Innermost Chorionic Layer Research Articles

Fine Structure of the Eggshell of the Blow Fly,Lucilia cuprina

The fine structure of the eggshell of blow fly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), was examined using scanning and transmission electron microscopy. Eggs, 1.09±0.07 mm in length and 0.25±0.05 mm in width, bore a relatively wide plastron that extending along almost the entire length. The polygonal pattern of chorionic sculpture was indistinct. The ultrathin section indicated a multi-layered eggshell having an exochorion, outer endochorion, pillars, an inner endochorion, innermost chorionic layer, and a wax layer. This study provides new information about the fine morphology of blow flies eggs. A key to differentiate the eggs of forensically important flies in Thailand is given.

The enzymatic component of Drosophila melanogaster chorion is the Pxd peroxidase

In the present study, we demonstrate the isolation and characterization of the Pxd cDNA clone, which codes for the Drosophila melanogaster chorion peroxidase. This specific peroxidase is involved in the chorion hardening process, through protein crosslinking mediated by the formation of di- and tri-tyrosine bonds. The Pxd gene product has been identified in crude protein extracts from adult flies as three immunoreacting, with the anti-rAePO polyclonal antibody, bands of 77, 67 and 55 kDa, while in larvae and purified chorions as a unique 55 kDa band. Moreover, the mature form of the Pxd recombinant protein was specifically recognized by the anti-rAePO antibody as a 77 kDa band, while in the presence of H 2O 2 was able to convert tyrosine residues to di-tyrosine moieties. Northern blotting analysis of total RNA preparations revealed distinct molecular weight patterns of the Pxd RNA transcripts among adult flies, ovaries and larvae. The in situ hybridization clearly shows that the Pxd mRNA is specifically expressed in follicle cells during the late stages of oogenesis 11–14, while the reverse transcription reactions dictate the stage-specific developmental regulation of the Pxd gene. The immunolocalization approach, using the anti-rAePO polyclonal antibody, has revealed that the Pxd peroxidase is selectively localized in the chorion structures and particularly in the endochorion and innermost chorionic layer (ICL).

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.

Mass Determination of the Unit Cell of the Innermost Chorionic Layer in Drosophilidae by Scanning Transmission Electron Microscopy

The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.

Effect of lufenuron on chorionic and cuticular structure of unhatched larval Ctenocephalides felis (Siphonaptera: Pulicidae).

When adult cat fleas, Ctenocephalides felis (Bouché), were fed concentrations of < or = 0.08 ppm lufenuron in cattle blood, egg hatch did not differ significantly from the controls. However, as the concentration of lufenuron in blood increased from 0.125 to 1.0 ppm egg hatch decreased to 64 and 2%, respectively. Most of these eggs contained fully developed larvae. Microscopic examination of unhatched larvae, revealed that the cuticle epidermal cells, chorion, and vitelline membrane all were affected by lufenuron treatment. Larvae often produced 2 separate cuticles in response to treatment. The 1st cuticle consisted of an indistinct layer of epicuticle and a procuticle composed of randomly deposited chitin microfibrils. After the 1st layer of procuticle separated from the epidermal cells, a 2nd layer of procuticle was deposited. It was not possible to determine whether the egg tooth was functional during larval hatch. The surface of the egg tooth appeared normal, but the cuticle may have had structural abnormalities similar to those seen in other areas of the exoskeleton. Structural defects appeared to be due to the cytotoxic effects of lufenuron. The epidermal cells of treated larvae showed evidence of disintegration (i.e., the nuclei and mitochondria appeared to be degenerating and the amount of endoplasmic reticulum and other cytoplasmic organelles was decreased). The chorion of lufenuron-treated larvae consisted of an outer layer, middle and inner layers that were thinner and less electron dense than those of controls, and lacked the innermost chorionic layer found in the control larvae. The vitelline membrane also was thinner than that of the controls. Larval hatching was prevented by ruptures in the cuticle, which opened during eclosion resulting in the loss of hemolymph and desiccation of the larva. Evidently, tearing of the cuticle was caused by abnormal formation of the procuticle that was not strong enough to withstand the cuticular expansion and muscular movement of the larva within the egg shell.

