Ultrastructural observations on oogenesis in Drosophila

The ultrastructure of the follicle cells and oocyte periplasm is described during the stages of oogenesis immediately prior to, during, and immediately subsequent to, vitellogenesis. A number of features have not been described previously in Drosophila. Some yolk appears prior to pinocytosis of blood proteins. However, most of the protein yolk forms while the periplasm is filled with micropinocytotic invaginations and tubules derived from the oolemma. These tubules retain the internal layer of material characteristic of coated vesicles and are found to fuse with yolk spheres. No accumulation of electron-dense material in the endoplasmic reticulum or Golgi of the oocyte is found. Both trypan blue and ferritin are accumulated by the oocyte. The follicle cells have an elaborate endoplasmic reticulum during the period of maximum yolk accumulation. Adjacent cells are joined at their base by a zonula adhaerens, forming a band around the cells, and by plaques of gap junctions. Gap junctions are also present between nurse cells and follicle cells. During chorion formation, septate junctions also appear between follicle cells, adjacent to the zonula adhaerens.     

PROTEIN UPTAKE IN THE OOCYTES OF THE CECROPIA MOTH

The formation of yolk spheres in the oocyte of the cecropia moth, Hyalophora cecropia (L.), is known immunologically to result largely from uptake of a sex-limited blood protein. Recent electron microscope analyses of insect and other animal oocytes have demonstrated fine structural configurations consistent with uptake of proteins by pinocytosis. An electron microscope analysis of the cecropia ovary confirms the presence of similar structural modifications. With the exception of two apparently amorphous layers, the basement lamella on the outer surface of the follicular epithelium and the vitelline membrane on the inner, there is free access of blood to the oocyte surface between follicle cells. Dense material is found in the interfollicular cell space and adsorbed to the outer surface of the much folded oocyte membrane. Pits in the oocyte membrane and vesicles immediately under it are lined with the same dense material not unlike the yolk spheres in appearance. Introduction of ferritin into the blood of a developing cecropia moth and its localization adsorbed to the surface of the oocyte, and within the vesicles and yolk spheres of the oocyte cortex, is experimental evidence that the structural modifications of the oocyte cortex represent stages in the pinocytosis of blood proteins which arrive at the oocyte surface largely by an intercellular route. Small tubules attached to the yolk spheres are provisionally interpreted as a manifestation of oocyte-synthesized protein being contributed to 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.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Studies on the ovotestis of the slug Agriolimax reticulatus (Müller)

Summary The ovarian oocytes of Agriolimax reticulatus (Müller) have been studied by light and electron microscopy and electron cytochemistry. The development of the oocyte in the ovotestis may be divided into three stages.During Stage I the oocyte cytoplasm contains mainly ribosomes and also strands of endoplasmic reticulum, scattered mitochondria and Golgi systems. The nucleus contains both a paranucleolus and an eunucleolus. By Stage II the oocyte has enlarged, especially in a plane parallel to the basement membrane. In addition to the above mentioned organelles, the cytoplasm contains lipid, glycogen and early yolk platelets. During Stage III, the oocyte continues to enlarge, but mainly in a plane perpendicular to the basement membrane. A considerable degree of cytoplasmic differentiation has also taken place. The plasma membrane of the oocyte has become specialized with the appearance of a polysaccharide-rich glycocalyx, microvilli and pinocytotic tubules. Elsewhere, much of the background cytoplasm, containing Golgi-derived, polysaccharide and acid phosphatase-rich multivesiculate bodies, lipid and glycogen, is sequestered by smooth membranes and ultimately fuses with the growing yolk platelets. The nucleus contains an amphinucleolus, characteristic of many gastropods.The findings of this study are discussed in relation to results from other studies on oogenesis.     

