Uncoupling of growth and cytological development in egg follicles ofHeteropeza pygmaea (Dipt., Cecidomyiidae)

Summary Oogenesis of egg follicles in paedogenetic reproduction of the gall midgeHeteropeza pygmaea is accompanied by a relatively slight growth of the oocyte. Egg growth takes place mainly during embryonic development. The nurse chamber in the egg follicle degenerates only at the beginning of embryogenesis. When ovaries of the female larvae are cultured in vitro under male-determining conditions, the ovaries produce mostly male-determined egg follicles. These follicles show nurse chamber degeneration and they grow to about the size of an egg in late cleavage division or blastoderm stage developing in situ, but cytological development invariably stops at the first or second meiotic division. Thus, growth and cytological development in such follicles are uncoupled. The presence of a meiotic block in paedogenetically developing follicles ofH. pygmaea gives a clue to the mode of evolution of paedogenetic reproduction.Dedicated to Professor Dr. Hans Bauer on the occasion of his 75th birthday     

Composite eggs in non-paedogenetic gall midges (Cecidomyiidae,Diptera)

Summary A new type of composite eggs was found in the non-paedogenetic gall midgesMikiola fagi andRhabdophaga rosaria. Composite eggs of this type contained two or three nurse chambers and one egg chamber with one oocyte nucleus. In all composite eggs examined only one nurse chamber developed normally, while the others, regardless of their number and position within a composite egg, were arrested in their growth. It is assumed that the arrested nurse chambers, contrary to normally developing ones, are deficient in generative nuclei and thus are derived exclusively from mesodermal cells.This work was supported in part under Contract DPKBN/52/76-II.1.3.10, with the Polish Academy of Sciences     

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.     

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.     

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     

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.     

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

 In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process. Received: 12 December 1997 / Accepted: 6 January 1998     

Phantom,a cytochrome P450 enzyme essential for ecdysone biosynthesis,plays a critical role in the control of border cell migration in Drosophila

The border cells of Drosophila are a model system for coordinated cell migration. Ecdysone signaling has been shown to act as the timing signal to initiate the migration process. Here we find that mutations in phantom (phm), encoding an enzyme in the ecdysone biosynthesis pathway, block border cell migration when the entire follicular epithelium of an egg chamber is mutant, even when the associated germline cells (nurse cells and oocyte) are wild-type. Conversely, mutant germline cells survive and do not affect border cell migration, as long as the surrounding follicle cells are wild-type. Interestingly, even small patches of wild-type follicle cells in a mosaic epithelium are sufficient to allow the production of above-threshold levels of ecdysone to promote border cell migration. The same phenotype is observed with mutations in shade (shd), encoding the last enzyme in the pathway that converts ecdysone to the active 20-hydroxyecdysone. Administration of high 20-hydroxyecdysone titers in the medium can also rescue the border cell migration phenotype in cultured egg chambers with an entirely phm mutant follicular epithelium. These results indicate that in normal oogenesis, the follicle cell epithelium of each individual egg chamber must supply sufficient ecdysone precursors, leading ultimately to high enough levels of mature 20-hydroxyecdysone to the border cells to initiate their migration. Neither the germline, nor the neighboring egg chambers, nor the surrounding hemolymph appear to provide threshold amounts of 20-hydroxyecdysone to do so. This “egg chamber autonomous” ecdysone synthesis constitutes a useful way to regulate the individual maturation of the asynchronous egg chambers present in the Drosophila ovary.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

F-actin distribution in the ovaries of pre-vitellogenic and vitellogenic black blowflies,Phormia regina (Meigen) (Diptera : Calliphoridae)

The spatial distribution of F-actin microfilaments in the ovaries of previtellogenic and vitellogenic female black blowflies, Phormia regina (Diptera : Calliphoridae), as the females shift from a sugar to a liver diet, is determined using rhodamine-labelled phalloidin (rh-phalloidin). During the pre-vitellogenic stages of ovarian development (i.e. corresponding to a sugar diet) a single bright fluorescent layer marks the interface between follicle cells and the oocyte. Fluorescence is also most evident at the inner surface of the ring canals of the nurse cells. This is observed in the nurse cells both in the distal part of the germarium, and in the vitellogenic growing oocyte. However, when liver-fed (i.e. necessary for vitellogenesis), 2 bright fluorescent layers are observed at the follicle cell-oocyte interface. In addition, the cytoplasm of the nurse cells during vitellogenesis appears full of fluorescent microfilaments and the actin rings are found to increase in size and thickness. The changing organization of the F-actin microfilaments in the follicles during the process of both egg chamber and oocyte formation is discussed and possible functions considered.     

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee . We named the allele chickadee^bin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadee^{wc57 and WF57} (2). In addition, the egg chamber of chickadee^bin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.     

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.     

The cytology of the vitellogenic stages of oogenesis in Drosophila melanogaster. I. General staging characteristics

The cytology of the vitellogenic stages in the development of the oocyte of Drosophila melanogaster has been studied using whole mounts and sections of plastic-embedded ovaries and single egg chambers for light microscopy and cytochemistry. The migrations, changes in morphology, and synthetic products of the follicle cells are described as a function of developmental stage. The follicle cells synthesize the egg coverings, the vitelline and chorionic membranes, and elaborate the micropyle and dorsal chorionic appendages. The changing structure of the nurse cell nucleus and changes in organelle composition of its cytoplasm are described. The nurse cells synthesize ribosomes, lipid droplets, and mitochondria. These components pass through the ring canal system into the oocyte, which increases in volume some 200,000 times during its 78 hours of development.     