Immunolocalization of the Temporally “Early” Secreted Major Structural Chorion Proteins, Dvs38 and Dvs36, in the Eggshell Layers and Regions of Drosophila virilis

We have shown by means of conventional electron microscopy that the eggshell of Drosophila virilisat the main body of the laid egg consists of the vitelline membrane and the multilayered chorion, which includes the wax layer, the innermost chorionic layer, the endochorion, and the exochorion, while several specialized regions of the eggshell are seen across the anterior–posterior axis of the egg. Biochemical analysis revealed the existence of six quantitatively enriched chorion proteins. Among them, Dvs38 and Dvs36 are synthesized when the innermost chorionic layer and the endochorion are assembled. Immunogold electron microscopy has shown that these two proteins are incorporated in the morphologically complete vitelline membrane apparently through an intercalation process and represent structural components of the endochorion in all the specialized regions of the eggshell. Additionally, by cytochemical means, the enzyme eggshell peroxidase, which is synthesized in parallel with Dvs38 and Dvs36, has been identified as a structural component of the innermost chorionic layer and the endochorion. These findings suggest a complex protein–protein recognition pattern during the formation of the eggshell since the cosecretion of its components (i.e., Dvs38, Dvs36 chorion proteins and eggshell peroxidase) does not recommend their colocalization in the eggshell sublayers and the timing of their synthesis is not related to their final position on the eggshell (i.e., the identification of Dvs38 and Dvs36 chorion proteins as vitelline membrane components).

The eggshell of drosophila melanogaster: IX. synthesis and morphogenesis of the innermost chorionic layer

Synthesis and morphogenesis of the innermost chorionic layer (ICL) was investigated by conventional EM methods, freeze-fracturing, tissue culture in Robb's medium, and EM autoradiography. Both autoradiography and fine structure results have shown that ICL-components are secreted prior to other chorion proteins. Their secretion starts on stage 12a but the first layer of ICL molecules is visible at stage 12b. Its thickness is gradually increased during the next stages, taking first, a bilaminar form along with the inner endochorion. Later, at the end of choriogenesis, ICL is detached from the endochorion and takes its final thickness and configuration, consisting of a 3-dimensional crystal, about 40 nm thick. The isolated ICL in conditions of air water interface is a monolayer crystal 10 nm thick. Studies on chorion mutants showed that the amount of protein secreted by the follicle cells is independent to the process of crystallisation. These data show how a proteinaceous extracellular substance is gradually assembled to form a 3-D crystal and how it can be organised to perform functions such as the physiological resistance of the insect eggs against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. These functions are performed by ICL in conjunction with the underlying wax layer.

Eggshell fine structure of Bradysia aprica (Winnertz) (Diptera : Sciaridae)

The eggshell fine structure of the dark-winged fungus-gnat Bradysia aprica (Winnertz) (Diptera : Sciaridae) was investigated by scanning and transmission electron microscopy. At the anterior pole of the ovoid egg is a single micropyle, centrally located in a well-defined micropylar area. The latter is covered by many long drumstick-like chorionic processes that are longer and more numerous than those of the rest of the egg surface. Cross-sections of the eggshell show 3 concentric envelopes: the vitelline envelope, wax layer and chorion. The chorion consists of 3 components with different morphological features: the inner, intermediate and outer chorion. The latter 2 layers, involved in the organization of the drumstick-like processes, have homogeneous features, whereas the former is crystalline and resembles the innermost chorionic layer of other Diptera.

Ultrastructure of the eggshell of Aeshna Juncea (L.) (Odonata : Aeshnidae)

The eggshell of Aeshna juncea L. (Odonata : Aeshnidae) is described using scanning and transmission electron microscopy. The vitelline envelope is homogenous. The endochorion is many-layered, the thickness of the sub-layers increasing from the inside and out. About 50 or more thin strata are arranged in a crystalline-like fashion, 0.17 μm thick, facing the vitelline envelope. This structure corresponds to the innermost chorionic layer (ICL) reported from Drosophila melanogaster. About 70 thin, bilaminar crystalline-like strata associated with a more electron-dense material, appear between the endochorionic sub-layers near the anterior end of the egg. The electron-dense exochorion is perforated with thin canals opening in small pores on the surface. The filamentous pre-chorion substance forming the exochorion, previously reported from Aeshna, is believed to form a paracrystalline region with a spherulite-like configuration comprising the inner half of the layer. At the anterior end of the egg, the endo- and the exochorion are anchored to each other in a regular manner. The micropylar chutes are 5 μm wide. Corresponding passages in the vitelline envelope are believed to close after fertilization. A standardized nomenclature for endophytic dragonfly eggs is proposed.

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs (1) 384

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit cell in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.

Structure and morphogenesis of the eggshell and micropylar apparatus in the olive fly, Dacus oleae (Diptera: Tephritidae)

The egg of the olive fly, Dacus oleae (Diptera, Tephritidae), is laid inside olives and the larva eventually destroys the fruit. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The chorion covering the main body of the egg outside of the vitelline membrane includes a "wax" layer, an innermost chorionic layer, an endochorion consisting of inner and outer layers separated by pillars and cavities similar to their counterparts in Drosophila melanogaster, as well as inner and outer exochorionic layers. The anterior pole is shaped like an inverted cup, which is chiefly hollow around its base and has very large openings communicating with the environment. Holes through the surface of the endochorion result from deposition of endochorionic substance around follicular cell microvilli. An opening at the apex of the cup provides an entrance for sperm entering the micropylar canal, which traverses the endochorion and continues into a "pocket" in a thickened vitelline protrusion. The micropylar canal is formed by deposition of endochorion and vitelline membrane around an elongated pair of follicular cell extensions. These extensions later degenerate and leave an empty canal about 5 microns in diameter and the narrower pocket about 1 micron in diameter. Respiration is thought to be facilitated by openings at the base of the anterior pole as well as by openings through the "plastron" around the main body of the shell.

