‘Endogenous yolk’ as the precursor of a possible fertilization envelope in a crab (Carcinus maenas)

After the egg attachment to a maternal ovigerous seta, the Carcinus maenas embryo is enclosed in a tripartite capsule. The innermost layer (envelope 2) which is also the main part of this capsule, is generally detected after egg-laying and is most probably closely related to the fecondation phenomenon. The precursor material of envelope 2, arising from the egg by a massive and very fast exocytosis process, appears as numerous ring-shaped granules. These granules, originated from numerous cortical vesicles perhaps intercommunicating with each others, are observed early in the ooplasm during oogenesis, These so-called ring-shaped granules seem very identical in form with the disc-shaped granules which are classically described as composing the endogenous or intracysternal yolk of many Decapoda crustacean oocytes. In view of our results the role of these granules, in endogenous yolk formation, is re-examined and discussed.     

The ultrastructure of oogenesis and yolk formation in Labidocera aestiva (Copepoda: Calanoida)

Yolk formation in the oocytes of the free-living, marine copepod, Labidocera aestiva (order Calanoida) involves both autosynthetic and heterosynthetic processes. Three morphologically distinct forms of endogenous yolk are produced in the early vitellogenic stages. Type 1 yolk spheres are formed by the accumulation and fusion of dense granules within vesicular and lamellar cisternae of endoplasmic reticulum. A granular form of type 1 yolk, in which the dense granules within the cisternae of endoplasmic reticulum do not fuse, appears to be synthesized by the combined activity of endoplasmic reticulum and Golgi complexes. Type 2 yolk bodies subsequently appear in the ooplasm but their formation could not be attributed to any particular oocytic organelle. In the advanced stages of vitellogenesis, a single narrow layer of follicle cells becomes more developed and forms extensive interdigitations with the oocytes. Extra-oocytic yolk precursors appear to pass from the hemolymph into the follicle cells and subsequently into the oocytes via micropinocytosis. Pinocytotic vesicles fuse in the cortical ooplasm to form heterosynthetically derived type 3 yolk bodies.     

Fertilization envelope in diapause eggs of Pontella mediterranea (Crustacea, Copepoda).

The sequence and timing of morphological changes during envelope formation was followed in diapause eggs of Pontella mediterranea (Crustacea, Copepoda). The multilayer coat enveloping these eggs resulted from the exocytosis of 4 types of cortical vesicles that sequentially released their contents in the perivitelline space. These included small high-density vesicles (hDV) with electron-dense material, vesicles (V) with dense ring granules and a uniform matrix contained within the same compartment, large high-density (HDV) vesicles, and large moderately dense (MDV) vesicles. All of these cortical vesicles were present in newly spawned, fertilized eggs. Their exocytosis resulted from egg activation. One of these cortical vesicles (V) was similar in morphology to the intracisternal granules precursors of endogenous yolk. Intracisternal granules, characteristic of previtellogenic oocytes of many crustaceans, were present in previtellogenic oocytes of P. mediterranea but disappeared in later stages of oocyte development once yolk formation was completed. We discuss the role of cortical vesicles in the formation of the complex extracellular coat enveloping copepod diapause eggs.     

Oogenesis in pig ovaries during the prenatal period: ultrastructure and morphometry