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

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

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Studies on the annual reproductive cycle of the female cobra,Naja naja. IV. OVARIAN HISTOLOGY

Morphological changes of the ovary of the Chinese cobra, Naja naja, throughout the annual reproductive cycle are described. A single clutch of between 6 and 22 eggs is produced in late June. From July to the following April the ovary remains quiescent and contains small previtellogenic, hydration stage follicles. The growth of an ovarian follicle from a primary oocyte to maturation and ovulation is estimated to take three years. The histology of the germinal epithelium and the follicular granulosa shows seasonal changes correlated with the growth of the oocyte. During the quiescent period, the germinal epithelium lacks mitotic activity, but during April, when yolk deposition and rapid growth of the preovulatory follicles take place, the germinal epithelium shows intense mitotic activity. The growth of the smallest hydration stage follicles, and the occurrence of cytoplasmic bridges between the pyriform cells of the granulosa and the developing oocyte, also appear to increase during this period. The possible function of the pyriform cell is discussed and the literature on the origin and fate of these cells in the squamate ovary is reviewed. Postovulatory follicles (corpora lutea) and two types of atresia are described and compared with what is known of these structures in other reptiles.     

Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. I. An histological and histochemical study of oocyte development

Histological and histochemical studies of oocyte development in the bass, Dicentrarchus labrax L., showed that three types of inclusions are formed during vitellogenesis. Lipid yolk accumulates first as lipid droplets, followed by protein yolk in the form of discrete protein yolk granules. The third type of inclusion are the small cortical alveoli (intravesicular yolk/yolk vesicles, i.e.'carbohydrate yolk') which form in the peripheral cytoplasm after both the lipid and protein yolk have started to accumulate. While the protein yolk granules maintain their structural integrity through to maturation, forming a densely packed zone in the mid-outer cortex, the lipid yolk droplets continually coalesce and migrate centripetally, forming a prominent zone of large lipid droplets in the inner-mid cortex. From the histological study of oocyte development, a number of distinct developmental stages are delineated, while gross examination of the paired ovary revealed that, depending on its stage of development, it can be placed into one of seven maturity stages.     

Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.     

Regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis

In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by the Drosophila melanogaster vitellogenin receptor gene yolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that yl RNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.     

YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L

Yolk proteins are thought to enter certain eggs by a process akin to micropinocytosis but the detailed mechanism has not been previously depicted. In this study the formation of protein yolk was investigated in the mosquito Aedes aegypti L. Ovaries were fixed in phosphate-buffered osmium tetroxide, for electron microscopy, before and at intervals after a meal of blood. The deposition of protein yolk in the oocyte was correlated with a 15-fold increase in 140 mµ pit-like depressions on the oocyte surface. These pits form by invagination of the oocyte cell membrane. They have a 20 mµ bristle coat on their convex cytoplasmic side. They also show a layer of protein on their concave extracellular side which we propose accumulates by selective adsorption from the extraoocyte space. The pits, by pinching off from the cell membrane become bristle-coated vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small crystalline yolk droplets, which subsequently coalesce to form the large proteid yolk bodies of the mature oocyte. Preliminary radioautographs, and certain morphological features of the fat body, ovary, and midgut, suggest that the midgut is the principal site of yolk protein synthesis in the mosquito.     

Regional differences in the pattern of vitellogenesis in the painted frog Discoglossus pictus

During oocyte growth in the frog Discoglossus pictus two patterns of vitellogenesis are described. The first one consists of the transformation of multivesicular bodies into yolk platelets; the second is the result of a typical endocytotic process, as described in other species. The peculiarity in Discoglossus vitellogenesis consists of a regional difference of these features of vitellogenesis in vitellogenic oocytes: the multivesicular bodies transforming into yolk platelets are found only in the germinative area—the central portion of the animal half—whereas deep crypts with numerous endocytotic pits are found only in the vegetal half. The probable meaning of this regional difference in vitellogenic oocytes is discussed.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

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.     