Ultrastructural features and formation of the micropylar apparatus in the cherry fly Rhagoletis cerasi.

The micropylar apparatus (MA) in Rhagoletis cerasi (Diptera, Tephritidae) is located at the anterior pole of the egg and consists of two parts: an outer chorion and an inner vitelline membrane. Sperm entry takes place through the micropylar canal, 2.0-2.5 μm in diameter, which penetrates the micropylar endochorion and terminates in the thick vitelline membrane, thus forming the "pocket." The pore of the micropylar canal, i.e., the micropyle, is covered by the exochorionic tuft. The formation of the MA is accomplished by 40 micropylar cells during oogenesis. These cells secrete the successive eggshell layers: the vitelline membrane, the wax layer, the innermost chorionic layer, the micropylar endochorion, and the exochorion. Two among 40 micropylar cells differentiate and form two tightly connected projections. The latter contain a bundle of parallel microtubules and participate in the formation of the micropylar canal and the pocket. At the tip of the projections there are two thin extensions full of microfilaments. In late developmental stages the two projections and the extensions degenerate and leave the canal and the pocket behind. We also discuss the structural features of the MA in relation to its physiology among Diptera.

A structural protein that plays an enzymatic role in the eggshell of drosophila melanogaster

E.S.P. is responsible for the hardering process of the egg-shell at the end of oogenesis (stage 14B) and constitutes a structural component. By immunoblotting, using polyclonal rabbit anti-HRP antibody and anti-rabbit IgG-HRP or Protein A-125I as second antibody, one major band with MW 38KD on nitrocellulose filter showed positive reaction. We conclude that the E.S.P. is identical to the S38 chorionic protein. Morphological immunogold staining, using pre-embedding procedure, revealed positive reaction in the innermost chorionic layer (ICL) and the endochorion of the eggshell. In addition, electron probe X-ray microanalysis revealed the existence of 37% calcium (explained since the enzyme is Ca2+ -activated) and 5% iron (explained due to the fact that it is a haemoprotein).

Choriogenesis in the medfly Ceratitis capitata (Wiedermann) (Diptera : Tephritidae)

Four chorionic stages (11, 12, 13, 14) can be discerned in the medfly Ceratitis capitata (Diptera : Tephritidae) by light microscope. More detailed staging (stages 11A, 11B, 12A, 12B, 12C, 13A, 13B, 13C, 14A and 14B) is possible only by electron microscope. Throughout these stages, the first chorionic layer (wax layer) is formed at stage 11A, followed by the formation of the innermost chorionic layer (stage 12A), the inner endochorion, the pillars and the cavities of the first trabecular layer (stage 12), creation of the second trabecular layer and the chorionic network (stage 13), and finally the secretion of the exochorionic layers (stage 14). Pulse-chase autoradiography has revealed that the follicle cells are responsible for the synthesis of all proteinaceous layers. No extensive regional complexity is observed besides the existence of the micropylar apparatus and aeropyles in the anterior pole. Biochemical analysis has revealed several eggshell proteins and their stage-specific synthesis: two intermediate molecular-weight proteins are the major chorion proteins; in addition, there are 2 more groups comprising 6 proteins, which might be characteristic for the different chorion layers as can be deduced by their stage-electrophoretic pattern.

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.

Fine structure and morphogenesis of the micropylar apparatus in the medfly Ceratitis capitata (Wiedemann) (Diptera : Tephritidae)

The micropylar apparatus (MA) in Ceratitis capitata (Diptera : Tephritidae) is a cone-like protrusion, 18 μm long, at the anterior pole of the egg, and exhibits about 40 follicle cell imprints externally. It consists of chorionic and vitelline membrane parts. The first contains at least a 3 μm wide micropylar canal; the tip of the MA is covered by a “tuft” and includes the micropyle, i.e. the entrance of the micropylar canal. The canal leads to the vitelline membrane part, where it forms a pocket. The sperm enters the oocyte by passing through the micropyle-micropylar canal-pocket route. At least 40 follicle cells participate in the formation of the micropylar apparatus. Two of these form 2 projections, which are tightly connected, and serve as a template for the formation of the canal and the pocket. Throughout their length, both projections have microtubules in parallel arrangement. During oogenesis, the remaining micropylar cells secrete the successive eggshell layers, i.e. the vitelline membrane, the wax layer, the innermost chorionic layer, the endochorion, and the exochorion. Towards the end of oogenesis, the 2 projections degenerate, and the canal becomes available for sperm passage.

