A stereological analysis of developing egg chambers in Ephestia kuhniella

A polytrophic ovariole of the flour moth, Ephestia kuhniella, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of seven nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each component as a function of the position of the egg chamber in the ovariole. Analysis of the growth curve of the Ephestia oocyte yields two possible correlations between accelerated oocyte growth and ultrastructural events enhancing the supply of yolk materials to the oocyte: the first is the initiation of yolk synthesis by the follicle cell layer and its transfer to the oocyte, and the second is the formation of channels between the follicle cells allowing hemolymph to gain access to the oocyte. An Ephestia oocyte increases in volume from approximately 2.5 × 10³ μm³ to approximately 2.0 × 10⁷ μm³ over an average series of 58 egg chambers.     

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.     

Comparative developmental physiology and molecular cytology of the polytrophic ovarian follicles of the blowfly Sarcophaga bullata and the fruitfly Drosophila melanogaster

1. The ovarian follicles of Sarcophaga and Drosophila consist of one oocyte and 15 nurse cells, the whole being surrounded by follicle cells. Although oocyte and nurse cells are genetically identical sibling cells, and although they are interconnected by cytoplasmic bridges, their physiology is very different. 2. The DNA content of the oocyte nucleus (germinal vesicle) never exceeds 4C, while values of polyploidisation up to 1024C have been measured in the nurse cells, this being dependent on their position within a follicle. 3. The nurse cell nuclei very actively synthesize RNA, while the germinal vesicle is almost completely inactive in this respect. 4. It has been possible to visualise the major cytoskeletal elements in the different ovarian cell types. Cellular markers of polarity and dorsoventral asymmetry have been described. 5. Electrophysiological measurements have been performed to find out whether or not the self-electrophoresis principle may be involved in polarised transport between nurse cells and oocyte. 6. Most of the vitellogenin is synthesized by the fat body but some follicle cells also synthesize small amounts. 7. The role of 20-OH ecdysone in the induction of vitellogenin synthesis in the fat body, as well as the presence of met-enkephalin like immunoreactivity in the gonads is well established in both species. Not so clear is the exact role of juvenile hormones and the nature of brain factors controlling ovarian development. 8. Drosophila has the advantage of its well documented genetics while the larger species Sarcophaga is preferable for the study of (electro-) physiological and cell biological mechanisms.     

Developmental stages of ovarian follicles of the silkworm,Bombyx mori L

Oogenesis in the silkworm, Bombyx mori, was studied by light and electron microscopy of sections of resin-embedded follicles. The development of the follicles was divided into a series of 12 distinctive stages based on various morphological criteria. Structural changes in the oocyte, nurse cells, and follicle cells are described and illustrated.     

Oogenesis of the mayfly Habrophlebia eldae: Synthesis of vitelline and chorionic envelopes

The ultrastructure of developing ovarian follicles inside the panoistic ovarioles of Habrophlebia eldae were examined to observe the events occurring during egg maturation up to the full formation of the chorionic envelopes. The early vitellogenic follicles are coupled by gap junctions and are extensively interlocked with the oocyte plasma membrane via microvilli. With the onset of vitellogenesis, coated pits and coated vesicles are precursors to yolk deposition and are visible at the follicle cell-oocyte interface. Postvitellogenic development entails the deposition of the egg envelopes. The vitelline envelope arises from the coalescence of rectangular plaques whose precursors are visible in Golgi complexes as heterogeneous electron-opaque granules. A chorionic pattern of ridges on the egg surface characterizes the shell of H. eldae. The fully developed chorion shows three distinct regions with differently organized patterns. A fine layer of fibrous material (a secretion of the follicle cells, Ephemeroptera devoid of accessory glands) adheres to the egg chorion and is probably involved in attachment to the substrate.     

