Ultrastructural studies of the formation of the egg capsule in the hermaphroditic species, Isohypsibius granulifer granulifer Thulin, 1928 (Eutardigrada: Hypsibiidae)

The egg capsule of Isohypsibius granulifer granulifer Thulin 1928 (Eutardigrada: Hypsibiidae) is composed of two shells: the thin vitelline envelope and the multilayered chorion. The process of the formation of the egg shell begins in middle vitellogenesis. The I. g. granulifer vitelline envelope is of the primary type (secreted by the oocyte), but the chorion should be regarded as a mixed type: primary (secreted by the oocyte), and secondary (produced by the cells of gonad wall). During early choriogenesis, the parts of the chorion are produced and then connected into a permanent layer. The completely developed chorion consists of three layers: (1) the inner, medium electron dense layer; (2) the middle labyrinthine layer; (3) the outer, medium electron dense layer. After the formation of the chorion, a vitelline envelope is secreted by the oocyte.     

Formation and structure of egg envelopes in Russian sturgeon Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The covering of the eggs in Russian sturgeon Acipenser gueldenstaedtii consists of three envelopes (the vitelline envelope, chorion and extrachorion) and is equipped with multiple micropyles. The most proximal to the oocyte is the vitelline envelope that consists of four layers of filamentous and trabecular material. The structural components of this envelope are synthesized by the oocyte (primary envelope). The chorion encloses the vitelline envelope. The extrachorion covers the external surface of the egg. Examination of the arrangement of layers that comprise the egg envelopes together with the ultrastructure of follicular cells revealed that the chorion and extrachorion are secondary envelopes. They are secreted by follicular cells and are built of homogeneous material. During formation of egg envelopes, the follicular cells gradually diversify into three morphologically different populations: 1) cells covering the animal oocyte region (cuboid), (2) main body cells (cylindrical) and (3) micropylar cells. The apical surfaces of follicular cells from the first two populations form processes that remain connected with the oocyte plasma membrane by means of gap junctions. Micropylar cells are located at the animal region of the oocyte. Their apical parts bear projections that form a barrier to the deposition of materials for egg envelopes, resulting in the formation of the micropylar canal.     

Ultrastructure de l'œuf de Triatoma infestans Klug

Summary The thick rigid chorion of the egg of Triatoma secreted by the follicle cells shows two porous layers: an aerial layer in the exochorion, an alveolar one in the endochorion. The anterior part of the eggshell is closed up by an operculum which is heaved up by the hatching larva. The operculum has no alveolar layer. The air enters through the numerous holes of the shell surface into the aerial layer and through the micropyles into the alveolar layer. The egg has no respiratory plastron.The follicle cells produce also a vitelline envelope whose structure shows a rapid condensation at fertilization time. During its development the embryo secretes two layers: serosal and embryonic cuticle.At high humidities, at low temperatures the egg is able to increase its weight during the early stages of embryogenesis, and this increase stops when the serosal cuticle is secreted. In a dry atmosphere the egg loses water but can develop if the temperature is higher than 20°C.The little permeability of the egg is related to the structure of its envelopes. The chorion and the vitelline envelope prevent the water from getting out of the egg. The serosal cuticle seems to be opposed to the penetration of the water into the egg. The role of the embryonic cuticle is probably limited in the transit of water.

Nous remercions Messieurs les Professeurs Maillet et Folliot qui ont mis le microscope R.C.A. à notre disposition, Madame Allo et Mademoiselle Le Gac, technicienne au microscope à balayage J.S.M. S1, pour leur collaboration technique.     

The ultrastructure and function of follicle cells in Foucartia squamulata (Herbst) (Curculionidae)

Summary The follicle cells of Foucartia squamulata are involved in the formation of both vitelline membrane and chorion. Precursors for these egg coverings are synthesized by the rough endoplasmic reticulum and condensed within dictyosomes. The vitelline membrane and the chorion appear on the oocyte surface simultaneously, which is an unusual phenomenon for insects. The follicular epithelium has not been found to contribute to vitellogenesis in the species under study.     