Early ultrastructural changes of developing oocytes in the dog

Many aspects of the developmental stages of the oocyte of the dog resemble those of other mammalian species. The oocyte of the dog, however, contains large amounts of lipid yolk material. A study of the ultrastructural morphology of early growth and maturation of dog oocytes was undertaken to clarify the nature and appearance of this yolk material. The lipid yolk first appears in early primary oocytes as aggregated dense bodies that gradually fill the ooplasm as the oocyte matures. The site of the yolk's initial appearance is consistently related to a single centriole and often to the lamellae of smooth endoplasmic reticulum that surrounds groups of forming lipid yolk bodies. Dense cortical granule-like vesicles are found to lie deep within the maturing oocyte and often are enclosed within the lamellar yolk space. Granules within this space undergo changes in size, matrix configuration, and vacuolization. These changes suggest a mechanism whereby material is added to the lipid yolk bodies. Light microscope histochemistry for lipid and polysaccharide material is described.     

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.     

Further morphological and histochemical studies on the yolk nucleus and associated cell components in the developing oocyte of the Indian wall lizard

In March through April when the oocyte growth in the ovaries of the wall lizard (Hemidactylus) is very rapid, the yolk nucleus continues to persist through various stages of previtellogenesis. This persisting yolk nucleus and associated cell components have been studied with histochemical techniques. The spherical and dense yolk nucleus stains for protein, lipoprotein and RNA. It does not form any close morphological association with the other cell components such as the mitochondria, lipid bodies (L₂), spaces or canals, diffuse sudanophilic substance and dense bodies, which are arranged into three zones round the yolk nucleus proper. The mitochondria stain for lipoprotein; the L₂ bodies consist of phospholipid; the spaces do not contain any material demonstrable with histochemical techniques; and the ooplasm containing the diffuse sudanophilic substance and dense bodies shows lipoprotein, protein and RNA. Eventually, the yolk nucleus disintegrates, and its substance as well as the other cell components are distributed in the cortical ooplasm of oocytes which are ready to form the yolk bodies. Concepts of the origin, morphology, cytochemistry and function of the yolk nucleus in the oocytes of invertebrates and vertebrates, which have come about recently through the application of cytochemical and submicroscopical techniques, are discussed.     

莫桑比克非鲫卵黄形成的电镜观察

运用透射电镜观察了莫桑比克非鲫卵母细胞的生长.根据卵母细胞的大小和内部结构特征,将其分为四个时期:卵母细胞生长早期:卵黄泡形成期:卵黄积累期:卵黄积累完成期.本文着重研究了主要卵黄成分--卵黄球的形成过程.卵黄球属外源性卵黄,由卵母细胞通过微胞饮作用吸收肝脏合成的卵黄蛋白原后形成的.在卵黄大量积累前,卵母细胞内的线粒体和多泡体聚集成团,构成卵黄核,继而线粒体大量增殖,线粒体形状发生改变,形成同心多层膜结构,为大量的卵黄物质积累提供场所.最终形成的卵黄球由被膜、卵黄结晶体和两者之间的非结晶区三部分组成.       

The regulation of the yolk protein genes,a family of sex differentiation genes in Drosophila melanogaster

There are many obvious morphological and behavioural differences between male and female Drosophila, whose differing phenotypes are produced by a hierarchy of sex determination genes. These genes have been well characterised at the genetic and molecular level. Similarly, a number of sex-specific differentiation genes have been characterised, such as the chorion and vitelline membrane genes in females and the sex peptide and other accessory gland proteins in males. Despite the depth of these parallel studies, there is only one example of a direct link between the sex determination pathway and the downstream sex differentiation genes, namely the regulation of the female-specific yolk protein genes. The yolk proteins are synthesised in the fat body and ovarian follicle cells of the adult female and are subsequently transported to the oocyte where they are stored for utilization during embrygenesis. The expression of the yolk protein genes is not entirely controlled by the sex determination hierarchy, as several different regulatory pathways must interact to direct their correct sexual, temporal and spatial regulation during development.