The eggshell of the cherry fly Rhagoletis cerasi

One of the major pests in Greek cherry orchards is the cherry fly Rhagoletis cerasi (Diptera: Tephritidae). In order to complete our comparative work on the chorion assembly of other representatives of the fruit flies (e.g. Ceratitis capitata and Dacus oleae) we studied eggshell morphogenesis in the cherry fly. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The eggshell consists of the vitelline membrane, a fibrous layer of possible water-proofing function, an innermost chorionic layer, endochorionic and exochorionic layers. The endochorion shows a branched configuration with irregular cavities, and the exochorion consists of inner and outer layers for better embryo protection. At the anterior region of the follicle, the hexagonal borders of the follicle cells are created by endochorionic material, covered by both inner and outer exochorion. This area resembles the D. melanogaster chorionic appendages and therefore can serve for plastron respiration. The structural results support the phylogenetic relationships among the tephritids (Rhagoletis is closer to Ceratitis than Dacus). The presence of peroxidase in the endochorion, detected by diaminobenzidine, is consistent with the eggshell hardening at the end of choriogenesis, following the same pattern with the other fruit flies studied so far. Two major chorionic proteins are found both in R. cerasi and in C. capitata and therefore general conclusions can be drawn from this study, concerning the pattern of choriogenesis, which all dipteran insects follow, in order to create a resistant and functional eggshell, and the high conservation of the proteinaceous components of the chorion among species in the order.     

Oogenesis of Cardiochiles nigriceps viereck (Hymenoptera : Braconidae): Histochemistry and development of the chorion with special reference to the fibrous layer

A fibrous layer on the surface of eggs of the parasitoid, Cardiochiles nigriceps (Hymenoptera : Braconidae), has been implicated by earlier studies in the evasion from encapsulation by host hemocytes. The present histochemical and ultrastructural study was undertaken to characterize fibrous layer material and to determine the source of fibrous layer and other components of the eggshell. The fibrous layer contains neutral glyco- or mucoprotein; acidic mucoproteins or glycosaminoglycans are absent. The mature eggshell is resolved into 5 morphologically distinct layers by electron microscopy: (from inner to outer) vitelline envelope, endochorion, an electron-dense “irregular layer”, papilliary layer and fibrous layer. During oogenesis each eggshell layer is laid down sequentially in the order mentioned above. Eggshell material appears to be produced by the follicle cells because these develop extensive rough endoplasmic reticulum and golgi apparatus and exhibit apparent exocytotic activity at the plasma membrane adjacent to the egg.     

The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 2. The micropylar appendage

Micropylar apparatuses in insects are specialized regions of the eggshell through which sperm enters the oocyte. This work is an ultrastructural study and deals with the structure and morphogenesis of the micropylar appendage in the hymenopteran Eurytoma amygdali. The micropylar appendage is a 130 mum long cylindrical protrusion located at the posterior pole of the egg, unlike other insects i.e. Diptera. in which the micropylar apparatus is located at the anterior pole. In mature eggs there is a 0.4 mum wide pore (micropyle) at the tip of the appendage leading to a 6 mum wide micropylar canal. The canal contains an electron-lucent substance, it travels along the whole appendage and finally reaches the vitelline membrane of the oocyte. The vitelline membrane is covered by a wax layer and an electron-lucent layer, whereas the chorion surrounding the canal consists of a granular layer (fine and rough) and a columnar layer. The morphogenesis of the appendage starts in immature follicles: four central cells located at the posterior tip of the oocyte near the vitelline membrane, differing morphologically from the adjacent follicle cells. These central cells degenerate during early chorionic stages, thus assisting in the formation of the micropylar canal. The adjacent, peripherally located cells secrete the electron-lucent substance which fills the canal and at the same time, the fine granular layer is formed starting from the base towards the tip of the appendage. The secretion persists at late chorionic stages and results in the formation of the chorion around the micropylar canal. The extremely long (compared to other insects) micropylar appendage seems to facilitate the egg passage through the very thin and long ovipositor. The structure and morphogenesis of this appendage differs significantly from the micropylar apparatuses studied so far in other insects i.e. Diptera, and may reflect adaptational and evolutionary relationships.     

Follicle duct cell ultrastructure and eggshell formation in Triops cancriformis (crustacea,notostraca)

Electron microscopy of the cells of the follicle duct of Triops cancriformis shows that the follicular ducts are lined by a single-layered epithelium which also produces the eggshell material. The cytoplasm is rich in rough endoplasmic reticulum that synthesizes the eggshell material which subsequently aggregates into preformed vacuoles. Newly formed spheres of eggshell material are then excreted into the lumen. At the end of vitellogenesis the oocytes descend toward the longitudinal oviduct and pass through the eggshell material which fills the follicle ducts. The production of the eggshell and its chemical composition in some Phyllopoda are compared. The paper discusses the relationship between the eggshell construction and the reproductive biology of the population.     

Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.     

Heterodera glycines: eggshell ultrastructure and histochemical localization of chitinous components

The eggshell in most nematodes consists of an outer vitelline layer, a middle chitinous and an inner lipid layer. Earlier work with eggs of Heterodera glycines suggests the presence of two chitinous layers but the vitelline layer was not observed. From our observation the outer chitin layer described in past literature is actually a vitelline layer. Histochemical analysis has demonstrated that chitin is absent from the outer envelope. Electron microscope observations of the eggshell show a waxy appearance and osmium staining consistent with that of the proteinaceous vitelline layer found in other nematodes. Lectin localization also shows that the eggshell continues to develop past fertilization with the delivery and integration of eggshell precursors. Contrary to previous reports, we propose that the ultrastructure of the eggshell H. glycines follows the common three-layer structure observed in other nematodes.     

Morphology and formation of the eggshell in the tarnished plant bug, Lygus lineloaris (Palisot de Beauvois) (Hemiptera: Miridae)

Scanning and transmission electron microscopy were used to study the morphology and formation of the eggshell in the tarnished plant bug, Lygus lineolaris. Eggs are bean-shaped, with an operculum at the anterior end surrounded by a row of 36-40 respiratory horns. Three micropylar openings are on the operculum, and are sealed in oviposited eggs. The chorion consists of the chorion proper and the innermost chorionic layer. An air layer composed of colonnades is present in the chorion. The innermost chorionic layer is homogeneous and electron lucent. The follicle cells secrete electron dense materials that later coalesced into the reticulated vitelline membrane. This is followed by the deposition of the innermost chorionic layer by the follicle cells. After the primordial innermost chorionic layer is formed, follicle cells at the anterior pole of the oocyte secrete the scaffold for the colonnades in the air layer. Later, the primordial scaffold matrix is redistributed and localized at the lateral and posterior end of the oocyte where it becomes secondarily modified. At the end of choriogenesis, follicle cells at the anterior pole secrete the operculum and respiratory horns.     

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.     

Laser-Raman spectroscopic studies of the eggshell (chorion) of Bombyx mori

Laser-Raman spectroscopic studies of the eggshell (chorion) of the silkmoth Bombyx mori reveal that its component proteins consist of 60–70% antiparallel β-pleated sheet and 30–40% of β-turns. The disulphide bonds, which crosslink the (extremely rich in cysteine)-proteins of the outer lamellar eggshell layer, are apparently found in G-G-G (gauche-gauche-gauche) and T-G-T (trans-gauche-trans) conformation; there is no evidence for the existence of free sulphydryls. The highly localized tyrosine residues appear to form hydrogen bonds, acting as weak proton donors or as acceptors.     

Hierarchical assembly of the eggshell and permeability barrier in C. elegans

In metazoans, fertilization triggers the assembly of an extracellular coat that constitutes the interface between the embryo and its environment. In nematodes, this coat is the eggshell, which provides mechanical rigidity, prevents polyspermy, and is impermeable to small molecules. Using immunoelectron microscopy, we found that the Caenorhabditis elegans eggshell was composed of an outer vitelline layer, a middle chitin layer, and an inner layer containing chondroitin proteoglycans. The switch between the chitin and proteoglycan layers was achieved by internalization of chitin synthase coincident with exocytosis of proteoglycan-containing cortical granules. Inner layer assembly did not make the zygote impermeable as previously proposed. Instead, correlative light and electron microscopy demonstrated that the permeability barrier was a distinct envelope that formed in a separate step that required fatty acid synthesis, the sugar-modifying enzyme PERM-1, and the acyl chain transfer enzyme DGTR-1. These findings delineate the hierarchy of eggshell assembly and define key molecular mechanisms at each step.     

The Drosophila embryonic patterning determinant torsolike is a component of the eggshell

The development of the head and tail regions of the Drosophila embryo is dependent upon the localized polar activation of Torso (Tor), a receptor tyrosine kinase that is uniformly distributed in the membrane of the developing embryo. Trunk (Trk), the proposed ligand for Tor, is secreted as an inactive precursor into the perivitelline fluid that lies between the embryonic membrane and the vitelline membrane (VM), the inner layer of the eggshell. The spatial regulation of Trk processing is thought to be mediated by the secreted product of the torsolike (tsl) gene, which is expressed during oogenesis by a specialized population of follicle cells present at the two ends of the oocyte. We show here that Tsl protein is specifically localized to the polar regions of the VM in laid eggs. We further demonstrate that although Tsl can associate with nonpolar regions of the VM, the activity of polar-localized Tsl is enhanced, suggesting the existence of another spatially restricted factor acting in this pathway. The incorporation of Tsl into the VM provides a mechanism for the transfer of spatial information from the follicle cells to the developing embryo. To our knowledge, Tsl represents the first example of an embryonic patterning determinant that is a component of the eggshell.     