Fine structure of the eggshell of Ommatissus binotatus Fieber (Homoptera,Auchenorrhyncha, Tropiduchidae)

The external morphology and fine structure of the eggshell of Ommatissus binotatus Fieber (Homoptera : Tropiduchidae) was investigated by light, scanning and transmission electron microscopy. The egg surface has 2 main regions: a specialized area and an unspecialized egg capsule. The specialized area is characterized by a large respiratory plate containing the operculum and a short respiratory horn. The latter consists of an external hollow tube and an internal coneshaped projection hosting a micropylar canal. The eggshell has 4 layers: the vitelline envelope, a wax layer, the chorion and an outer mucous layer. The chorion has inner, intermediate and outer parts. The functions of the different parts of the eggshell are discussed. Characters useful to define the eggs and the oviposition habit in the family Tropiduchidae were provided. The size and morphology of the egg, plate, respiratory horn and operculum are suggested as useful characters for ootaxonomic analysis.     

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.     

Annual fish oogenesis : I. Differentiation of the mature oocyte and formation of the primary envelope

The chorion surface in the eggs of the annual fishes Cynolebias melanotaenia and C. ladigesi contains an elaborate, three-dimensional species-specific pattern. Two concentric layers form the chorion. The pattern resides in the outer layer, the secondary envelope. It consists of closely packed tubules about 250 Å in diameter. A coat of electron dense “fuzzy” material increases this to 475 Å. The inner layer, the primary envelope, of uniformly low electron density possesses no obvious substructure. Oogenesis is divided into six stages. The oocyte increases in size from 10–20 μm in Stage 1 to 250 μm in Stage 3, 600 μm in Stage 4, and attains maximal size of 900 μm by Stage 6. Massive inclusions of protein and lipid yolk accumulate during Stages 4 and 5. Zone 1, one of the three zones of the primary envelope, first appears late in Stage 2. During Stage 3, Zone 1 is completed and Zone 2 appears between the oocyte surface and Zone 1. The oocyte cytoplasm increases in complexity. Material similar to Zone 1 (light, fibrillar) and Zone 2 (dark, compact) is present in the RER, Golgi, derivative vesicles, and apical pits. Micropyle formation also commences. The oocyte secretes Zone 3 during Stage 4 as thin filaments which consolidate into a highly ordered, transitional structure composed of tangentially oriented bundles of interwoven filaments. These partially fuse during Stage 5 except for fenestrations through which oocyte and follicle cell microvilli pass. Complete fusion during Stage 6 produces a continuous layer. Follicle cells retain an unspecialized structure from Stages 1 through 4. Secondary envelope material accumulates in the RER of the follicle cells during Stage 5. It is secreted and deposited during Stage 6.     

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.     

Egg Envelope Cytodifferentiation in the Colonial Ascidian Botryllus schlosseri (Tunicata)

Abstract The formation and cytodifferentiation of egg envelopes were studied at the ultrastructural level in blastozooids of Botryllus schlosseri. The process was divided into five recognized stages of oogenesis. First, the small young oocytes (stage 1) are contacted by scattered cells (primary follicle cells—PFC) which adhere to the oolemma at several junctional spots. PFC extend all around the growing oocyte, acquire polarity, and form a layer covered externally by a thin basal membrane (stage 2). At stage 3 isolated cells are recognizable between the PFC layer and oocyte. They never form junctions with the oocyte and represent prospective inner follicle cells (IFC) and test cells (TC), the latter being progressively received in superficial depressions in the oocyte. The layer of PFC, which maintains junctions with the oolemma, represents prospective outer follicle cells (OFC). PFC are considered to be the source of the three cellular envelopes because a contribution from mesenchymatous elements was not observed. At the beginning of vitellogenesis (stage 4), the vitelline coat (VC) becomes recognizable as a loose net covering the oocyte and TC. It is crossed by the oocyte microvilli and OFC projections which meet and form numerous small junctional plaques, some of them resembling gap junctions. IFC, VC and TC show marked signs of differentiation with approaching ovulation. OFC differentiate completely before ovulation (stage 5) and are engaged in intense synthesis of proteins which may be transferred and taken by endocytosis into the oocyte for yolk formation. Experiments with injected horseradish peroxidase also revealed that proteins present in the blood may reach the oocyte via the intercellular pathway, overcoming OFC and IFC. The possible roles of all the egg envelopes are discussed.     

