fertilization calcium wave.

Adenosine Diphosphate Ribose↗