Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

The effects of pH and weak bases on the in vitro endocytosis of vitellogenin by oocytes of Drosophila melanogaster

Summary The endocytosis of labeled vitellogenin by the developing oocytes of Drosophila melanogaster is pH dependent and inhibited in the presence of primary amines as determined by culturing whole ovaries in vitro. When the pH of the culture medium is adjusted to 6.8 or above, the vitellogenic oocytes sequester labeled vitellogenin synthesized by the follicle cells. The endocytosis of vitellogenin is shown autoradiographically by the accumulation of labeled yolk spheres within the oocytes. When the pH of the medium is reduced to 6.6 or below, the oocytes fail to sequester labeled vitellogenin, as demonstrated by an increase in immunoprecipitable vitellogenin in the culture medium and a concomitant reduction in the number of labeled yolk spheres within the oocytes. Vitellogenin endocytosis is also impaired by the addition of the primary amines methylamine or chloroquine to the culture medium. Monensin, a carboxylic ionophore, is shown to inhibit completely the secretion of labeled vitellogenin from the follicle cells.     

Effects of ionophores on vitellogenin uptake by Hyalophora oocytes

Oocytes of Hyalophora cecropia that were incubated in vitro with [³⁵S]vitellogenin incorporated label within 10 min into an intermediate-density compartment identified by sucrose density gradient centrifugation. During a subsequent 20-min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m-cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K⁺ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H⁺-K⁺ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.     

Incorporation de la vitellogénine tritiée dans les ovocytes du Crustacé Amphipode Orchestia gammarellus (Pallas) / Incorporation of tritiated vitellogenin into the oocytes of the crustacean amphipod Orchestia gammarellus (Pallus)

Summary

During the secondary vitellogenesis the oocytes of Orchestia gammarellus accumulate yolk spheres and lipid droplets. We studied the uptake of tritiated vitellogenin by the oocyte and its accumulation in the yolk spheres.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Localization of vitellogenin-related protein in the developing oocytes of winter flounder (Pleuronectes americanus) by protein A-gold immunocytochemistry

Vitellogenin-related proteins were localized within the oocytes of a marine teleost, the winter flounder, using protein A-gold immunocytochemical electron microscopy. A homologous vitellogenin antisera was used along with 15-nm protein-A gold on thin sections of oocytes. Localization was significantly confined to organelles described as yolk spheres within the oocyte cytoplasm. This technique provides specific identification of yolk-containing organelles within the developing winter flounder oocyte.     

家蝇卵巢摄取卵黄蛋白的机理

在家蝇Musca domestica viaina 的卵黄发生过程中,卵母细胞摄取卵黄原蛋白与滤泡开放是相关的。观察不同发育时期的家蝇滤泡结果表明,在摄取活动最旺盛的时期也就是卵黄发生的顶盛时期,其滤泡开放程度最大,而在卵黄发生前期和后期基本上没有摄取活动,此时的滤泡上皮细胞间不开放。卵巢体外培养的激素处理表明,JH可以促进滤泡开放。家蝇卵巢微粒体制备物的Na⁺-K⁺ATP酶活力在卵巢发育过程中存在着动态变化。羽化后24小时时有一定的酶活性,随着卵黄发生的进行,酶活力逐渐增加,到羽化48小时时酶活力最高,然后又开始下降,到羽化72小时时已经很小。羽化32小时的家蝇点滴或注躬 JH之后,测得的卵巢微粒体制备物的Na⁺-K⁺ATP酶活力比正常羽化36小时的高,羽化44小时的家蝇点滴和注射JH之后,测得酶活力比正常羽化48小时的低。羽化36小时和48小时的家蝇卵巢微粒体制备物与JH共同作用后,其Na⁺-K⁺ATP酶的活力分别增加2.95倍和3.50倍,羽化48小时的家蝇卵巢在含有JH的培养液中培养启,其匀浆液的酶活性为对照组的1.26倍。 由此我们可以推测在家蝇的卵黄发生过程中,JH通过促进滤泡开放和增加卵巢微粒体制备物Na⁺-K⁺ATP酶的活力,从而调控卵母细胞对卵黄蛋白的摄取。     

Vitellogenin transport and yolk formation in the quail ovary

Morphological and biochemical investigations were made on the yolk formation in ovaries of the quail Coturnix japonica. Morphologically, two ways of nutrient uptake were observed in follicles. In small oocytes of white follicles, vitellogenin (VTG) was taken up through fluid-phase endocytosis which was assisted by follicular lining bodies. The lining bodies were produced in follicle cells. They adhered to the lateral cell membrane, moved along the membrane in the direction of the enclosed oocyte and were posted to the tips of the microvilli. These tips, now with lining bodies, were pinched off from the main cell body, engulfed by indented cell membranes of the oocyte, and transported to yolk spheres. In large oocytes of yellow follicles, VTG and very-low-density lipoproteins (VLDL) were taken up through receptor-mediated endocytosis. The VTG and VLDL particles diffused through the huge interspaces between follicle cells, and once in oocytes were transported to yolk spheres via coated vesicles. Immunohistochemistry showed that the VTG resides on or near the surface of the follicle cell membrane at the zona radiata whereas the cathepsin D resides at or near the oocytic cell membranes. Tubular and round vesicles in the cortical cytoplasm of oocytes were also stained with both antisera, suggesting that these vesicles are the sites where the VTG is enzymatically processed by cathepsin D. Upon analysis by SDS-PAGE, a profile similar to that of yolk-granule proteins was produced by incubating VTG with a quail cathepsin D of 40 kD.     

