Isolation of plasma membrane complexes fromXenopus oocytes

Summary A method for the isolation of plasma membrane fractions fromXenopus oocytes has been developed, and the membranes have been characterized biochemically and morphologically. Plasma membrane complexes prepared by this procedure consisted of large sheets of the membrane, with associated vitelline envelope (a nonmembranous meshwork of fibers) and cortical (secretory) granules still attached. The morphology of cell surface microvilli and coated pits was well preserved. Cortical granules were removed by gentle homogenization in a low ionic strength medium, and integral and peripheral membrane proteins were then separated from vitelline envelopes by detergent extraction and phase separation in Triton-X-114. Biochemical characterization of the plasma membrane fractions indicated substantial levels of 5-nucleotidase and alkaline phosphodiesterase activity associated with the oocyte cell surface, with 44–66% recovery of these markers in the final membrane preparations. Lectin blotting and lectin affinity chromatography with Concanavalin A and wheat germ agglutinin were used to characterize the major glycoprotein species associated with the plasma membrane complexes. Plasma membrane fractions prepared by this procedure should be very useful in both biochemical and morphological studies of membrane protein sorting in theXenopus oocyte system.     

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.     

Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries

Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. Here we show that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally we used cell markers to examine the distribution of these proteins at the follicle cells and show they accumulated in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. We propose that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.     

Yolk polypeptide secretion and vitelline membrane deposition in a female sterile Drosophila mutant

Ovarian follicle cells of wild type Drosophila melanogaster simultaneously secrete yolk polypeptides (YP1, YP2 and YP3) and vitelline membrane proteins. In order to understand the relationship between these two secretory activities, we have investigated the ultrastructure of a female sterile mutation that alters YP1 secretion and vitelline membrane deposition. Homozygous fs(1)1163 females lay eggs that collapse and contain reduced quantities of YP1. Secretory granules in follicle cells contain an electron-translucent component that is assembled into the developing vitelline membrane in both mutant and wild-type ovaries, and an electron-dense component that disperses after secretion in wild-type ovaries. Mutant ovaries differ from wild-type by (1) having larger secretory granules (2) forming clumps of the dense secretory component within the developing vitelline membrane (3) accumulating more tubules in the cortical ooplasm of vitellogenic oocytes, and (4) possessing altered yolk spheres. Mutant ovaries implanted into wild-type hosts showed no improvement in the secretory granules and slight improvement in the vitelline membrane clumps but amelioration of the oocyte phenotypes. Since genetic evidence suggests that the fs(1)1163 mutation resides in or near the Yp1 gene and biochemical data show that the mutation alters YP1 structure, we conclude that the ultrastructural phenotypes are due to a structurally abnormal YP1 in the mutant. The alteration in vitelline membrane structure caused by the dense clumps could account for collapsed eggs and, hence, the female sterility of the mutant.     

Egg-shells in mites

Summary This communication presents results of studies on the formation and structure of the vitelline envelopes in three species of mites: Euryparasitus emarginatus (Gamasida), Erythraeus phalangoides (Actinedida), and Hafenrefferia gilvipes (Oribatida). In E. emarginatus and E. phalangoides, in which the oocytes are not covered with follicular cells, the material of the vitelline envelope appears first in vesicles under the surface of the oocytes prior to secretion by exocytosis. The formed vitelline envelope is built of a homogeneous material which is perforated by numerous channels containing oocyte microvilli. Later, as the microvilli are retracted, the channels disappear. In both of these species the formed vitelline envelope is incomplete and the micropylar orifice occurs as a transitional structure.In H. gilvipes follicular cells encircling the oocyte contain granules filled with material that is subsequently secreted into the perivitelline space forming the vitelline envelope on the oocyte surface. The inner layer of the vitelline envelope is granular, whereas the outer part is more homogeneous. Both lack channels containing microvilli and micropyle.     

