Osmolarity-regulated swelling initiates egg activation in Drosophila

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na⁺ channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.     

Revealing phosphoproteins playing role in tobacco pollen activated in vitro

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co‐exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.     

Modulation of MAPK activities during egg activation in Drosophila

The mitogen-activated protein kinases (MAPKs) play essential roles during oocyte maturation and egg activation and are also active in somatic cell cycle regulation in many animals. In clams, starfish, ascidians, mice, and frogs, the species-specific timing of MAPK activity during oocyte maturation and egg activation correlates with the different meiotic arrest points of these various organisms. Furthermore, MAPKs have been shown to regulate the meiotic cell cycle in marine invertebrates and vertebrates. The initial trigger for egg activation in insects is different from that of marine invertebrates and vertebrates, and it was not previously known whether changes in MAPK activity accompany egg activation in insects. To examine the regulation of MAPKs during Drosophila egg activation and early embryogenesis, we quantified the levels of phosphorylated (active) forms of ERK, p38 and JNK by western blotting with antibodies specific to the phospho-forms of these kinases. Levels of phospho-ERK, phospho-p38 and phospho-JNK are high in Drosophila oocytes. Upon egg activation, levels of all these phospho- (active) forms of MAPKs decrease. Fertilization is not required for this decrease, consistent with the independence of egg activation from fertilization in Drosophila. The decrease in levels of phospho-MAPK occurs normally in embryos laid by sterile females mutant in the egg activation genes cortex, sarah, and prage. We present a model in which the decrease in MAPK activity is an intermediate step in the pathway leading from the calcium signal that initiates egg activation to the downstream events of activation.     

Modulation of MAPK Activities During Egg Activation in Drosophila

The mitogen-activated protein kinases (MAPKs) play essential roles during oocyte maturation and egg activation and are also active in somatic cell cycle regulation in many animals. In clams, starfish, ascidians, mice, and frogs, the species-specific timing of MAPK activity during oocyte maturation and egg activation correlates with the different meiotic arrest points of these various organisms. Furthermore, MAPKs have been shown to regulate the meiotic cell cycle in marine invertebrates and vertebrates. The initial trigger for egg activation in insects is different from that of marine invertebrates and vertebrates, and it was not previously known whether changes in MAPK activity accompany egg activation in insects. To examine the regulation of MAPKs during Drosophila egg activation and early embryogenesis, we quantified the levels of phosphorylated (active) forms of ERK, p38 kinase, and JNK by western blotting with antibodies specific to the phospho- forms of these kinases. Levels of phospho-ERK, phospho-p38 kinase, and phospho-JNK are high in Drosophila oocytes. Upon egg activation, levels of all these phospho- (active) forms of MAPKs decrease. Fertilization is not required for this decrease, consistent with the independence of egg activation from fertilization in Drosophila. The decrease in levels of phospho-MAPK occurs normally in embryos laid by sterile females mutant in the egg activation genes cortex, sarah, and prage. We present a model in which the decrease in MAPK activity is an intermediate step in the pathway leading from the calcium signal that initiates egg activation to the downstream events of activation.     

Biochemical analysis of torso and D-raf during Drosophila embryogenesis: implications for terminal signal transduction.

Determination of anterior and posterior terminal structures of Drosophila embryos requires activation of two genes encoding putative protein kinases, torso and D-raf. In this study, we demonstrate that Torso has intrinsic tyrosine kinase activity and show that it is transiently tyrosine phosphorylated (activated) at syncytial blastoderm stages. Torso proteins causing a gain-of-function phenotype are constitutively tyrosine phosphorylated, while Torso proteins causing a loss-of-function phenotype lack tyrosine kinase activity. The D-raf gene product, which is required for Torso function, is identified as a 90-kDa protein with intrinsic serine/threonine kinase activity. D-Raf is expressed throughout embryogenesis; however, the phosphorylation state of the protein changes during development. In wild-type embryos, D-Raf is hyperphosphorylated at 1 to 2 h after egg laying, and thereafter only the most highly phosphorylated form is detected. Embryos lacking Torso activity, however, show significant reductions in D-Raf protein expression rather than major alterations in the protein's phosphorylation state. This report provides the first biochemical analysis of the terminal signal transduction pathway in Drosophila embryos.     

