Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites.

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster larvae, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure (WC), another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveal that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal WC or hemocyte spreading.

Proteomics support the threespine stickleback egg coat as a protective oocyte envelope.

Ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Ecological and behavioral factors have been suggested to underlie stickleback reproductive isolation and incipient speciation, but reproductive proteins mediating gamete recognition during fertilization have so far remained unexplored. To begin to investigate the contribution of reproductive proteins to stickleback reproductive isolation, we have characterized the stickleback egg coat proteome. We find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3, as in other teleost fish. Our molecular evolutionary analyses indicate that across teleosts, ZP3 but not ZP1 has experienced positive Darwinian selection. Mammalian ZP3 is also rapidly evolving, and surprisingly some residues under selection in stickleback and mammalian ZP3 directly align. Despite broad homology, however, we find differences between mammalian and stickleback ZP proteins with respect to glycosylation, disulfide bonding, and sites of synthesis. Taken together, the changes we observe in stickleback ZP protein architecture suggest that the egg coats of stickleback fish, and perhaps fish more generally, have evolved to fulfill a more protective functional role than their mammalian counterparts.

The subcortical maternal complex: emerging roles and novel perspectives.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

Receptor-mediated yolk uptake is required for oskar mRNA localization and cortical anchorage of germ plasm components in the Drosophila oocyte.

The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. Here, we report that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte.

Maternal Larp6 controls oocyte development, chorion formation and elevation.

La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus.

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

ZAR1 and ZAR2 are required for oocyte meiotic maturation by regulating the maternal transcriptome and mRNA translational activation.

Zar1 was one of the earliest mammalian maternal-effect genes to be identified. Embryos derived from Zar1-null female mice are blocked before zygotic genome activation; however, the underlying mechanism remains unclear. By knocking out Zar1 and its homolog Zar2 in mice, we revealed a novel function of these genes in oocyte meiotic maturation. Zar1/2-deleted oocytes displayed delayed meiotic resumption and polar body-1 emission and a higher incidence of abnormal meiotic spindle formation and chromosome aneuploidy. The grown oocytes of Zar1/2-null mice contained decreased levels of many maternal mRNAs and displayed a reduced level of protein synthesis. Key maturation-associated changes failed to occur in the Zar1/2-null oocytes, including the translational activation of maternal mRNAs encoding the cell-cycle proteins cyclin B1 and WEE2, as well as maternal-to-zygotic transition (MZT) licensing factor BTG4. Consequently, maternal mRNA decay was impaired and MZT was abolished. ZAR1/2 bound mRNAs to regulate the translational activity of their 3'-UTRs and interacted with other oocyte proteins, including mRNA-stabilizing protein MSY2 and cytoplasmic lattice components. These results countered the traditional view that ZAR1 only functions after fertilization and highlight a previously unrecognized role of ZAR1/2 in regulating the maternal transcriptome and translational activation in maturing oocytes.

Migration of chicken egg-white protein ovalbumin-related protein X and its alteration in heparin-binding affinity during embryogenesis of fertilized egg.

Ovalbumin-related protein X (OVAX) is a 50 kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis.

Progression of Meiosis Is Coordinated by the Level and Location of MAPK Activation Via OGR-2 in Caenorhabditis elegans.

During meiosis, a series of evolutionarily conserved events allow for reductional chromosome division, which is required for sexual reproduction. Although individual meiotic processes have been extensively studied, we currently know far less about how meiosis is regulated and coordinated. In the Caenorhabditis elegans gonad, mitogen-activated protein kinase (MAPK) signaling drives oogenesis while undergoing spatial activation and deactivation waves. However, it is currently unclear how MAPK activation is governed and how it facilitates the progression of oogenesis. Here, we show that the oocyte and germline-related 2 (ogr-2) gene affects proper progression of oogenesis. Complete deletion of ogr-2 results in delayed meiotic entry and late spatial onset of double-strand break repair. Elevated levels of apoptosis are observed in this mutant, independent of the meiotic canonical checkpoints; however, they are dependent on the MAPK terminal member MPK-1/ERK. MPK-1 activation is elevated in diplotene in ogr-2 mutants and its aberrant spatial activation correlates with stages where meiotic progression defects are evident. Deletion of ogr-2 significantly reduces the expression of lip-1, a phosphatase reported to repress MPK-1, which is consistent with OGR-2 localization at chromatin in germ cells. We suggest that OGR-2 modulates the expression of lip-1 to promote the timely progression of meiosis through MPK-1 spatial deactivation.

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.

The eggs of the freshwater Pomacea apple snails develop above the water level, exposed to varied physical and biological stressors. Their high hatching success seems to be linked to their proteins or perivitellins, which surround the developing embryo providing nutrients, sunscreens and varied defenses. The defensive mechanism has been unveiled in P. canaliculata and P. maculata eggs, where their major perivitellins are pigmented, non-digestible and provide a warning coloration while another perivitellin acts as a toxin. In P. scalaris, a species sympatric to the former, the defense strategy seems different, since no toxin was found and the major perivitellin, PsSC, while also colored and non-digestible, is a carbohydrate-binding protein. In this study we examine the structure and function of PsSC by sequencing its subunits, characterizing its carbohydrate binding profile and evaluating its effect on gut cells. Whereas cDNA sequencing and database search showed no lectin domain, glycan array carbohydrate binding profile revealed a strong specificity for glycosphingolipids and ABO group antigens. Moreover, PsSC agglutinated bacteria in a dose-dependent manner. Inspired on the defensive properties of seed lectins we evaluated the effects of PsSC on intestinal cells both in vitro (Caco-2 and IEC-6 cells) and in the gastrointestinal tract of rats. PsSC binds to Caco-2 cell membranes without reducing its viability, while a PsSC-containing diet temporarily induces large epithelium alterations and an increased absorptive surface. Based on these results, we propose that PsSC is involved in embryo defenses by altering the gut morphophysiology of potential predators, a convergent role to plant defensive lectins.

The Fish Egg's Zona Pellucida.

All fish eggs are surrounded by an envelope, called the zona pellucida (ZP), that plays various roles during oogenesis, egg deposition, fertilization, and embryogenesis. The fish egg ZP consists of only a few proteins that are homologs of mammalian ZP proteins ZP1, ZP3, and ZP4. Unlike the situation in mammals, in fishes there are often multiple copies of ZP genes, perhaps a consequence of ancient polyploidization, gene amplification, and mutation. Like mammalian ZP proteins, fish egg ZP1-like proteins exhibit conserved organization with distinct domains and motifs, but unlike mammalian ZP1 and ZP4 have a glutamine (Q)- and/or proline (P)-rich stretch as an N-terminal extension. Such extensions may play a role in assembly of ZP fibrils and/or account for certain properties of the fish egg ZP, such as elasticity. Recent proposals suggest that fish egg ZP proteins can adopt amyloid-like structures, serve as antifreeze proteins in Antarctic icefishes, and protect eggs subjected to desiccating conditions in small shallow pools. In this chapter, these and other aspects of fish egg ZP proteins are presented.

Egg-Coat and Zona Pellucida Proteins of Chicken as a Typical Species of Aves.

Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.

Conceptus Coats of Marsupials and Monotremes.

Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT).

Egg Coat Proteins Across Metazoan Evolution.

All animal oocytes are surrounded by a glycoproteinaceous egg coat, a specialized extracellular matrix that serves both structural and species-specific roles during fertilization. Egg coat glycoproteins polymerize into the extracellular matrix of the egg coat using a conserved protein-protein interaction module-the zona pellucida (ZP) domain-common to both vertebrates and invertebrates, suggesting that the basic structural features of egg coats have been conserved across hundreds of millions of years of evolution. Egg coat proteins, as with other proteins involved in reproduction, are frequently found to be rapidly evolving. Given that gamete compatibility must be maintained for the fitness of sexually reproducing organisms, this finding is somewhat paradoxical and suggests a role for adaptive diversification in reproductive protein evolution. Here we review the structure and function of metazoan egg coat proteins, with an emphasis on the potential role their evolution has played in the creation and maintenance of species boundaries.

Embryonic and post-embryonic development inside wolf spiders' egg sac with special emphasis on the vitellus.

The development of Pardosa saltans wolf spiders inside an egg sac includes two periods: an embryonic period and a post-embryonic period after hatching. We investigated spiderlings' energy expenditure to assess energetic costs during the different embryonic and post-embryonic developmental stages during which they are confined within their egg sac. We focused on the following developmental stages: egg, embryonic stages 1 and 2, and two stages, separated by a moult, during post-embryogenesis inside the egg sac: "juvenile instars 1 and 2" until emergence of 2 instar juveniles from their egg sac. We present the first biochemical characterization of the vitellus of wolf spiders' eggs, embryos and juveniles. Lipovitellins (LV) are composed of four apolipoproteins of 116, 87, 70 and 42 kDa, respectively, and LV represent 35-45% of total protein during development. The principal LV lipids are triglycerides, phospholipids, free fatty acids and sterols. Egg caloric content averaged 127 cal/g (proteins: 91 cal/g, lipids: 33 cal/g, carbohydrates: 3 cal/g). During development from undivided egg to emerged "juvenile 2", 67% of proteins, 51% of carbohydrates and 49% of triglycerides stocks were depleted. At the end of the post-embryonic period, at emergence from egg sac, body energy stock of "juveniles 2" was 38% of the initial calorie stocks in the eggs.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Translation repression by maternal RNA binding protein Zar1 is essential for early oogenesis in zebrafish.

A large amount of maternal RNA is deposited in oocytes and is reserved for later development. Control of maternal RNA translation during oocyte maturation has been extensively investigated and its regulatory mechanisms are well documented. However, translational regulation of maternal RNA in early oogenesis is largely unexplored. In this study, we generated zebrafish zar1 mutants that result in early oocyte apoptosis and fully penetrant male development. Loss of p53 suppresses the apoptosis in zar1 mutants and restores oocyte development. zar1 immature ovaries show upregulation of proteins implicated in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). More importantly, loss of Zar1 causes marked upregulation of zona pellucida (ZP) family proteins, while overexpression of ZP proteins in oocytes causes upregulation of stress-related activating transcription factor 3 (atf3), arguing that tightly controlled translation of ZP proteins is essential for ER homeostasis during early oogenesis. Furthermore, Zar1 binds to ZP gene mRNAs and represses their translation. Together, our results indicate that regulation of translational repression and de-repression are essential for precisely controlling protein expression during early oogenesis.

The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease.

Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3'-5' EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3' untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.

Regulation of Exacerbated Immune Responses in Human Peripheral Blood Cells by Hydrolysed Egg White Proteins.

The anti-allergic potential of egg white protein hydrolysates (from ovalbumin, lysozyme and ovomucoid) was evaluated as their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of reactive oxygen species from Th1-stimulated peripheral blood leukocytes (PBLs). The binding to IgE of egg allergic patients was determined and the peptides present in the hydrolysates were identified. The hydrolysates with alcalase down-regulated the production of Th2-biased cytokines and the secretion of IgE to the culture media of Th2-skewed PBMCs, and they significantly neutralized oxidative stress in PBLs. The hydrolysates of ovalbumin and ovomucoid with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. The hydrolysates with alcalase, in addition to equilibrating Th2 differentiation, exhibited a low IgE-binding. Therefore, they would elicit mild allergic reactions while retaining T cell-stimulating abilities, which might correlate with an anti-allergic benefit.

Immune-Relevant and Antioxidant Activities of Vitellogenin and Yolk Proteins in Fish.

Vitellogenin (Vtg), the major egg yolk precursor protein, is traditionally thought to provide protein- and lipid-rich nutrients for developing embryos and larvae. However, the roles of Vtg as well as its derived yolk proteins lipovitellin (Lv) and phosvitin (Pv) extend beyond nutritional functions. Accumulating data have demonstrated that Vtg, Lv and Pv participate in host innate immune defense with multifaceted functions. They can all act as multivalent pattern recognition receptors capable of identifying invading microbes. Vtg and Pv can also act as immune effectors capable of killing bacteria and virus. Moreover, Vtg and Lv are shown to possess phagocytosis-promoting activity as opsonins. In addition to these immune-relevant functions, Vtg and Pv are found to have antioxidant activity, which is able to protect the host from oxidant stress. These non-nutritional functions clearly deepen our understanding of the physiological roles of the molecules, and at the same time, provide a sound basis for potential application of the molecules in human health.