Effects of starvation on reproduction of the predacious mite <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae)

Effects of starvation on gravid females of Neoseiulus californicus were investigated at 20°C and 85% RH. When females that had been reared with abundant prey were swapped, just after laying their first egg, to conditions without any prey and water, they laid 1.8 eggs and survived for 4.3 days. In the body of well-fed females, an egg with eggshell and/or two oocytes were observed in the ventral and dorsal regions, respectively. The larger oocyte had two roundish nuclei and abundant yolk granules, and was enveloped with a vitelline membrane. These two nuclei were not fused but were just close to each other. The smaller oocyte had a nucleus, but had not yet formed yolk granules and vitelline membrane. Females after 12 h starvation had an egg in the ventral region and an oocyte in the dorsal region of the body. After more than 24 h starvation females maintained an oocyte in the dorsal region of the body, but had no egg in the ventral region. The oocyte was filled with abundant yolk granules and contained two irregular nuclei when females were starved for 24 h, but when starved for more than 36 h it contained one irregular nucleus. These findings suggest that (1) gravid females maintained an oocyte in the dorsal region after laying two eggs during starvation, (2) the oocyte was not absorbed during starvation, (3) the oocyte advanced vitellogenesis and the fusion of two nuclei, and (4) the vitellogenic oocyte was not enveloped with an eggshell and had not started embryogenesis.     

closca, a new gene required for both Torso RTK activation and vitelline membrane integrity. Germline proteins contribute to Drosophila eggshell composition

The Drosophila eggshell is a specialised extracellular matrix (ECM) that surrounds and protects the oocyte and the embryo until its eclosion. In addition, the vitelline membrane, the innermost layer of the eggshell, holds the local determinant required to activate the Torso RTK pathway, which establishes the embryonic terminal regions. Here we report the identification and characterisation of closca, a gene encoding a new member of a group of proteins that act non-redundantly in vitelline membrane biogenesis and in Torso signalling. We also show that the Nasrat protein, another member of this group, is incorporated into the vitelline membrane, thereby indicating that the eggshell is a shared ECM that receives contributions from both follicle cells and the germline. This observation also provides a new scenario that accounts for the long known contribution of germline products to vitelline membrane biogenesis and to the follicle cell-dependent activation of the Torso receptor.     

Stages in follicle cell/oocyte interface during vitellogenesis in caecilians <Emphasis Type="Italic">Ichthyophis tricolor</Emphasis> and <Emphasis Type="Italic">Gegeneophis ramaswamii</Emphasis>: a transmission electron-microscopic study

We describe the ultrastructural organization of the vitellogenic follicle stages in two caecilian species. Monthly samples of slices of ovary of Ichthyophis tricolor and Gegeneophis ramaswamii from the Western Ghats of India were subjected to transmission electron-microscopic analysis, with special attention to the follicle cell/oocyte interface. In order to maintain uniformity of the stages among the amphibians, all the stages in the caecilian follicles were assigned to stages I–VI, the vitellogenic and post-vitellogenic follicles being assigned to stages III–VI. Stage III commences with the appearance of precursors of vitelline envelope material in the perivitelline space. Stages IV and V have been assigned appropriate substages. During the transition of stage III to stage VI oocytes, a sequential change occurs in the manifestations of follicle cells, perivitelline space, vitelline envelope and oocyte cortex. The vitelline envelope becomes a tough coat through the tunnels of which the macrovilli pass to interdigitate between the microvilli. The oocyte surface forms pinocytic vesicles that develop into coated pits and, later, coated vesicles. Contributions of the oocyte cortex to the vitelline envelope and of the follicle cells to yolk material via synthesis within them are indicated. The follicle cell/oocyte interface of vitellogenic follicles of these two caecilians resembles that in anurans and urodeles, with certain features being unique to caecilians. Thus, this paper throws light on the possible relationships of caecilians to anurans and urodeles with special reference to ovarian follicles. This research was supported by funds from the Kerala State Council for Science, Technology and Environment (KSCSTE), through the SARD facility, and by the FIST scheme of Department of Science and Technology, Government of India, New Delhi, to the Department of Zoology, University of Kerala, Thiruvananthapuram, and to the Department of Animal Science, Bharathidasan University, Thiruchirapalli (SR/FST/LSI-233/2002).     