Oogenesis in fetal pig ovaries comprises the successive changes from the primordial germ cells to the dictyotene oocytes in primordial ovarian follicles. In this study the observations were carried out with an electron microscope and stereological analysis was performed. At the ultrastructural level there are no differences between the primordial germ cells and oogonia, but oogonia are connected with the intercellular bridges. The onset of the dictyotene phase was accompanied by the changes in the cytoplasm of oocytes. Near the nucleus, the yolk nucleus is formed containing numerous Golgi bodies, endoplasmic reticulum (ER), mitochondria and granules. ER proliferates in contact with the external leaflet of the nuclear envelope forming the narrow ER cisterns. Between the nuclear envelope and ER cisterns, the vesicles with grey content are visible. The proliferating ER forms numerous concentric cisterns around the nucleus. Next, the most external cisterns fragment, detach, and then form the cup-like structures. These structures separate the distinct areas of cytoplasm-compartments, which contain mitochondria, ribosomes and lipid droplets. The cells of cortical sex cords of the ovary, which encloses the oocyte, form the follicles. The volume of oocytes in forming follicle increases due to the increase in the number of the cell inclusions: lipid droplets, vacuoles and yolk globules. In the oocytes of primordial ovarian follicles, the compartments are transformed into the yolk globules, which are encountered by a sheath of ER cisterns and the grey vesicles; they contain the mitochondria, lipid droplets and light vacuoles. The role of the compartments and yolk globules as metabolic units is discussed in comparison with similar structures of the mature eggs of pigs and other mammal species.     

Mechanisms of protein yolk synthesis and deposition in crustacean oocytes

Summary Electron microscope studies on the oocytes of several crustacean species demonstrate that the protein yolk arises within vesicular and lamellar forms of the rough-surfaced endoplasmic reticulum. The vesicular form of the endoplasmic reticulum may have its origin from a blebbing process of the outer layer of the nuclear envelope. Disc-shaped granules, representing precursor elements of the yolk granules, appear within the vesicular and lamellar profiles of endoplasmic reticulum. Autoradiographic results suggest that the ribosomes attached to the endoplasmic reticulum take part in the biosynthesis of yolk proteins. Numerous disc-shaped granules accumulate within the cisternae of the endoplasmic reticulum, but eventually they undergo a transformation into a finely granular yolk granule. Thus, both the origin and growth of protein yolk granules occur within membranes constituting the endoplasmic reticulum. The results provide evidence that intra-ooplasmic synthesis of yolk protein occurs in these oocytes.This investigation was supported by research grants (HD-00699; GM-09229) and a Career Development Award (GM-11,524) from the National Institutes of Health, U.S. Public Health Service.     

Electron microscope studies on developing oocytes of a coelenterate medusa with special reference to vitellogenesis

In a hydrozoan jellyfish, the female gonad is differentiated from a specialized region of the epidermis near the manubrium. Changes in the oocytes during growth and vitellogenesis are described as observed with electron microscopic and cytochemical techniques. Three major types of yolk are formed; these include lipid, glycogen, and membrane-bound granules consisting of both protein and carbohydrate. The latter first appear evident within vesicular and cisternal elements of the numerous Golgi complexes. The orientation and structural variations noted between the endoplasmic reticulum and forming face of the Golgi complexes suggest that the protein component of the yolk granules may be transferred from the cisternae of the endoplasmic reticulum to the Golgi complex where it is joined to carbohydrate perhaps synthesized by the Golgi complexes. Stages in the release of the precursor yolk material sequestered in cisternal elements of the Golgi complexes are illustrated. The presence of coated and uncoated vesicles in the Golgi regions and their possible role in intracellular transport are described and discussed. The presence and possible method of morphogenesis of vesiculate yolk bodies are also described. What appear to represent invaginations of the oolemma extend into the ooplasm and display a special orientation with respect to lamellae of the rough-surfaced endoplasmic reticulum. Intraooplasmic synthesis appears to constitute the major pathway for protein-carbohydrate yolk deposition.     

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation

Morphological changes in the growing and maturing oocytes of Patiria ( Asterina ) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.     