The rôle of juvenile hormone in housefly ovarian follicle morphogenesis

Oögenesis in the housefly, Musca domestica, was divided into a series of 10 stages where stage 1 was the germarium, stage 4 was the beginning of yolk deposition, stage 7 was characterized by maximal nurse cell development, stage 9 by the degeneration of the nurse cells and chorion formation, and stage 10 was the mature egg. It required 69 hr from eclosion at 27°C to develop mature eggs. This represented an oöcyte volume increase of 3700-fold, a seventeenfold increase in follicle length, and a sevenfold increase in weight. The application of 2 μg of isopropyl (E,E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate (ZR-515) to allatectomized (-CA) flies stimulated egg development, which progressed at the same rate as the controls. The -CA flies did not develop eggs past stage 4, which represented a cessation of development at a volume of 1·4 per cent that of a mature egg and an ovarian dry weight of 11 per cent that of a mature ovary. The follicle cells from -CA flies did not differentiate into the squamous condition over the nurse chamber, did not become columnar over the oöcyte, did not produce the chorion or vitelline membrane, and did not decrease in number as they did on the stage 10 follicles. Endomitosis in the nurse cell nuclei of -CA flies stopped development at 290 c, but maximum development of 2400 c occurred in stage 7 follicles from controls, and then the nurse cells began to disintegrate.     

Peculiarities of the organization of egg chambers in carabid ground beetles and their phylogenetic implications

The ovaries of 31 species of the coleopteran familyCarabidae have been studied by light and electron microscopy. Ovarioles of all the examined insects are of the polytrophic type. In the majority of the species a constant number of nurse cells per egg chamber has been observed. However, several species do not obey the 2(n) rule and the number of nurse cells varies considerably even in the consecutive egg chambers of the same ovariole. In spite of the differences, the number of intercellular bridges connecting nurse cells to the oocyte is fixed and species specific. InCarabidae seven types of egg chambers have been characterized regarding the number of divisions, the number of nurse cells and the way the nurse cells are bridged to the oocyte. Some phylogenetic implications are considered.     

Structure of ovarioles in adult queens and workers of the common wasp, Vespula germanica (Hymenoptera: Vespidae)

The ovaries of the common wasp, Vespula germanica are polytrophic-meroistic and consist of 2-3 (workers) or 7 (queens) ovarioles. The ovarioles are differentiated into three regions: a terminal filament, a germarium, and a vitellarium. The germaria of both castes consist of two zones: an anterior zone of germ-cell cluster formation and a posterior one of germ-cell cluster differentiation. The vitellaria comprise 4-6 (workers) or 7-10 (queens) ovarian follicles (egg chambers). Each chamber consists of an oocyte and about 60 isodiametric nurse cells (trophocytes). The egg chambers have been arbitrarily classified into four developmental categories: early and late previtellogenic, vitellogenic, and choriogenic. The process of oogenesis in workers proceeds only up to the onset of the late previtellogenesis. Neither vitellogenic nor choriogenic egg chambers were observed in this caste. During early and late previtellogenesis the envelope of the oocyte nucleus proliferates and becomes highly folded. This process leads to the formation of characteristic organelles, termed accessory nuclei (AN). Although AN arise in the oocytes of both queens and workers, their number in the latter caste is always considerably lower. At the onset of the late previtellogenesis AN start to migrate towards the periphery of the oocyte where they reside till the end of oogenesis. The physiological state of the worker ovaries is discussed in the light of the presented results.     

Observations on the polarity of mutant Drosophila follicles lacking the oocyte

Summary Homozygous females of the mutantsegalitarian andBicaudal-D ^R26produce follicles in which the oocyte is replaced by an additional nurse cell. Normal morphological markers for polarity can be identified in mutant follicles but the normal spatial organization of these markers is disturbed. For example, nurse-cell nuclei of different ploidy classes are present but, contrary to wild-type follicles, the nuclei show no anteroposterior ploidy gradient. The two cells with four intercellular bridges, one of which should have developed into the oocyte rather than a nurse cell, are located at the posterior pole only in young follicles (up to about stage 5), whereas during later stages they are more often found at lateral or intermediate positions. This disturbed polarity correlates with a variable aberrant pattern of extracellular ionic currents. Moreover, in the mutant follicles patches of columnar follicular epithelium differentiate locally although this type of epithelium forms normally only around the oocyte. The follicle cells at both follicle poles possess anterior quality since they migrate from both poles towards the centre of the follicle, as do the border cells restricted to the anterior pole in wild-type follicles. Our analysis indicates that in the mutants the follicular polarity is normal at first but becomes disturbed during stages 5 to 6. The secondary breakdown of polarity is likely to follow on from the absence of the oocyte.     

A stereological analysis of developing egg chambers in the honeybee queen,Apis mellifera

Summary A polytrophic ovariole of the queen honeybee, Apis mellifera, is composed of a linear series of increasingly mature egg chambers, each consisting of an oocyte, an interconnected cluster of nurse cells, and a covering layer of follicle cells. This study describes changes in the volume of each of these components, as a function of the position of the egg chamber in the ovariole. An oocyte increases in volume from approximately 8.9 × 10³ m³ to approximately 9.6 × 10⁶ m³ over an average series of 20 egg chambers.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.