不同发育时期哲罗鱼卵黄的超微结构

应用透射电镜观察了不同发育时期哲罗鱼(Hucho taimen)卵黄的超微结构.根据哲罗鱼卵黄物质在卵母细胞中的加工合成、积累以及卵母细胞中参与卵黄颗粒形成的细胞器的变化,可将该鱼卵黄发生分为4个特征时期,即卵黄发生前期、卵黄泡期、卵黄积累期和卵黄积累完成期.卵黄发生前期是指卵母细胞发育过程中的卵黄物质开始积累前的时期,此时期核仁不断分裂,出现线粒体云和早期的滤泡细胞层、基层和鞘细胞层;卵黄泡期特点主要是细胞器不断变化产生卵黄泡和皮层泡;卵黄积累期的滤泡膜由内向外依次为放射带、颗粒细胞层、基层和鞘细胞层,此时外源性卵黄前体物质不断经过血液汇集于鞘细胞层,后经微胞饮作用穿过胶原纤维组成的基层,经过多泡体作用转运至颗粒细胞内,在细胞内经过加工和修饰形成小的卵黄蛋白颗粒,卵黄蛋白颗粒经微胞饮穿过放射带进入卵母细胞边缘形成的空泡中,不断积累形成卵黄球;进入卵黄积累完成期,卵黄球体积变大,向细胞中心聚集,填满大部分卵母细胞,卵黄积累完毕.     

Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes

We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional “Two Electrode Voltage Clamp” (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or “macropatch” techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between −100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration–inhibition curve displaying an inhibition constant IC₅₀ of about 50 nM, comparable to previously published values obtained with the TEVC technique.     

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.     

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

Oocyte development in four species of scyphomedusa in the northern Adriatic Sea

Oocyte development was followed in 4 species of Scyphomedusae. In Pelagia noctiluca a centrifugal maturity gradient is present. Vitellogenesis may be related first to exogenous endodermal production and later to oocyte endogenous activity. Simultaneously, the paraovular body (POB) develops from the secondary endoderm; it is connected to the oocyte and controls secretion of mucus, which envelops the oocyte during spawning. In Aurelia aurita, there is no maturity gradient, nor any differentiated structures of endodermal origin, associated with oocytes. In Discomedusa lobata a maturity gradient is absent. Its vitellogenesis is similar to that in P. noctiluca, and is associated with structural modification of the secondary endoderm in the area contacting the oocyte. This structure is cytologically similar to the POB of P. noctiluca, though less differentiated. Spawning is similar to that of P. noctiluca, with fenestration of the surrounding endodermal cells as the oocyte passes from the ovary to the genital sinus. In Rhizostoma pulmo a maturity gradient is absent. An early and fairly evident development of the vitelline membrane was observed.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

中华蚱蜢卵子发生中花生凝集素受体的初步鉴定和发育表达

The distributions of PNA binding glycoconjugates in the plasma membrane of Acrida cinerea Thunberg germ cells were detected using biotin labeled PNA, for better understanding of the formation and changes of glycoconjugates during oogenesis. The ultrastructure of vitellogenesis also was observed by electron microscopy for detection of the origin and track of vitelline material. In the ovary, PNA receptors appeared in the oocyte cytoplasm of the second phases of oogenesis; positive granules gradually increased from the third phase to the fourth, and they exhibited a maximum expression before the vitellogennic stage in the cytoplasm of the oocyte. From the vitellogennic to chorionation stage, positive granules gradually declined. Binding sites on follicle cells were changed with their morphological variation in every stage of oogenesis. The vitelline of A. cinerea formed within the oocyte by degrees. The results suggest that PNA receptors and yolk materials are synthesized by the oocytc at an early period. With the development of the oocyte, some exogeous materials from two sources act as PNA receptors and others take part in vitelline synthesis. One is blood lymph that offers some useful materials to the oocyte directly through follicle cell gaps; the other are follicle cells that produce and transmit some materials to oocyte to support vitellogenesis. In addition, PNA receptors secreted by follicle cells participate in the formation of yolk membrane [ Acta Zoologica Sinica 5 l （5） ： 932 - 939, 2005 ].     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