Oogenesis of the mayfly Habrophlebia eldae: Synthesis of vitelline and chorionic envelopes

The ultrastructure of developing ovarian follicles inside the panoistic ovarioles of Habrophlebia eldae were examined to observe the events occurring during egg maturation up to the full formation of the chorionic envelopes. The early vitellogenic follicles are coupled by gap junctions and are extensively interlocked with the oocyte plasma membrane via microvilli. With the onset of vitellogenesis, coated pits and coated vesicles are precursors to yolk deposition and are visible at the follicle cell-oocyte interface. Postvitellogenic development entails the deposition of the egg envelopes. The vitelline envelope arises from the coalescence of rectangular plaques whose precursors are visible in Golgi complexes as heterogeneous electron-opaque granules. A chorionic pattern of ridges on the egg surface characterizes the shell of H. eldae. The fully developed chorion shows three distinct regions with differently organized patterns. A fine layer of fibrous material (a secretion of the follicle cells, Ephemeroptera devoid of accessory glands) adheres to the egg chorion and is probably involved in attachment to the substrate.     

Retinal-tectal connections in the embryonic chick: evidence for regionally specific cell surface components which mimic the pattern of innervation.

The chorion surface in the eggs of the annual fishes Cynolebias melanotaenia and C. ladigesi contains an elaborate, three-dimensional species-specific pattern. Two concentric layers form the chorion. The pattern resides in the outer layer, the secondary envelope. It consists of closely packed tubules about 250 Å in diameter. A coat of electron dense “fuzzy” material increases this to 475 Å. The inner layer, the primary envelope, of uniformly low electron density possesses no obvious substructure. Oogenesis is divided into six stages. The oocyte increases in size from 10–20 μm in Stage 1 to 250 μm in Stage 3, 600 μm in Stage 4, and attains maximal size of 900 μm by Stage 6. Massive inclusions of protein and lipid yolk accumulate during Stages 4 and 5. Zone 1, one of the three zones of the primary envelope, first appears late in Stage 2. During Stage 3, Zone 1 is completed and Zone 2 appears between the oocyte surface and Zone 1. The oocyte cytoplasm increases in complexity. Material similar to Zone 1 (light, fibrillar) and Zone 2 (dark, compact) is present in the RER, Golgi, derivative vesicles, and apical pits. Micropyle formation also commences. The oocyte secretes Zone 3 during Stage 4 as thin filaments which consolidate into a highly ordered, transitional structure composed of tangentially oriented bundles of interwoven filaments. These partially fuse during Stage 5 except for fenestrations through which oocyte and follicle cell microvilli pass. Complete fusion during Stage 6 produces a continuous layer. Follicle cells retain an unspecialized structure from Stages 1 through 4. Secondary envelope material accumulates in the RER of the follicle cells during Stage 5. It is secreted and deposited during Stage 6.     

Studies on the formation of germ cells in a compound ascidian Botryllus,primigenus oka

Gametogenesis of a compound ascidian Botryllus primigenus was studied histologically. On either side of the zooid (stage 9), in the gonadal space between the epidermis and the atrial epithelium, either a single testis or a complex of an egg follicle and a testis can be formed. The egg follicle consists of a single ovum (occasionally two ova) and its accessory cells and is connected with the atrial epithelium by the follicle stalk. The egg follicle is always accompanied by the brood pouch, a diverticulum of the atrial cavity. The testis is equipped with a vestigial spermiduct and is attached to the atrial epithelium. Buds of stage 8 comprise, besides the developing testes and, egg follicles, loose aggregations of hemoblasts and oocytes of early developmental stages, which are already accompanied by primary follicular cells. Both the oocytes and the primary follicular cells seem to arise from the hemoblasts. The young oocytes are isolated in the gonadal space of the buds nnd are transferred to buds of the succeeding generations until they finally mature. In the bud of stage 3, a compact mass of cells appears, attaching to tbe inner vesicle on either side of the body. It is derived from the hemoblasts lodged there in the preceding generation and presumably also from the circulating hemoblasts. When the cell mass receives a large oocyte derived from the preceding generation, part of the cell mass differentiates into egg envelopes, forming an egg follicle, and a follicle stalk and the remainder into a testis. When the cell mass receives no oocyte, it differentiate as a whole into a testis. In the egg follicle thus formed the outer and inner follicular cells increase in number by mitotic division. Subsequently, initial test cells are derived from the inner follicle by migration across the developing chorion; then they increas2 in number by mitosis. In the testis, meiosis and spermiogenesis take place.     