Ultrastructural studies of differentiation in the oocyte of the polyclad turbellarian,Prostheceraeus floridanus

Oocyte differentiation in the polyclad turbellarian Prostheceraeus floridanus has been examined to determine the nature of oogenesis in a primitive spiralian. The process has been divided into five stages. (1) The early oocyte: This stage is characterized by a large germinal vesicle surrounded by dense granular material associated with the nuclear pores and with mitochondria. (2) The vesicle stage: The endoplasmic reticulum is organized into sheets which often contain dense particles. Vesicles are found in clusters in the cytoplasm, some of which are revealed to be lysosomes by treatment with the Gomori acid phosphatase medium. (3) Cortical granule formation: Cortical granules are formed by the fusion of filled Golgi vasuoles which have been released from the Golgi saccules. The association between the endoplasmic reticulum and Golgi suggests that protein is synthesized in the ER and transferred to the Golgi where polysaccharides are added to form nascent cortical granules. (4) Yolk synthesis: After a large number of cortical granules are synthesized, yolk bodies appear. They originate as small membrane-bound vesicles containing flocculent material which subsequently increase in size and become more compact. Connections between the forming yolk bodies and the endoplasmic reticulum indicate that yolk synthesis occurs in the ER. (5) Mature egg: In the final stage, the cortical granules move to the periphery and yolk platelets and glycogen fill the egg. At no time is there any evidence of uptake of macromolecules at the oocyte surface. Except for occasional desmosomes between early oocytes, no membrane specialization or cell associations are seen throughout oogenesis. Each oocyte develops as an independent entity, a conclusion supported by the lack of an organized ovary.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).     

Oocyte-follicle cell differentiation in two species of amphineurans (Mollusca), Mopalia mucosa and Chaetopleura apiculata

Differentiating oocytes and associated follicle cells of two species of amphineurans (Mollusca) Mopalia muscosa and Chaetopleura apiculata have been studied by techniques of light and electron microscopy. In addition to the regularly occurring organelles, the ooplasm of young oocytes contains large, randomly situated, basophilic regions. These regions are not demonstrable in mature eggs. As oocytes differentiate, lipid, pigment and protein-carbohydrate yolk bodies accumulate within the ooplasm. Concomitant with the appearance of pigment and the protein carbohydrate containing yolk bodies, the saccules of the Golgi complex become filled with a dense material. Associated with the Golgi complex are cisternae of the rough endoplasmic reticulum which are filled with an electron opaque substance which is thought to be composed of protein synthesized by this organelle. That portion of the cisternae of the endoplasmic reticulum facing the Golgi complex shows evaginations. These evaginations are thought to finalize into protein containing vesicles that subsequently fuse with the Golgi complex. Thus, the Golgi complex in these oocytes might serve as a center for packaging and concentrating the protein used in the construction of the protein containing pigment or protein-carbohydrate yolk bodies. The suggestion is made that the Golgi complex may also synthesize the carbohydrate portion of the formentioned yolk bodies. In an adnuclear position in young oocytes are some acid mucopolysaccharide containing vacuolar bodies. In mature eggs, these structures are found within the peripheral ooplasm and we have referred to them as cortical granules. There is no alteration of these cortical granules during sperm activation.     

Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1–1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0–2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.     

Endoplasmic reticulum whorls as a source of membranes for early cytaster formation in parthenogenetically stimulated sea urchin eggs

Summary Sea urchin eggs exposed to a continuous hypertonic treatment rapidly form many concentric whorls of endoplasmic reticulum (ER) during the pre-activation period of the parthenogenetic development. These whorls, however, are only a temporary configurational alteration of ER which begin to break up just prior to egg activation. The conversion back to normal vesicles and lamellae occurs not only concurrently with the appearance of early cytastral areas, but also frequently in close association with the formation of these membranous areas. It is revealed here that membrane elements from disrupting whorls may become incorporated into adjacent, developing clear areas, early cytastral areas, and that this ER constitutes an initial major source of membranes for these early astral areas. Having previously suggested that the actual formation of ER whorls occurs in direct response to released intracellular calcium in hypertonic stressed eggs, the new findings, along with other related data and correlations, further suggest that whorl disruption and the formation of associated astral areas can be correlated with a corresponding decrease in the concentration of this released calcium in the cytoplasm.     