Follicle cell development is partly independent of germ-line cell differentiation in Drosophila oogenesis

Summary The developmental potential of the cells of the somatic follicular epithelium (follicle cells) was studied in mutants in which the differentiation of the germ-line cells is blocked at different stages of oogenesis. In two mutants, sn ^36a and kelch, nurse cell regression does not occur, yet the follicle cells around the small oocyte continue their normal developmental program and produce an egg shell with micropylar cone and often deformed operculum and respiratory appendages. Neither the influx of nurse cell cytoplasm into the oocyte nor the few follicle cells covering the nurse cells are apparently required for the formation of the egg shell. In the tumor mutant benign gonial cell neoplasm (bgcn) the follicle cells can also differentiate to some extent although the germ-line cells remain morphologically undifferentiated. Vitelline membrane material was synthesized by the follicle cells in some bgcn chambers and in rare cases a columnar epithelium, which resembled morphologically that of wild-type stage-9 follicles, formed around the follicle's posterior end. The normal polarity of the follicular epithelium that is characteristic for mid-vitellogenic stages may, therefore, be established in the absence of morphologically differentiating germ-line cells. However, the tumorous germ-line cells do not constitute a homogeneous cell population since in about 30% of the analyzed follicles a cell cluster at or near the posterior pole can be identified by virtue of its high number of concanavalin A binding sites. This molecular marker reveals an anteroposterior polarity of the tumorous chambers. In follicles mutant for both bgcn and the polarity gene dicephalic the cluster of concanavalin A-stained germ-line cells shifts to more anterior positions in the follicle.     

The vitelline envelope,chorion, and micropyle of Fundulus heteroclitus eggs

The architecture and transformation of the vitelline envelope of the developing oocyte into the chorion of the mature egg of Fundulus heteroclitus have been examined by scanning and transmission electron microscopy. The mature vitelline envelope is structurally complex and consists of about nine strata. The envelope is penetrated by pore canals that contain microvilli arising from the oocyte and macrovilli from follicle cells. During the envelope's transformation into the chorion, the pore canals are lost and the envelope becomes more fibrous and compact and its stratified nature less apparent. The micropyle, of pore, through which the sperm gains access to the enclosed egg is located at the bottom of a small funnel-shaped depression in the envelope. Internally, the micropyle opens on the apex of a cone-like elevation of the chorion. During the development of the envelope, structured chorionic fibrils, the components of which are presumed to be synthesized by the follicle cells, become attached to its surface. These chorionic fibrils are though to aid in the attachment of the egg to the substratum and perhaps to help prevent water loss during low tides when the egg may be exposed.     

Regulation of growth of nurse nuclei in the development of egg follicles inCecidomyiidae (Diptera)

InCecidomyiidae the number of trophocytes derived from the somatic tissue of the ovary and forming nutritive chambers of egg follicles is variable. The regulation of growth of the whole nutritive chambers and of the nurse nuclei was investigated in two species of the gall midges,Mikiola fagi andBoucheella artemisiae, at two different stages of the egg follicle development during the second period of the oocyte growth. The volume of a nutritive chamber is correlated with the size of the egg follicle as a whole and is not dependent on the number of nurse nuclei it contains. The total volume of nurse nuclei at each stage under investigation was found to have a constant value which is independent of their number. It was established that the growth of the nurse nuclei takes place through endomitosis, and that at a given stage of the egg follicle development the constant value of the total volume of the nurse nuclei reflects the constancy of degree of their total polyploidy. The results obtained indicate that at the early stages of the egg follicle development the rates of growth of the nurse nuclei and of the whole nutritive chambers in the egg follicles differing with respect to the number of their nurse nuclei must be different; the greater the number of nurse nuclei in a given nutritive chamber the slower the rate of growth of the chamber and their nuclei. As a result of this differential rate of growth the volumes of the nutritive chambers and total volumes of nurse nuclei reach at a certain stage of the egg follicle development certain values common for all egg follicles, irrespective of the number of the nurse nuclei they contain. Beginning with this stage the dependence between the endomitotic activity of the nurse nuclei and the rate of growth of the whole nutritive chamber on the one hand, and the number of the nurse nuclei in the chamber on the other, evidently disappears. The available evidence supports the hypothesis that in the egg follicle ofCecidomyiidae the growth regulation of nurse nuclei and, indirectly, also of whole nutritive chambers results from developmental interrelationships between the oocyte and the nutritive chamber, and that the oocyte plays a leading role in this process. In view of a syncytial character of the nutritive chambers inCecidomyiidae and distinctly expressed asynchrony of the growth-duplication cycles of nurse nuclei belonging to a given chamber it is concluded that the control mechanism for DNA synthesis and endomitosis in nurse nuclei must possess the property of a rapid switch. Processes of the growth regulation of the nurse nuclei are discussed in connection with the role of the nutritive chamber in production of RNA and its supply to the growing oocyte. It is suggested that in the egg follicles ofCecidomyiidae there exists a complex interrelationship between the control mechanism for DNA synthesis and endomitosis in the nurse nuclei and the synthetic processes regulated by the supply of the growing oocyte with RNA produced by the nuclei of the nutritive chamber.     

