Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation.
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ABSTRACT:
SUBMITTER: Hu Q
PROVIDER: S-EPMC7594917 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.
| S-EPMC6754564 | biostudies-literature
Project description:Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca(2+) concentration in mammals, ascidians and polychaete worms and a single Ca(2+) peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca(2+) levels occur. Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca(2+) wave requires an intact actin cytoskeleton and an increase in intracellular Ca(2+) can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca(2+) at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca(2+) channels; a single Ca(2+) wave then propagates in an actin dependent manner; this Ca(2+) wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.
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Project description:Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. We used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, we showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. We show that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, we show that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, we present a model in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins.
| S-EPMC3761299 | biostudies-literature
Project description:Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.
| S-EPMC7044060 | biostudies-literature
Project description:Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.
| S-EPMC7330606 | biostudies-literature
Project description:Rhythmic behaviors are ubiquitous phenomena in animals. In C. elegans, defecation is an ultradian rhythmic behavior: every ?50 s a calcium wave initiating in the posterior intestinal cells triggers the defecation motor program that comprises three sequential muscle contractions. Oscillatory calcium signaling is central to the periodicity of defecation. The posteriormost intestinal cells function as the pacemaker for this rhythmic behavior, although it is unclear how the supremacy of these cells for calcium-wave initiation is controlled.We describe how the loss of the mir-240/786 microRNA cluster, which results in arrhythmic defecation, causes ectopic intestinal calcium-wave initiation. mir-240/786 expression in the intestine is restricted to the posterior cells that function as the defecation pacemaker. Genetic data indicate that mir-240/786 functions upstream of the inositol 1,4,5-trisphosphate (IP(3)) receptor. Through rescue analysis, it was determined that miR-786 functions to regulate defecation. Furthermore, we identified elo-2, a fatty-acid elongase with a known role in defecation cycling, as a direct target for miR-786. We propose that the regulation of palmitate levels through repression of elo-2 activity is the likely mechanistic link to defecation.Together, these data indicate that miR-786 confers pacemaker status on posterior intestinal cells for the control of calcium-wave initiation through the regulation of elo-2 and, subsequently, palmitate levels. We propose that a difference in fatty-acid composition in the posterior intestinal cells may alter the activities of membrane proteins, such as IP(3)-receptor or TRPM channels, that control pacemaker activity in the C. elegans intestine.
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Project description:The GLD-2 class of poly(A) polymerases regulate the timing of translation of stored transcripts by elongating the poly(A) tails of target mRNAs in the cytoplasm. WISPY is a GLD-2 enzyme that acts in the Drosophila female germline and is required for the completion of the egg-to-embryo transition. Though a handful of WISPY target mRNAs have been identified during both oogenesis and early embryogenesis, it was unknown whether WISP simply regulated a small pool of patterning or cell cycle genes, or whether, instead, cytoplasmic polyadenylation was widespread during this developmental transition. To identify the full range of WISPY targets, we carried out microarray analysis to look for maternal mRNAs whose poly(A) tails fail to elongate in the absence of WISP function. We examined the polyadenylated portion of the maternal transcriptome in both stage 14 (mature) oocytes and in early embryos that had completed egg activation. Our analysis shows that the poly(A) tails of thousands of maternal mRNAs fail to elongate in wisp-deficient oocytes and embryos. Furthermore, we have identified specific classes of genes that are highly regulated in this manner at each stage. Our study shows that cytoplasmic polyadenylation is a major regulatory mechanism during oocyte maturation and egg activation.
| S-EPMC3821703 | biostudies-literature
Project description:In animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transits into an actively developing embryo, initiates with an increase of Ca2+ in the oocyte’s cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepare the oocyte to undertake embryogenesis. Calcineurin is a highly conserved phosphatase that is activated directly by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fzy, Greatwall (Gwl) and Endosulfine (Endos), protein translation modulators including PNG, NAT, eIF4G and eIF4B, and important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo.
2018-11-29 | PXD010991 | Pride
Project description:In animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transits into an actively developing embryo, initiates with an increase of Ca2+ in the oocyte’s cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepare the oocyte to undertake embryogenesis. Calcineurin is a highly conserved phosphatase that is activated directly by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fzy, Greatwall (Gwl) and Endosulfine (Endos), protein translation modulators including PNG, NAT, eIF4G and eIF4B, and important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo
2019-03-13 | PXD011930 | Pride
Project description:Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKII? activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKII? in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKII? that are required for second polar body emission.
| S-EPMC3306664 | biostudies-literature