Ultrastructural and Morphometric Observations of Cortical Endoplasmic Reticulum in Arbacia,Spisula and Mouse Eggs*

Results of morphometric investigations indicate that Arbacia eggs possess a network of cortical endoplasmic reticulum equal in volume and surface area to that within the subcortex. The cortical endoplasmic reticulum surrounds individual cortical granules and forms associations with the plasma membrane reminiscent of junctions shared by the sarcolemma and sarcoplasmic reticulum. Mouse eggs, which also exhibit a cortical granule reaction, possess endoplasmic reticulum that is associated with cortical granules and the plasmalemma. The same relative volume of cortical endoplasmic reticulum is present in mouse eggs as in Arbacia. Significantly less cortical endoplasmic reticulum is present in Spisula eggs which do not undergo cortical granule discharge upon activation. These observations are discussed in light of the hypothesis that the cortical endoplasmic reticulum transduces the interaction of the gametes into an intracellular calcium release which initiates the cortical granule reaction and the activation of development.     

Cortical localization of a calcium release channel in sea urchin eggs

We have used an antibody against the ryanodine receptor/calcium release channel of skeletal muscle sarcoplasmic reticulum to localize a calcium release channel in sea urchin eggs. The calcium release channel is present in less than 20% of immature oocytes, where it does not demonstrate a specific cytoplasmic localization, while it is confined to the cortex of all mature eggs examined. This is in contrast to the cortical and subcortical localization of calsequestrin in mature and immature eggs. Immunolocalization of the calcium release channel reveals a cortical reticulum or honeycomb staining network that surrounds cortical granules and is associated with the plasma membrane. The network consists of some immunoreactive electron-dense material coating small vesicles and elongate cisternae of the endoplasmic reticulum. The fluorescent reticular staining pattern is lost when egg cortices are treated with agents known to affect sarcoplasmic reticulum calcium release and induce cortical granule exocytosis (ryanodine, calcium, A-23187, and caffeine). An approximately 380-kD protein of sea urchin egg cortices is identified by immunoblot analysis with the ryanodine receptor antibody. These results demonstrate: (a) the presence of a ryanodine-sensitive calcium release channel that is located within the sea urchin egg cortex; (b) an altered calcium release channel staining pattern as a result of treatments that initiate the cortical granule reaction; and (c) a spatial and functional dichotomy of the ER which may be important in serving different roles in the mobilization of calcium at fertilization.     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.     

Oogenesis in Centropages typicus (Copepoda, Calanoida)

We carried out a complete study of oogenesis in Centropages typicus using structural, ultrastructural and cytochemical data. The usual stages of oogenesis, i.e. germinative phase, premeiosis, primary and secondary vitellogenesis, were found. The latter two stages were the most typical. Primary vitellogenesis consisted of endogenous yolk accumulations; these substances, probably of lipoprotein or lipoglycoprotein nature, were produced at the granular endoplasmic reticulum level and then stocked in the reticulum cavities. During secondary vitellogenesis, endogenous yolk production continued, but we mainly observed the development of exogenous yolk accumulation (lipid droplets and protein globules) in the ooplasm. These accumulations resulted from the fusion of very numerous pinocytotic vesicles arising from the oolemma and containing substances probably brought to the oocytes by the hemolymph. The effect of various proteases on the vitellus globules caused a more or less marked digestion of their contents, tending to prove their protein nature. The end of vitellogenesis was marked by the appearance of vacuolar formations with dense lamellae which could correspond to cortical granules.     