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.     

The eggshell in the C. elegans oocyte-to-embryo transition

In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development.     

fc177, a minor dec-1 proprotein,is necessary to prevent ectopic aggregation of the endochorion during eggshell assembly in Drosophila

The Drosophila eggshell is a highly specialized extracellular matrix that forms between the oocyte and the surrounding epithelial follicle cells during late oogenesis. The dec-1 gene, which is required for proper eggshell assembly, produces three proproteins that are cleaved within the vitelline membrane layer to multiple derivatives. The different spatial distributions of the cleaved derivatives suggest that they play distinct roles in eggshell assembly. Using extant dec-1 mutations in conjunction with genetically engineered dec-1 transgenes, we show that, although all three dec-1 proproteins, fc106, fc125, and fc177, are required for female fertility, gross morphological abnormalities in the eggshell are observed only in the absence of fc177. The coalescence of the roof, pillar, and floor substructures of the tripartite endochorion suggested that quantitatively minor fc177 derivatives are necessary to prevent ectopic aggregation of endochorion proteins during the assembly process. Expression of a fc177 cDNA in dec-1 null mutants was sufficient to restore spaces within the endochorion layer. Fc177 may function as a scaffolding protein akin to those utilized in viral morphogenesis.     

Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

Background

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated; one reason is that there is no follicle culture system that can reproduce theca cell layer formation in vitro. Therefore, a three-dimensional follicle culture system that can reproduce theca cell layer formation is required.

Methods

A collagen gel was used in the follicle culture system. To determine the optimum conditions for follicle culture that can reproduce theca cell layer formation, the effects of hormonal treatment and cell types co-cultured with follicles were examined. In addition, immunohistochemistry was used to examine the properties of the cell layers formed in the outermost part of follicles.

Results

Follicles maintained a three-dimensional shape and grew in collagen gel. By adding follicle-stimulating hormone (FSH) and co-culturing with interstitial cells, the follicles grew well, and cell layers were formed in the outermost part of follicles. Immunohistochemistry confirmed that the cells forming the outermost layers of the follicles were theca cells.

Conclusion

In this study, follicle culture system that can reproduce theca cell layer formation in vitro was established. In our opinion, this system is suitable for the analysis of theca cell layer formation and contributes to our understanding of the mechanisms of folliculogenesis.     

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster egg' s during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the 10b of lipid vesicles during static 10b. During secretion (stages 10b, 11 and 12) the lipid vcsicles are accumulated on the vitelline membrane surface and become flat. At the late stage of choriogenisis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in crossfractured views.     

Differential expression of Paragonimus westermani eggshell proteins during the developmental stages

Eggs of trematode parasites are comprised of numerous vitelline cells and one fertilized ovum, and are encapsulated within a protein shell provided by the vitellocytes. In this study, we isolated two full-length cDNA clones that showed substantial levels of sequence identity with trematode-specific eggshell precursor proteins from the human lung fluke, Paragonimus westermani. These cDNAs, designated Pw-Vit20 (868-bp-long) and Pw-Vit36 (883-bp-long), shared a 76% identity with one another at the nucleotide level, and each encoded a 261-amino acid (aa) polypeptide. The deduced aa sequences contained a N-terminal hydrophobic segment, as well as a sequence motif of Gly-Gly-Gly-Tyr-Asp-Asn/Thr-Tyr-Gly-Lys/Gln, which is highly homologous with the eggshell proteins of Fasciola hepatica. With the high frequencies of tyrosine, glycine and lysine, the positions occupied by tyrosine, which has been proved to be converted into dihydroxyphenylalanine, were well preserved. Pw-Vit20 and Pw-Vit36 were found to be monoexonic genes with variably diverged variants scattered into multiple genomic loci. Their protein products were localized in the vitelline follicles and eggshells. Expression of Pw-Vit20 was restricted to the egg and adult stages, thus suggesting a critical involvement of Pw-Vit20 in the parasite's fecundity activity. Conversely, Pw-Vit36 was constitutively expressed in the metacercariae and juvenile stages in the vitelline follicles and ducts, which suggested that the prepositioning of stem or primordial vitelline cells within the juveniles prior to sexual maturation. Pw-Vit36 might acquire a unique or additional function relevant to the maturation and/or development of the vitelline cells/follicles during the evolutionary period of P. westermani. Differential biological implications of multiple eggshell precursor proteins may provide insight into the molecular mechanism of eggshell formation and the developmental process of the vitelline follicles in the parasitic trematode.