L'action exercée par l'appareil endocrine sur les phases tardives de l'ovogenèse chez Locusta migratoria

Most studies dealing with the endocrine control of oogenesis are focused on vitellogenesis, but the later events of oogenesis: vitelline membrane formation and choriogenesis have received less atention. In Locusta migratoria, the corpora allata evidently control vitellogenesis, but seem to have no bearing upon the formation of the vitelline membrane or the chorion. If the insects are deprived of their CA at the time when the oocyte length is around 3.5 to 4 mm, (normal length at egg laying: 6.5 mm) vitelline membrane formation and the chorion production are unaffected. In the same species, the electrocoagulation of the pars intercerebralis or excision of corpora cardiaca disturb oogenesis; vitelline membrane material is synthesized and secreted, but the primary granules fail to join each other and to transform into vitelline membrane and the production of chorionic material is disturbed.The rER of the follicle cell seems quite active but the substance elaborated accumulates in the Golgi saccules, and therefore the cytoplasm contains only very few secretory granules. The implantation of supernumerary pars intercerebralis and corpora cardiaca into immature females induces precocious deposition of the vitelline membrane which is complete in 5.0 mm oocytes (instead of 5.6 to 6.0 mm) and chorion formation is completed in 5.5 mm oocytes (instead of 6.0 to 6.3 mm). Ecdysone is secreted in the ovaries at the end of oogenesis. We tested the result of injecting ecdysone to immature females. It results in hastening oocyte growth during 24 hr, but this accelerated growth is followed by degeneration.     

The <Emphasis Type="Italic">Drosophila</Emphasis> RNA-binding protein Lark is required for localization of Dmoesin to the oocyte cortex during oogenesis

The RNA-binding protein Lark has an essential maternal role during Drosophila oogenesis. Elimination of maternal expression results in defects in cytoplasmic dumping and actin cytoskeletal organization in nurse cells. The function of this protein is dependent on the activity of one or more N-terminal RNA-binding domains. Here, we report the identification of Dmoesin (Dmoe) as a candidate RNA target of Lark during oogenesis. In addition to actin defects in the nurse cells of lark mutant ovaries, we observed mislocalization of posteriorly localized mRNAs including oskar and germ cell less in the developing oocyte. Anteriorly and dorsally localized mRNAs were not affected. In addition, we observed displacement of the actin cytoskeleton from the oocyte plasma membrane. These phenotypes are reminiscent of mutations in Dmoe and suggested that this RNA maybe a potential target of Lark. We observed a significant decrease in Dmoe protein associated with the membrane of the developing oocyte with no changes in expression or localization within the nurse cells. Evidence for an association between Lark protein and moe RNA during oogenesis comes from results of a microarray-based Ribonomics approach to identify Lark RNA targets. Thus, our results provide evidence that Dmoe RNA is a target of Lark during oogenesis and that it likely regulates either the splicing or translation of this RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The vitelline membranes of Aedes aegypti and Drosophila melanogaster: A comparative review

Summary

The insect eggshell provides a model system for the study of gene regulation because several proteins are synthesized in an ordered spatial and temporal pattern within a single tissue, the follicular epithelium. Progress is being made towards an understanding of Aedes aegypti and Drosophila melanogaster vitelline membrane formation. The vitelline membrane is the innermost layer of the eggshells of A. aegypti and D. melanogaster. Genes encoding three A. aegypti and four D. melanogaster vitelline membrane proteins have been cloned and sequenced. Significant similarity is observed between the A. aegypti and D. melanogaster vitelline membrane genes. Both families contain highly conserved regions of 34 and 38 amino acids in A. aegypti and D. melanogaster respectively. The protein composition of the two families are both rich in proline and alanine, but differ in their serine and histidine compositions. The regulation of vitelline membrane gene expression in A. aegypti and D. melanogaster is compared.