Immunocytochemical localization of BiP to the rough endoplasmic reticulum: evidence for protein sorting by selective retention

Immunoglobulin heavy chain binding protein (BiP) (also known as GRP 78) is a protein of the endoplasmic reticulum (ER) which has been shown to be involved in post-translational processing of nascent membrane and secretory proteins. To determine BiP's location in the exocytic pathway, we localized BiP at the electron microscopic level in mouse myeloma cell lines by immunoperoxidase cytochemistry. BiP was found to be present within the cisternal spaces of the RER and nuclear envelope but was not detected in the cisternae of the Golgi complex. BiP reaction product was also found within transitional elements of the RER but was absent from smooth-surfaced vesicles found between the ER and the Golgi complex. Immunoperoxidase staining of BiP was reduced or absent in regions of a smooth ER membrane system in myelomas that contained endogenous murine retrovirus A particles. All compartments of the exocytic pathway, including the virus-containing smooth ER, stained for IgG, a secretory protein. These observations suggest that BiP is selectively retained in the cisternae of the ER and is not free to enter Golgi-directed transport vesicles. These studies suggest that BiP's subcellular localization may occur by selective interaction with component(s) of the ER.     

凡纳滨对虾卵母细胞卵黄发生的超微结构

利用电镜研究凡纳滨对虾卵母细胞卵黄发生的全过程。结果表明 :凡纳滨对虾卵黄的发生是双源性的。卵黄发生早、中期是内源性卵黄大量合成的阶段 ,卵黄发生中、后期则以外源性卵黄的合成为主。内源性卵黄主要由内质网、线粒体、核糖体、溶酶体、高尔基器等多种胞器活跃参与形成。其中数量众多的囊泡状粗面内质网是形成内源性卵黄粒的最主要的细胞器 ;部分线粒体参与卵黄粒的合成并自身最终演变为卵黄粒 ;丰富的游离核糖体合成了大量致密的蛋白质颗粒并在卵质中直接聚集融合成无膜的卵黄粒 ;溶酶体通过吞噬、消化内含物来形成卵黄粒和脂滴 ,且方式多样 ;高尔基器不直接参与形成卵黄粒。外源性卵黄主要通过卵质膜的微吞饮活动从卵周隙或卵泡细胞中摄取外源物质来形成     

Morphology and ultrastructure of the adult ovarian cycle in Mithracidae (Crustacea,Decapoda, Brachyura,Majoidea)

The ultrastructure of the ovary during development and yolk production is poorly known in Brachyura and Majoidea in particular. Here, we describe the histology, histochemistry and ultrastructure of the adult ovarian cycle in four Mithracidae species from three different genera: Mithrax hispidus, Mithrax tortugae, Mithraculus forceps and Omalacantha bicornuta. All species showed a similar pattern of ovarian development and vitellogenesis. Macroscopically, we detected three stages of ovarian development: rudimentary (RUD), developing (DE) and mature (MAT); however, in histological and ultrastructural analyses, we identified four stages of development. The oocytes of the RUD stage, during endogenous vitellogenesis, have basophilic cytoplasm filled with dilated rough endoplasmic reticulum. The reticulum lumen showed many granular to electron-dense materials among the different stages of development. The Golgi complexes were only observed in the RUD stage and are responsible for releasing vesicles that merge to the endogenous or immature yolk vesicles. At the early DE stage, the oolemma showed many coated and endocytic vesicles at the cortex. The endocytic vesicles merge with the endogenous yolk to form the exogenous or mature yolk vesicles, always surrounded by a membrane, characterizing exogenous vitellogenesis. The exogenous yolk vesicles comprise glycoproteins, showing only neutral polysaccharides. At the late DE stage, endocytosis still occurs, but the amount of endogenous yolk decreases while the exogenous yolk increases. The late DE stage is characterized by the beginning of chorion production among the microvilli. The MAT stage is similar to the late DE, but the endogenous yolk is restricted to a few cytoplasmic areas, the ooplasma is filled with exogenous yolk, and the oolemma has very few coated vesicles. In the MAT stage, the chorion is fully formed and shows two electron-dense layers. The ovarian development of the species studied has many similarities with the very little known Majoidea in terms of the composition, arrangement and increment of the yolk vesicles during oocyte maturation. The main differences are in the vitellogenesis process, where immature yolk formation occurs without the direct participation of the mitochondria but with the participation of the rough endoplasmic reticulum in the endogenous phase.     

Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica

The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well‐defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER–Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin‐rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica.