Yolk organelles and their membranes during vitellogenesis ofXenopus oocytes

Summary The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Throughout vitellogenesis, large yolk platelets are in close contact with smaller nascent yolk organelles. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets was not observed. On freezeetching replicas, yolk-platelet membranes present fracture faces with intramembranous particles (IMP) of various sizes and a heterogeneous distribution of approximately 200–600 IMP/μm² at the E face, and 1200–2100 IMP/μm² at the P face. Again, this presentation of the membrane exhibits neither anastomoses to the endoplasmic reticulum, nor caveolae that exclude the uptake of yolk-containing vesicles into these yolk organelles. Proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.     

Yolk nucleus – The complex assemblage of cytoskeleton and ER is a site of lipid droplet formation in spider oocytes

Oocytes (future egg cells) of various animal groups often contain complex organelle assemblages (Balbiani bodies, yolk nuclei). The molecular composition and function of Balbiani bodies, such as those found in the oocytes of Xenopus laevis, have been recently recognized. In contrast, the functional significance of more complex and highly ordered yolk nuclei has not been elucidated to date. In this report we describe the structure, cytochemical content and evolution of the yolk nucleus in the oocytes of a common spider, Clubiona sp. We show that the yolk nucleus is a spherical, rather compact and persistent cytoplasmic accumulation of several different organelles. It consists predominantly of a highly elaborate cytoskeletal scaffold of condensed filamentous actin and a dense meshwork of intermediate-sized filaments. The yolk nucleus also comprises cisterns of endoplasmic reticulum, mitochondria, lipid droplets and other organelles. Nascent lipid droplets are regularly found in the cortical regions of the yolk nucleus in association with the endoplasmic reticulum. Single lipid droplets become surrounded by filamentous cages formed by intermediate filaments. Coexistence of the forming lipid droplets with the endoplasmic reticulum in the cortical zone of the yolk nucleus and their later investment by intermediate-sized filamentous cages suggest that the yolk nucleus is the birthplace of lipid droplets.     

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H⁺-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H⁺-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg²⁺ and inhibited by NaF, as well as by IDP and Ca²⁺. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H⁺-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.     

Cytochemistry of yolk elements in the egg of the tunicate Molgula

Summary Cytochemical techniques demonstrate two types of yolk elements (compound yolk and lipid yolk) in the egg of the tunicate (Molgula manhattensis). The compound yolk elements consisting of proteins, carbohydrates, lipoproteins and triglycerides arise under the influence of cell organelles. A few lipid yolk elements staining moderately for phospholipids are also formed. The distribution and cytochemistry of cell organelles have also been described briefly in growing oocytes, test cells and follicular epithelial cells.     

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.     

Lipid yolk synthesis in a marine teleost,Blennius pholis L.

Summary The site of lipid yolk synthesis in the marine teleost Blennius pholis L. is examined. The relative distributions of cellular organelles and inclusions in the developing oocytes are analysed by morphometric analysis, which indicates that synthesis is probably endogenous occurring in the perinuclear cytoplasm. The role of lipid yolk in this species is discussed.     

Verteilungsmuster und Ultrastruktur des Glykogens in der Oocyte vonMusca domestica

Summary Fully grown oocytes ofMusca domestica contain large amounts of glycogen distributed in a characteristic pattern. Three cytoplasmic layers can be distinguished: 1. The periplasm which is free of carbohydrates and merely contains some lipid and protein yolk. 2. A zone of large glycogen clods. 3. Adjacent to this the central ooplasm where numerous lipid droplets and protein yolk spheres are found beside medium size glycogen clods. The glycogen areas are not surrounded by membranes, in contrast to the other yolk inclusions. Some possible interpretations of this ooplasmic pattern, which is already established during oogenesis, are discussed.     

Distribution of incorporated and synthesized protein among cell fractions of Xenopus oocytes

Developing oocytes of Xenopus laevis were isolated, pulsed for 10 minutes with either vitellogenin-³H, ³²P or a mixture of l-leucine-³H and ³²P_i, and subsequently incubated for various lengths of time in unlabeled medium. Homogenates were then prepared and centrifuged on 20–60% sucrose gradients.Vitellogenin-³H, ³²P was found to associate initially with membranous material, but within 45 min more than half the label was associated with the yolk platelets. Since it takes at least 60–120 min for vitellogenin to be converted into lipovitellin and phosvitin, this transformation must occur within the platelets rather than at the oocyte surface or within pinosomes.Eighteen hours after a pulse with l-leucine-³H and ³²P_i, neither the yolk nor the mitochondria fraction became significantly labeled; this indicates that the macromolecular components of these structures are not synthesized or phosphorylated by the oocyte to any great extent during vitellogenesis. Instead, various components within the membranous and “soluble” regions became labeled.     