The egg-shell of Drosophila melanogaster VI, structural analysis of the wax layer in laid eggs

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 Å thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5–1 γm in diameter), which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.

A cell biological study of the micropylar apparatus in dipterans of economic importance

The micropylar apparatus is a specialized region of the eggshell, serving as a passage (micropylar canal) to the spermatozoon. The entire eggshell is secreted by the follicle cells during oogenesis, in successive layers, in the following order: vitelline membrane, wax layer, innermost chorionic layer, endochorion, exochorion. This study is an E.M. investigation of the structure and morphogenesis of the micropylar apparatus at the follicles of Dipterans of economic importance, such asCeratitis capitata,Dacus oleaeandRhagoletis cerasi. These insects affect the fruits of orange, olive and cherry trees respectively.The micropylar apparatus of the DipteransC. capitata,D. oleaeandR. cerasi. forms a protrusion at the anterior part of the egg. This apparatus consists of the vitelline membrane (Vm), which is surrounded by the wax layer (wl); the innermost chorionic layer (icl); the endochorion (En); and the exochorionic “tuft”. The endochorion has many cavities which probably take part in the air storage, in order to facilitate the embryo’s respiratory needs (Figs. 1,3,5). The micropylar canal is covered by the characteristic tuft, resulting in the vitelline membrane, where it becomes thinner and forms the pocket.

The innermost chorionic layer of Drosophila: I. The role of chorin octamers in the formation of a family of interdigitating crystalline plates

The innermost chorionic layer (ICL) within egg shells of Drosophila melanogaster is composed of thin, abutting three-dimensional crystalline plates which form a closed, membrane-like sheath. Collectively, the crystals within the sheath appear to form a family of related three-dimensional crystals in space group C222; however, specimens prepared for electron microscopy are actually two-dimensional crystals in c222. The projected structures of the negatively stained crystals have been studied by minimal dose electron microscopy employing image reconstruction methods. Thin sections indicate that unit cells within the ICL are composed of paired layers; top and bottom layers are related by centrally located 2-fold axes, aligned parallel to the surface of the ICL. The most probable structural unit of the crystals is a tetramer of chorin dimers with a point group symmetry of 222, which is denoted a chorin octamer. Projection maps were computed from average transforms of two-dimensional crystals for Δ (the primitive unit cell angle) equal to 84 °, 90 ° and 97 ° (±1.5 °). The maps indicate that the molecular transitions responsible for the observed family of crystals involve concerted intramolecular rearrangements about molecular 2-fold axes. The significance in vivo of the family of crystals within the ICL is not known; however, structural considerations suggest that the observed polymorphism may reflect one facet of an intrinsic bonding flexibility of the ICL octamer that may play a role in the formation of interplate junctions and the assembly of a continuous closed sheath. The ICL may therefore serve as a structural bridge between the vitelline membrane-wax layer and the endochondrial floor, allowing the larva to shed the inner egg shell layers during hatching.

The innermost chorionic layer of Drosophila: II. Three-dimensional structure determination of the 90 ° crystal form by electron microscopy

The innermost chorionic layer (ICL) within egg shells of Drosophila is composed of a family of related, thin three-dimensional crystals that form a continuous sheath encapsulating the egg shell lumen. Junctions formed by interdigitating lattices play a central role in the construction of this macroscopic assembly. The three-dimensional structure of a two-dimensional crystal isolated from the ICL, with a primitive lattice angle Δ of 90 °, has been determined from a complete tilt series of a negatively contrasted specimen at a resolution of 25 Å. Inspection of the three-dimensional transform after data merging revealed that the space group is c222 and this symmetry was employed to generate a three-dimensional structure. The basic structural unit of the ICL is an octamer, described formally as a tetramer of dimers with point group symmetry 222. There are two classes of dimer in the octamer designated α and β. The chorin octamer is composed of two classes of bent dimers, which make intramolecular contacts at the top and bottom of the molecule. The α-dimers are curved outwards away from the crystallographic 2-fold axis, while the β-dimers are curved towards the molecular center. In addition, lattice contacts are formed primarily by β-chorin dimers at both the top and bottom surfaces of the unit cell. The molecular weight of a chorin octamer determined from the analysis is about 6 × 10 5. The conformation of the chorin octamer determined here suggests that permutations of a basic molecular mechanism may be adequate to explain both the observed lattice polymorphisms of the ICL and the formation of interplate junctions necessary for the assembly of the macroscopic sheath.