Dependence of removal of sperm-specific proteins from Xenopus sperm nuclei on the phosphorylation state of nucleoplasmin

In Xenopus laevis , nucleoplasmin from fully grown oocytes is not highly phosphorylated, but is more extensively phosphorylated during oocyte maturation to retain this state until mid-blastula transition. Incubation of demembranated sperm with nucleoplasmin from oocytes or mature eggs revealed that egg nucleoplasmin is twice as potent as oocyte nucleoplasmin in removing sperm-specific basic proteins from chromatin (protamine-removing activity: PRA). Dephosphorylation of egg nucleoplasmin by alkaline phosphatase induced a remarkable decline of PRA in nucleoplasmin. Treatment of oocyte nucleoplasmin with cdc2 protein kinase induced an increase of the extent of phosphorylation, but to a level lower than that exhibited by egg nucleoplasmin, suggesting the involvement of other unspecified kinase(s) in phosphorylating nucleoplasmin during oocyte maturation. Incubation of sperm with cdc2 kinase induced selective phosphorylation of sperm-specific basic proteins, accompanied by their enhanced removal from sperm chromatin upon exposure to high-salt solutions. These results suggest that removal of sperm-specific basic proteins from sperm chromatin in fertilized eggs is facilitated by phosphorylation of both nucleoplasmin and sperm-specific basic proteins.     

Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation

The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho‐proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. Here, we show that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. We show that neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.     

Phosphorylation-dependent interaction of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHA) with PADI6 following oocyte maturation in mice

Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation.     

Use of combined in silico expression data and phylogenetic analysis to identify new oocyte genes encoding RNA binding proteins in the mouse

During folliculogenesis, oocytes accumulate maternal mRNAs in preparation for the first steps of early embryogenesis. The processing of oocyte mRNAs is ensured by heterogeneous nuclear ribonucleoproteins (hnRNPs) genes that encode RNA binding proteins implied in mRNA biogenesis, translation, alternative splicing, nuclear exportation, and degradation. In the present work, by combining phylogenetic analyses and, when available, in silico expression data, we have identified three new oocyte-expressed genes encoding RNA binding proteins by using two strategies. Firstly, we have identified mouse orthologs of the Car1 gene, known to be involved in regulation of germ cell apoptosis in C. elegans, and of the Squid gene, required for the establishment of anteroposterior polarity in the Drosophila oocyte. Secondly, we have identified, among genes whose ESTs are highly represented in oocyte libraries, a paralog of Poly(A) binding protein--Interacting Protein 2 (Paip2) gene, known to inhibit the interaction of the Poly(A)-Binding Protein with Poly(A) tails of mRNAs. For all of these genes, the expression in oocyte was verified by in situ hybridization. Overall, this work underlines the efficiency of in silico methodologies to identify new genes involved in biological processes such as oogenesis.     

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.     

Further analysis of cytoplasmic polyadenylation in Xenopus embryos and identification of embryonic cytoplasmic polyadenylation element-binding proteins.

Early development in Xenopus laevis is programmed in part by maternally inherited mRNAs that are synthesized and stored in the growing oocyte. During oocyte maturation, several of these messages are translationally activated by poly(A) elongation, which in turn is regulated by two cis elements in the 3' untranslated region, the hexanucleotide AAUAAA and a cytoplasmic polyadenylation element (CPE) consisting of UUUUUAU or similar sequence. In the early embryo, a different set of maternal mRNAs is translationally activated. We have shown previously that one of these, C12, requires a CPE consisting of at least 12 uridine residues, in addition to the hexanucleotide, for its cytoplasmic polyadenylation and subsequent translation (R. Simon, J.-P. Tassan, and J.D. Richter, Genes Dev. 6:2580-2591, 1992). To assess whether this embryonic CPE functions in other maternal mRNAs, we have chosen Cl1 RNA, which is known to be polyadenylated during early embryogenesis (J. Paris, B. Osborne, A. Couturier, R. LeGuellec, and M. Philippe, Gene 72:169-176, 1988). Wild-type as well as mutated versions of Cl1 RNA were injected into fertilized eggs and were analyzed for cytoplasmic polyadenylation at times up to the gastrula stage. This RNA also required a poly(U) CPE for cytoplasmic polyadenylation in embryos, but in this case the CPE consisted of 18 uridine residues. In addition, the timing and extent of cytoplasmic poly(A) elongation during early embryogenesis were dependent upon the distance between the CPE and the hexanucleotide. Further, as was the case with Cl2 RNA, Cl1 RNA contains a large masking element that prevents premature cytoplasmic polyadenylation during oocyte maturation. To examine the factors that may be involved in the cytoplasmic polyadenylation of both C12 and C11 RNAs, we performed UV cross-linking experiments in egg extracts. Two proteins with sizes of ~36 and ~45 kDa interacted specifically with the CPEs of both RNAs, although they bound preferentially to the C12 CPE. The role that these proteins might play in cytoplasmic polyadenylation is discussed.     

Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation

Egg activation is the process that modifies mature, arrested oocytes so that embryo development can proceed. One key aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. wispy (wisp) maternal-effect mutations in Drosophila block development during the egg-to-embryo transition. We show here that the wisp gene encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). The WISP protein is required for poly(A) tail elongation of bicoid, Toll, and torso mRNAs upon egg activation. In Drosophila, WISP and Smaug (SMG) have previously been reported to be required to trigger the destabilization of maternal mRNAs during egg activation. SMG is the major regulator of this activity. We report here that SMG is still translated in activated eggs from wisp mutant mothers, indicating that WISP does not regulate mRNA stability by controlling the translation of smg mRNA. We have also analyzed in detail the very early developmental arrest associated with wisp mutations. Pronuclear migration does not occur in activated eggs laid by wisp mutant females. Finally, we find that WISP function is also needed during oogenesis to regulate the poly(A) tail length of dmos during oocyte maturation and to maintain a high level of active (phospho-) mitogen-activated protein kinases (MAPKs).     

2DE identification of proteins exhibiting turnover and phosphorylation dynamics during sea urchin egg activation

The animal egg is a unique quiescent cell, prepackaged with maternal mRNAs and proteins that have functions in early development. Rapid, transient signaling at fertilization alters egg physiology, resulting in Ca²⁺ release from the endoplasmic reticulum (ER) and cytoplasmic alkalinization. These events trigger the zygote developmental program through initiation of DNA synthesis and entry into mitosis. Post-translational modifications of maternal proteins are responsible for the spatio-temporal regulation that orchestrates egg activation. We used functional proteomics to identify the candidate maternal proteins involved in egg activation and early development. As the first step of this analysis, we present the data on the baseline maternal proteome, in particular, on proteins exhibiting changes in abundance and in phosphorylation state upon egg activation. We identify 94 proteins that were stable, reproducibly displayed a shift in isoelectric point, or changed in relative abundance at specific times after activation. The identities of these proteins were determined by quadrupole time-of-flight tandem mass spectrometry. The set of the most dynamic proteins appear to be enriched in intermediary metabolism proteins, cytoskeletal proteins, gamete associated proteins and proteins that have Ca²⁺ mediated activities.     

Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway

LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor (EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNFalpha protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.     

XGef mediates early CPEB phosphorylation during Xenopus oocyte meiotic maturation

Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process.     

EGG-3 regulates cell-surface and cortex rearrangements during egg activation in Caenorhabditis elegans

Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family, is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition, whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition.     

Spatial distribution of abundant proteins in oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum)

Two-dimensional protein patterns were compared from sections along the longitudinal axis of oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum). Only a few differences were observed between four different sections through both oocyte and fertilized eggs. A set of proteins (14 out of 120 proteins) were found that reside only in the germinal vesicles (GV) of the fully grown oocyte. Two of these were observed exclusively in the vegetal half, and one in the animal half after GV breakdown, while other proteins were randomly distributed within the fertilized egg. One cytoplasmic protein was present only in the vegetal half of the mature oocyte and became present also in the animal half of the fertilized egg. Additional proteins were observed in all transverse sections of both mature oocyte and fertilized eggs. It is proposed that these proteins are modified rather than newly synthesized proteins.