Onset of vitellogenin production and vitellogenesis,and their relationship to changes in the midgut epithelium and oocytes in the tickDermacentor variabilis

InDermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (up-take of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.     

Asymmetry in the cytoplasm of oocytes of largescale yellowfish Labeobarbus marequensis Smith 1841 (Teleostei: Cypriniformes: Cyprinidae)

The ovaries of the largescale yellowfish, Labeobarbus marequensis (Teleostei: Cypriniformes: Cyprinidae), are made up of the germinal epithelium, nests of late chromatin nucleolus stage oocytes, and ovarian follicles. Each follicle is composed of a single oocyte, which is surrounded by somatic follicular cells and a basal lamina covered by thecal cells. We describe polarization and ultrastructure of oocytes during the primary growth stage. The oocyte nucleus contains lampbrush chromosomes, nuclear bodies and fibrillar material in which multiple nucleoli arise. Nuage aggregations composed of material of a nuclear origin are present in the perinuclear cytoplasm. The Balbiani body (Bb) contains aggregations of nuage, rough endoplasmic reticulum, individual mitochondria and complexes of mitochondria with nuage (cement). Some mitochondria in the Bb come into close contact with endoplasmic reticulum cisternae and vesicles that contain granular material. At the start of primary growth, the Bb is present in the cytoplasm close to the nucleus. Next, it expands towards the oocyte plasma membrane. In these oocytes, a spherical structure, the so-called yolk nucleus, arises in the Bb. It consists of granular nuage in which mitochondria and vesicles containing granular material are immersed. Later, the Bb becomes fragmented and a fully grown yolk nucleus is present in the vegetal region. It contains numerous threads composed of granular nuage, mitochondria, lysosome-like organelles and autophagosomes. We discuss the formation of autophagosomes in the cytoplasm of primary growth oocytes. During the final step of primary growth, the cortical alveoli arise in the cytoplasm and are distributed evenly. The eggshell is deposited on the external surface of the oocyte plasma membrane and is made up of two egg envelopes that are pierced by numerous pore canals. The external egg envelope is covered in protuberances. During primary growth no lipid droplets are synthesized or stored in the oocytes.     

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.     

THE ROUTE OF ENTRY AND LOCALIZATION OF BLOOD PROTEINS IN THE OOCYTES OF SATURNIID MOTHS

The oocytes of saturniid moths take up proteins selectively from the blood. The distribution of blood proteins in the ovary during protein uptake was investigated by staining 2 µ sections of freeze-dried ovaries with fluorescein-labeled antibodies. The results indicate that blood proteins occur primarily in the intercellular spaces of the follicle cell layer, in association with a brush border at the surface of the oocyte, and within the oocyte in the yolk spheres. That proteins derived from the blood are associated with the yolk spheres was confirmed by isolating these bodies and showing that lysis, which can be induced by any of a number of mechanical means, causes them to release immunologically defined proteins known to be derived from the blood. That the level of blood proteins in the cytoplasm is low relatively to that in the yolk spheres was confirmed by the observation that the yellow pigments associated with several blood proteins, although conspicuous in the yolk spheres, are not visible in the translucent layer of centrifuged oocytes. From these and previous physiological observations, it is proposed that blood proteins reach the surface of the oocyte by an intercellular route, that they combine with some component of the brush border, and that they are transformed into yolk spheres by a process akin to pinocytosis.     

Fine structure of the developing oocytes in adultArgas (Persicargas) arboreus (Ixodoidea: Argasidae)

The structure of the developing oocytes in the ovary of unfed and fed femaleArgas (Persicargas) arboreus is described as seen by scanning (SEM) and transmission (TEM) electron microscopy. The unfed female ovary contains small oocytes protruding onto the surface and its epithelium consists of interstitial cells, oogonia and young oocytes. Feeding initiates oocyte growth through the previtellogenic and vitellogenic phases of development. These phases can be observed by SEM in the same ovary.The surface of isolated, growing oocytes is covered by microvilli which closely contact the basal lamina investing the ovarian epithelium and contains a shallow, circular area with cytoplasmic projections and a deep pit, or micropyle, at the epithelium side. In more advanced oocytes the shell is deposited between microvilli and later completely covers the surface.Transmission EM of growing oocytes in the previtellogenic phase reveals nuclear and nucleolar activity in the emission of dense granules passing into the cytoplasm and the formation of surface microvilli. The cell cytoplasm is rich in free ribosomes and polysomes and contains several dictyosomes associated with dense vesicles and mitochondria which undergo morphogenic changes as growth proceeds. Membrane-limited multivesiculate bodies, probably originating from modified mitochondria, dictyosomes and ribosomal aggregates, are also observed. Rough endoplasmic reticulum is in the form of annulate lamellae. During vitellogenesis, proteinaceous yolk bodies are formed by both endogenous and exogenous sources. The former is involved in the formation of multivesicular bodies which become primary yolk bodies, whereas the latter process involves internalization from the haemolymph through micropinocytosis in pits, vesicles and reservoirs. These fuse with the primary yolk bodies forming large yolk spheres. Glycogen and lipid inclusions are found in the cytoplasm between the yolk spheres.     