An electron microscope study on the developing oocytes of the crab Cancer pagurus L. with special reference to yolk formation (Crustacea)

Summary The developing oocytes of the crab Cancer pagurus L. were studied with the light and electron microscope.Protein yolk formation was found to take place in two different ways. Yolk precursors of type 1 accumulate within the cisternae of an extensively developed granular endoplasmic reticulum. Also further growth and transformation into the definite yolk body occur within the reticular membranes. There is no structural indication that any other cell organelle contributes to the synthesis of this type of yolk building.Protein yolk formation of type 2 involves accumulation and transformation of material within a limiting membrane of the smooth type. The enclosed material is presumably derived from micropinocytosis, enclosed cellular elements and vesicles originating from the Golgi complex.It thus appears that the cell organelles play an important role in the process of drotein yolk formation in the growing oocytes of Cancer pagurus.     

Unusual Leucophore‐Like Cells Specifically Appear in the Lineage of Melanophores in the Periodic Albino Mutant of Xenopus laevis

In the periodic albino mutant (a^p/a^p) of Xenopus laevis, peculiar leucophore‐like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild‐type (+/+). These leucophore‐like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of α‐melanocyte stimulating hormone (αMSH), and (4) containing an abundance of bizarre‐shaped, reflecting platelet‐like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore‐like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to αMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore‐like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore‐like cells are essentially similar to melanophores of the wild‐type with respect to their localization in the skin and manner of response to αMSH, (3) the leucophore‐like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet‐like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore‐like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

Subcortical microtubule network separates the periplasm from the endoplasm and is responsible for maintaining the position of accessory nuclei in hymenopteran oocytes

Oocytes of hymenopterans are equipped with peculiar organelles termed accessory nuclei. These organelles originate from the germinal vesicle (oocyte nucleus) and gather preferentially at the anterior pole. To gain insight into the mechanism of uneven (asymmetrical) distribution of accessory nuclei, the organization of the microtubule cytoskeleton in the oocytes of two hymenopterans Chrysis ignita and Cosmoconus meridionator has been studied. It is shown that during late previtellogenesis two networks of microtubules are present along the contact zone between the oocyte and enveloping follicular epithelium. The external one is associated with belt desmosomes connecting neighbouring follicular cells. The internal network is composed of randomly orientated microtubules and separates transparent, organelle-free periplasm from the endoplasm. All cellular organelles and the germinal vesicle are localized in the endoplasm. Accessory nuclei are accumulated in the anterior endoplasm; they always lie in direct contact with the subcortical network. Treatment with colchicine results in the disappearance of the periplasm as well as in the redistribution of cellular organelles including accessory nuclei. Presented findings suggest that subcortical microtubules play an important role in the positioning of accessory nuclei throughout the ooplasm.     

Lipovitellin 2β is the 31 kD Ni2+-binding protein (pNiXb) in Xenopus oocytes and embryos

An Ni²⁺-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with ⁶³Ni²⁺, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2β, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2β agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metalblot competition assay, the abilities of metal ions to compete with ⁶³Ni²⁺ for binding to pNiXb were ranked: Zn²⁺ ≈ Cu²⁺ ≈ Co²⁺ > Cd²⁺ ≈ Mn²⁺ > Sn²⁺. This study shows that Xenopus lipovitellin 2β is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo. © 1994 Wiley-Liss, Inc.     

Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals

Oogenesis in the anuran Xenopus laevis can be divided into six stages based on the anatomy of the developing oocyte. Stage I consists of small (50 to 100 μ) colorless oocytes whose cytoplasm is transparent. Their large nuclei and mitochondrial masses are clearly visible in the intact oocyte. Stage II oocytes range up to 450 μ in diameter, and appear white and opaque. Stage I and II are both previtellogenic. Pigment synthesis and yolk accumulation (vitellogenesis) begins during Stage III. Vitellogenesis continues through Stage IV (600 to 1000 μ), the oocytes grow rapidly, and the animal and vegetal hemispheres become differentiated. By Stage V (1000 to 1200 μ) the oocytes have nearly reached their maximum size and yolk accumulation gradually ceases. Stage VI oocytes are characterized by the appearance of an essentially unpigmented equatorial band. They range in size from 1200 to 1300 μ, are postivtellogenic and ready for ovulation. These stages of oocyte development have been correlated with physiological and biochemical data related to oogenesis in Xenopus.     

A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K⁺ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1–S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.     

