Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan Aurelia aurita (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages ofoocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the zona pellucida type.     

Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan <Emphasis Type="Italic">Aurelia aurita</Emphasis> (Cnidaria: Semaeostomae)

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages of oocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the Zona Pellucida type.     

Mesogleal cells of the jellyfish Aurelia aurita are involved in the formation of mesogleal fibres

The extracellular matrix of the jellyfish Aurelia aurita (Scyphozoa, Cnidaria), known as the mesoglea, is populated by numerous mesogleal cells (Mc). We determined the pattern of the Mc and the mesoglea, raised polyclonal antibodies (RA47) against the major mesogleal protein pA47 (47 kDa) and checked their specificity. In the mesoglea, RA47 stains pA47 itself. In immunoblots of Mc, RA47 stains bands of 120 kDa and 80 kDa; weaker staining is observed at pA47. The same staining pattern is seen on blots of jellyfish epidermal cells and of whole Hydra (Hydrozoa) or isolated mesoglea of Hydra. Our data indicate that pA47 is synthesized by Mc and epidermal cells as high molecular precursors. Using immunostaining techniques, we showed Mc to be involved in the formation of mesogleal non-collagenous (called "elastic" in classic morphological studies) fibres. The biochemical and morphological data suggest that Mc originate from the epidermis.     

Protein composition of mesoglea and mesogloeal cells of medusa Aurelia aurita

Protein composition of mesoglea of the scyphomedusa Aurelia aurita was revealed in SDS-PAGE. Some major bands are visible in mesoglea of a mature medusa: 30, 45-47, 85 kDa, three bands between 100-200 kDa, and several bands with molecular weights > 300 kDa. Polyclonal antisera RA45/47 against protein 45 kDa were raised. RA45/47 react with 45-47 kDa protein in mesogleal sample and protein 120 kDa in mesogleal cells on immunoblot. Immunohistochemical analysis of A. aurita histological sections of young and mature medusae showed antigen localization in mesogleal cell granules and in the apical part of ectodermal cells. In mature medusae, the antigen was localized also in elastic fibers. We can conclude that in A. aurita mesogleal cells, along with ectodermal cells, take part in the formation of extracellular matrix of mesoglea.     

Plate in the zone of oocyte and germinal epithelium contact in Scyphomedusa Aurelia aurita binds antibodies to ZP-domain protein mesoglein

The morphological features of oocyte and germinal epithelium (epithelial wall of germinal sinus) contact area in Scyphomedusa Aurelia aurita are described. Growing oocytes were divided into seven stages based on oocyte size. The structure revealed the area of contact between the oocyte and the germinal epithelium called the contact plate. During oocyte growth, single granules are fused into the homogeneous mass area of oocyte contact with epithelium. Plate components bound antibodies to mesoglein. It was assumed that the plate material contained ZP-domain proteins. Electrophoresis and immunoblotting results show that proteins immunologically similar to mesoglein have higher molecular masses, probably due to post-translation modifications, which are common for extracellular proteins. On the other hand, however, gonad proteins may be other representatives of jellyfish ZP-domain proteins. Further experiments should be conducted to clarify which alternative is true.     

Differential gene expression in the jellyfish Aurelia aurita

The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium.     

Regeneration in Medusa buds and Medusae of Hydrozoa

Isolated medusa buds of Podocoryne carnea (Anthomedusa) caneither develop autonomously to functional medusae or transformto stolons and polyps. Up to stage 8 the percentage of transformaiioncan be idised 10 100% by repeated traumatization of the isolatedbuds. From stage 9 onwards, even traumatized buds always regeneratemedusae and never transform to polyps. At stage 8 the bud isdifferentiated, lacking only the swelling of the outer mesogleato become a normal medusa. A correlation between the swellingof this mesoglea and the decrease of the percentage of transformationto zero can be observed between stages 8 and 9. The presenceof a swollen mesoglea is not the only factor in stabilizingmedusa structures, since, despite removal of the mesoglea bythe dissociation method, aggregates of dissociated buds of stages7 and 8 always formed medusa structures and never transformedto stolons or polyps. Regeneration of the manubrium in adult Hydromedusae shows agraded distribution, being highest in the center of the umbrellaand decreasing rapidly towards the circular canal. The extentof manubrium regeneration is not influenced by the amount ofwound material or the components of the gastrovascular system.     

Do respiratory metabolic rates of the scyphomedusa Aurelia aurita scale isometrically throughout ontogeny in a sexual generation?

We measured the respiration rate of Aurelia aurita payingspecial attention to the relationship between its metabolic ratesand body mass throughout ontogeny of the jellyfish in a sexualgeneration. Two different regression lines between respirationrates and body dry weight were obtained in ephyra to young medusa(bell diameter 4.2–19 mm, 0.07–14 mg dry weight) and medusa(BD17-120 mm, 12-2100 mg DW), at respective temperatures of 10 and15 °C. The cut off point of the metabolic rates was foundat the developmental stage just being metamorphosed into medusashape (BD 12–20 mm). The slope value of medusa respiration ratewas close to isometric scaling (0.9), whereas that of ephyra toyoung medusa was lower (0.6). Ecological implications of allometricscaling in the early developmental stages of ephyrae and metephyraeare discussed.     

Scyphomedusae and their polyps are the same immunologically: implications for systematics.

1. Polyp and medusa of the scyphozoans Aurelia aurita and Pelagia colorata (phylum Cnidaria) are indistinguishable by radioimmunoassay of whole animals, yet differ from other cnidarians against which they were tested. 2. We infer that proteins distinguishing species swamp those that differentiate the two (very distinct) life history phases. 3. Thus, at least for some taxa and some systematic techniques analyzing proteins, using organisms at the same developmental phase may be unnecessary, contrary to conventional wisdom.     

The ZP domain is a conserved module for polymerization of extracellular proteins

Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), alpha- and beta-tectorins, transforming growth factor (TGF)-beta receptor III and endoglin, DMBT-1 (deleted in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of alpha-tectorin.     

Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins

A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.     

Egg extracellular coat proteins: from fish to mammals

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.     

Compartments in Scyphozoa

Polyps of Scyphozoa have a cup-shaped body. At one end is the mouth opening surrounded by tentacles, at the other end is an attachment disc. The body wall consists of two tissue layers, the ectoderm and the endoderm, which are separated by an extracellular matrix, the mesoglea. The polyp's gastric cavity is subdivided by septa running from the apical end to the basal body end. The septa consist of two layers of endoderm and according to biology textbooks the number of septa is four. However, in rare circumstances Aurelia produces polyps with zero, two, six, or eight septa. We found that the number was always even. Therefore we propose that two types of endoderm exist, forming alternating stripes running from the oral body end to the aboral end. The stripes have some properties of developmental compartments. Where cells of different compartments meet, they form a septum. We also propose that the ectoderm is subdivided into compartments. The borders of the ectodermal and endodermal compartments are perpendicular to each other. Tentacles of the polyp and rhopalia (sense organs) of the ephyra (young medusa), respectively, develop at the border between two ectodermal compartments. The number can be even or odd. Rhopalia formation is particularly favored where two ectodermal and two endodermal compartments meet.     

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.