Oocyte growth in the sheepshead minnow: Uptake of exogenous proteins by vitellogenic oocytes

The structure of the vitellogenic follicle of the sheepshead minnow, Cyprinodon variegatus, is described. Follicles enlarge primarily by protein yolk accumulation (vitellogenesis) and subsequently increase in size by hydration. This study uses the electron-dense tracer, horseradish peroxidase, and a larger heterologous protein,Xenopus laevis [³H]vitellogenin, to follow the fate of exogenous proteins from the maternal circulation to yolk spheres of the growing oocyte. Materials appear to leave the perifollicular capillaries via an interendothelial route, traverse the theca and the patent intercellular channels of the follicular epithelium and the pore canals of the vitelline envelope. At the oocyte surface they are incorporated via micropinocytosis and translocated to growing yolk spheres in the peripheral ooplasm. In contrast to other studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study substantiates the importance of heterosynthetic processes during oocyte growth in C. Variegatus.     

Activin incorporation into vitellogenic oocytes of Xenopus laevis.

Activin uptake into Xenopus oocytes was studied by several complementary methods. Immunocytochemistry of adult ovary localized activin and follistatin in the cytoplasm of vitellogenic oocytes and surrounding follicle cells. Surface plasmon resonance analysis of protein interaction kinetics indicated that while follistatin or a complex of activin-follistatin bound to yolk vitellogenin, activin alone did not. Radioactive tracer analysis measured specific incorporation of activin by viable oocytes in vitro. Together, the results suggest that vitellogenic oocytes can import activins from follicle cells and that follistatin may act as a chaperone for binding activin to vitellogenin in yolk platelets.     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.     

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.     

Intracellular transport of vitellogenin in Xenopus oocytes: An autoradiographic study

The role of primordial yolk platelets (PYPs) in the transport of the yolk precursor vitellogenin to the yolk platelets in Xenopus laevis oocytes has been demonstrated by electron microscopic autoradiography. Within 20 min after exposure of the oocyte to ³H-labeled-vitellogenin, silver grains are associated with small PYPs which are formed by the fusion of endosomes. At 40 min after incorporation of ³H-labeled vitellogenin, autoradiographic silver grains are associated with larger PYPs and with the superficial layer of yolk platelets. Thus, the results demonstrate that PYPs are an intermediate in the transport of vitellogenin from endosomes to yolk platelets. These observations are consonant with the general hypothesis that vitellogenin first associates (binds?) with the plasma membrane, then is incorporated by endocytosis into endosomes which fuse to form PYPs, and finally the contents of the PYPs are eventually deposited into yolk platelets.     

Ferromagnetic isolation of endosomes involved in vitellogenin transfer into Xenopus oocytes

The transport pathway of the yolk precursor vitellogenin (VTG) has been followed using the techniques of ferrolabeling and ferromagnetic sorting, coupled with electron microscopic visualization. Vitellogenin conjugated to colloidal ferric particles of ca. 11 nm is selectively transported from the oolemma to the yolk platelets of vitellogenic Xenopus oocytes after gonadotropin stimulation of the female. Several cortical membrane compartments, labeled or unlabeled with ferric particles, are involved in the internalization and the transfer of vitellogenin to the yolk platelets. 1) Coated pits apparently fuse with coated vesicles, and coated vesicles fuse with each other in the outermost cortical cytoplasm. 2) Vesicles, depleted of their clathrin coat, fuse with cortical tubular endosomes and discharge their contents into yolk endosomes. 3) These endosomes are the direct precursors of the yolk organelles. 4) Endocytic vesicles fuse only with primordial yolk platelets of type I and not with type II or fully grown yolk platelets. After pulse-chase loading with ferric particles conjugated to vitellogenin and subsequent subcellular fractionation of the oocytes, ferromagnetic sorting of the various vesicle populations has been performed by using a "free-flow magnetic chamber". This novel method enables specification and characterization of purified endosomal compartments that accumulate protein yolk in Xenopus oocytes.     

Effects of ablation of the pars intercerebralis on ecdysteroid quantities and yolk protein expression in the blowfly Protophormia terraenovae

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau‐Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham‐operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.