The ultrastructure of the ovarian follicle of medaka,Oryzias latipes

Summary The follicular cells in the oocytes of Oryzias latipes were studied by electron microscopy in order to clarify the fine structure, and the role of the cells during yolk formation and ovulation. The smallest follicles were observed during the early phase of peri-nucleolus stage of the oocyte. The cells have flattened nuclei, and perikarya with undeveloped organelles. But when the oocytes attain diameter of about 250 (yolk vesicle stage), both types of endoplasmic reticula are present. Moreover, the microvilli of the plasma membrane of oocyte as well as the follicles protrude into the pore canals of the zona radiata. In the oocytes of yolk stage the rough-surfaced endoplasmic-reticulum is typically developed and observed around the nuclei. Other organelles (lysosomes, mitochondria and Golgi) increase in number. The relation between the changes of cytoarchitecture in the follicles and yolk formation is discussed.At 17.00 p.m. on the day preceding ovulation the microvilli withdraw somewhat. Ribosomes are attached to the vesicular and cisternal endoplasmic reticula. When the oocytes attain complete maturation (24.00 p.m. at near ovulation), striking changes of the follicles are observed. The microvilli are almost withdrawn. In the degenerating follicles the lamellar structure is formed, and lipids are deposited at the center. At this time the contents of lysosomes have mostly disappeared.     

Melanogenesis in oocytes of wild-type and mutant albino axolotls

Melanogenesis during oogenesis in the wild-type and albino (a/a) axolotl was compared. Tyrosine-dopa oxidase activity, melanin accumulation, and melanosome development were correlated and the effect of the a gene on these biochemical and morphological events was examined. Studies of wild-type oocytes at the electron and light microscope level revealed that premelanosomes first appear in stage 2 oocytes. Mature melanosomes are present in stage 3 oocytes and steadily increase in number, reaching a maximum level in stage 6 oocytes. Melanosomes were detected in the albino. No obvious structural abnormalities were observed in these organelles, although they fail to accumulate melanin. Tyrosine-dopa oxidase (TDO) activity assayed radiometrically is at a very low level in stages 1 and 2 oocytes, reaches a maximum level in stage 3 oocytes, and declines to zero activity in stage 6 oocytes. In contrast to the finding with albino skin homogenates (Harsa-King, 1978), TDO activity was detected in albino oocytes. This activity never declined from its maximal stage 3 level. The addition of an inhibitor of proteolytic enzymes, phenylmethyl sulfonyl fluoride (PMSF), to the oocyte homogenization buffer completely blocks TDO activity in albino samples and reduces it somewhat in wild-type samples. It is suggested that TDO activity eliminated by PMSF represents TDO existing in an inactive form in vivo which is activated by proteolytic enzymes released upon homogenization. These results suggest that TDO is found only in an inactive state in albinos, a conclusion in agreement with the earlier work on albino skin melanocytes (Harsa-King, 1978). There is an inverse relationship between TDO activity and melanization in the wild type. The greater the amount of melanin deposited within the premelanosomes, the less enzyme activity is present. It is suggested, as it has been by others, that as melanin is synthesized within the confines of the oocyte melanosome, the active sites of the enzyme are covered up, resulting in its inactivation. The findings with the albino mutant support this hypothesis. No melanin deposition occurs in the albino, and TDO activity in PMSF-untreated samples does not decline from its maximal stage 3 level.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Do fast increasing food conditions promote the midsummer decline of Daphnia galeata?

Ovaries from mature giant red shrimp Aristaeomorpha foliacea were investigated histochemically and ultrastructurally. Four growing stages of the oocytes were distinguished: premeiosis stage, previtellogenetic stage, early vitellogenic stage and late vitellogenic stage. In addition, occasional resorptive oocytes were found. Oogonia and premeiotic oocytes were found in germinative zones. Previtellogenic and vitellogenic oocytes were localized in maturative zones. As vitellogenesis proceeded, oocytes showed a progressive development in the number of lipid droplets as well as in the extension of RER, constituted of dilated cisternae, uniformely scattered throughout the cytoplasm. The RER produced yolk granules and a lampbrush-like substance. The latter was released under the oolemma and constituted a characteristic cortical zone. The oolemma did not develop microvilli or micropinocytotic vesicles to incorporate yolk precursors. Thus, the protein yolk appeared to be of endogenous origin. Few somatic cells were found around the oocytes, but they never gave place to a continuous epithelial layer around oocytes, thus it is not possible to speak of ovarian follicle. The cytoplasm of these mesodermal-oocyte associated cells (MOAC) was characterized by a typical steroidogenic apparatus. Few resorptive immature oocytes were found inside late vitellogenic oocytes. Since the ovaries were packed with late vitellogenic oocytes and the few immature oocytes were hardly detectable, oocyte maturation occurred in a synchronous way.