An EM autoradiographic study on ovarian follicle cells of Drosophila melanogaster with special reference to the formation of egg coverings.

Ovarian follicle cells of Drosophila melanogaster have been studied by ultrastructural and autoradiographic analyses. During their migration through the germarium, follicle cells undergo several structural changes and, of these, the most conspicuous one occurs at the level of the nucleolus. By the time the first ovarian chamber is formed, follicle cells have formed a layer of uniform thickness all around a cluster or nurse cells and the oocyte. Following the initiation of vitellogenesis, the follicle cells overlaying the oocyte become columnar while those over the nurse cells become very thin. During stages 9-10, the columnar follicle cells are involved in the formation of the vitelline membrane, while from stages 11 to 13 these cells produce the endochorion. An EM autoradiographic analysis has shown that the rate of 3H-uridine incorporation in follicle cells nuclei is low in previtellogenic chambers, while it becomes very high in nuclei of stage 9-10 chambers. After short exposure to uridine, silver grains are located predominantly over nucleoli. Evidence from incorporation studies with 3H-lysine indicates that the columnar follicle cells and the region of the various egg coverings are highly labelled within an hour of incubation in the tracer. The observations confirm that columnar follicle cells are the only cells in the chamber involved in the formation of materials which make up the egg coverings.     

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

Summary The follicle cells, nurse cells and germinal epithelia, which are closely associated with the oocyte of Agriolimax reticulatus (Müller) during its development in the ovotestis, have been studied using light and electron microscopy. The various secretory, digestive and phagocytic activities of these cells have also been investigated using electron cytochemical tests for oxidisable polysaccharide, acid phosphatase and electron-opaque tracer molecules. The oocyte lies initially between the germinal epithelia and a layer of nurse cells but, as oocyte vitellogenesis proceeds, it becomes encapsulated by a layer of follicle cells. Both the follicle and the nurse cells are active in secretion and digestion and contain Golgi apparatus, granular endoplasmic reticulum and acid phosphatase-rich digestive vacuoles. The significance of these activities is discussed in relation to oocyte vitellogenesis, secondary envelope formation and the digestion and recycling of cellular material.     

An EM autoradiographic study on ovarian follicle cells of Drosophila melanogaster with special reference to the formation of egg coverings

Summary Ovarian follicle cells of Drosophila melanogaster have been studied by ultrastructural and autoradiographic analyses.During their migration through the germarium, follicle cells undergo several structural changes and, of these, the most conspicuous one occurs at the level of the nucleolus. By the time the first ovarian chamber is formed, follicle cells have formed a layer of uniform thickness all around a cluster or nurse cells and the oocyte. Following the initiation of vitellogenesis, the follicle cells overlying the oocyte become columnar while those over the nurse cells become very thin. During stages 9–10, the columnar follicle cells are involved in the formation of the vitelline membrane, while from stages 11 to 13 these cells produce the endochorion.An EM autoradiographic analysis has shown that the rate of ³H-uridine incroporation in follicle cell nuclei is low in previtellogenic chambers, while it becomes very high in nuclei of stage 9–10 chambers. After short exposure to uridine, silver grains are located predominantly over nucleoli.Evidence from incorporation studies with ³H-lysine indicates that the columnar follicle cells and the region of the various egg coverings are highly labelled within an hour of incubation in the tracer.The observations confirm that columnar follicle cells are the only cells in the chamber involved in the formation of materials which make up the egg coverings.This work was partly supported by C.N.R. (Italy)I am indebted to Dr. J. Jacob from the Institute of Animal Genetics (Edinburgh) for introducing me to the use of EM autoradiography     

INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER : An Electron Microscopic Study

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.     

Structural and functional dynamics of oogenesis in Glossina austeni: vitellogenesis with special reference to the follicular epithelium.

Morphological changes of the oocyte, follicle cells and nurse cells of the ovaries of the viviparous fly Glossina austeni during vitellogenesis and postvitellogenesis are outlined. During vitellogenesis, material is pinocytosed and incorporated into yolk spheres by subsequent fusions. Various lines of evidence are presented that indicate much of this material is derived from the follicular epithelium. The ultrastructure of the follicular cells throughout the 9 day cycle and their role in protein synthesis is presented. Subsequent to vitellogenesis, the follicle cells synthesize the secondary envelopes.     

Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.     

Oogenesis in a paedogenetic dipteran insect under normal conditions and after experimental elimination of the follicular epithelium

Summary Paedogenetically developing eggs of the gall midgeHeteropeza pygmaea are not deposited, but develop in the hemocoel of the mother larva. The nurse chamber remains present in the cleaving egg, and the follicular epithelium does not form a chorion but envelops the growing egg during embryonic development. It is possible to obtain naked eggs, i.e. eggs lacking the follicular epithelium, which are able to develop up to the blastoderm stage but remain spherical instead of assuming an elongated shape. Oogenesis of normal and naked eggs has been studied at the ultrastructural level with special reference to the nurse chamber. It is shown that the nurse chamber nuclei develop large nucleoli during oogenesis, indicating that the nurse chamber supplies the oocyte with ribosomal RNA (rRNA). The dense bodies in the nurse chamber may represent an intermediate stage in the transport of the rRNA from the nurse chamber to the oocyte; they are probably not related to the polar granules in the oocyte. It is also shown that the intercellular bridge joining the nurse chamber to the oocyte disappears shortly before cleavage initiation. During egg cleavage the follicular epithelium surrounds the nurse chamber, which degenerates and is gradually absorbed by the growing egg plasmodium. Naked cleaving eggs are never attached to a nurse chamber or to relics of it. Naked oocytenurse chamber complexes frequently aggregate, which may indicate a role of the follicular epithelium in follicle separation during normal development.     

chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis.

The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis. chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport. We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin. The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly. In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm. We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells.     

The origin and early differentiation of the egg chamber of Drosophila melanogaster

The egg chamber of Drosophila melanogaster consists of 16 interconnected cells surrounded by a monolayer of follicle cells. Each 16 cell cluster (from which the oocyte and 15 nurse cells differentiate) arises within the germarial region of an ovariole. To study the ultrastructure of the early stages in the formation and differentiation of egg chambers, a three dimensional reconstruction was made from serial thin sections through a germarium from a 24-hour old, virgin female. The germarium was found to be subdivided into three regions: (1) The mitotically active area where clusters of 16 cells originate from a series of cystocyte divisions, (2) the region where these cells interact with mesodermal cells, and (3) the region where the germarial cyst is transformed into the first egg chamber in the vitellarium. Since cystocytes were found to decrease in size with each division, the possibility exists that cell size may determine when the divisions cease. Models are presented which mimic with varying degrees of success the developmental changes the germarial cells undergo with time. Hypothesis are developed to explain why stem line oogonia are restricted to the anterior portion of the germarium, why mesodermal cells first interact with cystocytes in region 2, and why the oocyte is oriented posteriorly. The nuclear differentiations of the component cells of the chamber are described and correlated with observed differences in radiosensitivity. Symbionts were observed in the germaria of several strains of Drosophila, and the bearing of these findings upon nutritional studies is discussed.     

Morphogenesis of the micropylar apparatus in ovarian follicles of the fungus gnatBradysia tritici (syn.Sciara ocellaris)

Summary The ultrastructure and morphogenesis of the micropylar apparatus (MPA) have been studied in follicles of the fungus gnatBradysia tritici. The MPA is formed by a group of follicle cells located at the anterior pole of the single large nurse cell. In principle, the MPA consists of two thickened plates made of vitelline membrane material, the lower (LMP) and upper micropylar plate (UMP). The former is synthesized by 3 follicle cells, the latter by 4 different follicle cells. The micropylar channel system consists of a central channel with a single outer orifice and three branches which reach the plasma membrane of the oocyte. The branches are moulded by cellular extensions of the LMP-forming cells which are sandwiched between the two growing micropylar plates. Microtubuli and microfilaments were identified parallel to the long axis of the cellular extensions. At the time of MPA synthesis the nurse cell is still large and hence the MPA-forming cells have no contact to the oocyte. At the end of oogenesis when the regression of the nurse cell is completed, the MPA becomes connected to the other parts of the egg shell. At this time an ultrastructurally homogeneous region forms in the adjacent ooplasm (cytoplasmic cone). The possible relevance of these cytological observations for the control of development is discussed.