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.     

Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

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.     

Ultrastructure and resistance to water loss in eggs of Acanthoscelides obtectus Say (Coleoptera: Bruchidae)

The mature oöcyte of Acanthoscelides obtectus is surrounded by three envelopes: an external layer, a chorion and a vitelline membrane. The external layer is secreted by the walls of the lateral oviducts. The chorion and vitelline membrane are secreted by the follicular cells. The vitelline membrane becomes very compact during the hour following fertilization and laying. The chorion is composed of three layers, one of which has a paracrystalline ultrastructure.Mature, unfertilized, chorion-containing oöcytes, whose vitelline membranes are loose, dehydrate rapidly in a dry atmosphere after laying or after removal from the lateral oviducts. Fertilized eggs are quite resistant to desiccation: after 12 days at 25°C and 5% relative humidity, viable larvae are obtained.The compact vitelline membrane is the most effective protection against dehydration. The chorion and the external layer are much less effective in preventing water loss from the egg.The retention of eggs in the lateral oviducts does not seem to lead to any modification of the structure of their envelopes.     

Ultrastructural and histochemical study of previtellogenic oogenesis in the desert lizard Scincus mitranus (Squamata,Sauropsida)

The structure of the granulosa in reptilian sauropsids varies between groups. We investigated the follicle development in the desert lizard Scincus mitranus. In the germinal bed, oogonia, and primary oocytes were identified and found to be interspersed between the epithelial cells. Previtellogenesis was divided into three stages: early, transitional, and late previtellogenic stages. During the early previtellogenic stage (diplotene), the oocyte is invested by small epithelia cells that formed a complete single layer, which may be considered as a young follicle. The transitional previtellogenic stage was marked by proliferation and differentiation of the granulosa layer from a homogenous layer consisting of only small cells to a heterogeneous layer containing three cell types: small, intermediate, and large cells. The late previtellogenic stage was marked by high-synthetic activity of large cells and the initiation of cytoplasmic bridges between large granulosa cells and the oocyte. Small cells were the only type of granulosa cells that underwent division. Thus, these cells may be stem cells for the granulosa cell population and may develop into intermediate and subsequently large cells. The intermediate cells may be precursors of large cells, as suggested by their ultrastructure. The ultrastructure of the large granulosa was indicative of their high synthetic activity. Histochemical analysis indicated the presence of cholesterol and phospholipids in the cytoplasm of large cells, the zona pellucida, among the microvilli, in the bridges region, and in the cortical region of the oocyte cytoplasm. These materials may be transferred from large cells into the oocyte through cytoplasmic bridges and provide nutritive function to large cells rather than functioning in steroidogenesis or vitellogenesis.     

Egg development in the octopus Eledone cirrhosa

The development of egg/follieular cell complexes is described in maturing females of the octopus Eledone cirrhosa. Follicle cells proliferate to enclose the oocyte in a single epithelial layer which becomes deeply infolded. Active cell division of the follicle cells and recruitment of cells from an outer (thecal) layer generate this expansion of follicle cell epithelium. The onset of the main phase of vitellogenesis, secretion of protein yolk, occurs when eggs reach about 2 mm in length and is marked by the columnar appearance of the follicle cells and an increased number of larger and more complex nuclei. A significant proportion of the egg population fails to develop beyond 2–3 mm in length and these eggs subsequently degenerate.     

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.     

A stereological analysis of developing egg chambers in Ephestia kuhniella

A polytrophic ovariole of the flour moth, Ephestia kuhniella, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of seven nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each component as a function of the position of the egg chamber in the ovariole. Analysis of the growth curve of the Ephestia oocyte yields two possible correlations between accelerated oocyte growth and ultrastructural events enhancing the supply of yolk materials to the oocyte: the first is the initiation of yolk synthesis by the follicle cell layer and its transfer to the oocyte, and the second is the formation of channels between the follicle cells allowing hemolymph to gain access to the oocyte. An Ephestia oocyte increases in volume from approximately 2.5 × 10³ μm³ to approximately 2.0 × 10⁷ μm³ over an average series of 58 egg chambers.