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.     

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     

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.     

Effects of Gamma-Radiation in Oogenesis of Drosophila Mutants Defective for Repair and Meiotic Recombination

Effects of mutations rad201, mei-9, and mei-41on cell sensitivity to gamma-radiation in Drosophilaoogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201 ^G1increased the frequency of DLs and egg chamber degeneration, mei-41 ^D5affected only the DL frequency, and mei-9 ^a, in addition to enhancing the chamber degeneration frequency, promoted radiation rescue of some oocytes from the DL induction.     

Direct evidence for aerial egg deposition in the burrows of the Malaysian mudskipper, <Emphasis Type="Italic">Periophthalmodon schlosseri</Emphasis>

The presence of mudskipper eggs in an air-filled chamber was confirmed by direct endoscopic observation of intact burrows of Periophthalmodon schlosseri in a mudflat in Penang, Malaysia. For all five burrows from which video images of egg chambers were successfully obtained, the presence of air was unequivocally demonstrated by the existence of an air–water interface inside the chambers. Of these burrows, eggs were found in two, but not in the others. Eggs were laid uniformly in a monolayer on the inner top surface of the chamber. The much brighter color of the surface mud of the egg chambers than the surrounding mud, irrespective of the presence or absence of the eggs, suggested that the surface mud had been oxidized by deposited air.     

Effects of elimination of Wolbachia on oogenesis of the uzifly Exorista sorbillans,a parasitoid of the silkworm Bombyx mori

Maternally inherited bacteria of the genus Wolbachia are known to affect the physiology and reproduction of several arthropod and nematode hosts. This paper presents the effects of using tetracycline to eliminate Wolbachia on the oogenesis of its host uzifly Exorista sorbillans (Diptera: Tachinidae), a parasitoid of silkworm Bombyx mori L. Earlier we reported cytoplasmic incompatibility, nucleo‐cytoplasmic conflict, male biased sex ratio and reduction in fecundity upon curing of Wolbachia infection using an antibiotic. This paper presents the abnormal growth and proliferation of oocytes in the uninfected/cured uziflies. The anomalies include abnormal/arrested oocyte production with inhibition of normal functioning of nurse cell nuclei, malformation and agglutination in egg chambers, empty egg chambers due to arrested vitellogenesis and undeveloped ovarian tubules. These anomalies further lead to reduction in growth of the ovarian tubules, which resulted in a 20% reduction of fecundity. It is hypothesized that normal oocyte production in uzifly requires the infection of Wolbachia bacteria. Further, in the uzifly, infection is at transition stage from a facultative to an obligatory mode of symbiosis. In light of this, this paper discusses the relationship between Wolbachia and oocyte production.     

Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

The effects of nurse eggs and sibling interactions on the larval development of the poecilogonous annelid Boccardia proboscidea (Spionidae)

In poecilogony, different types of larvae are produced within the same species. Previous studies have suggested maternal control of the production of larval types; however, it is not clear which factors or mechanisms generate contrasting developmental patterns among siblings. The spionid polychaete Boccardia proboscidea produces within the same capsule adelphophagic larvae that eat nurse eggs and siblings and complete all or most of their development inside the capsule (Type A larvae), and larvae with little growth until they hatch as planktotrophic larvae (Type B larvae). In this study, we manipulated capsule content to explore the factors determining larval type in B. proboscidea and the role of extra‐embryonic maternal nutrition and sib–sib interaction in the developmental fate of offspring. When early larval stages were grown individually in vitro, with nurse eggs as the only food source, some of them remained small, while others continue developing into larger pre‐competent larvae by feeding on nurse eggs. This suggests that larval types in B. proboscidea are determined very early in development and are not solely the product of sib–sib interaction inside the capsule. However, our data also suggest that hatching size variability within larval types of a clutch depends on nurse egg availability. Type B larvae grew normally to metamorphosis when phytoplankton was available, but suffered high rates of cannibalism by Type A larvae. These results are consistent with the hypothesis that individual larval fates are determined very early in development and that once their fate is determined, hatching size and intracapsular survival are affected by maternal food provisioning and sibling interaction.     

Development of ovarioles and nurse-cell cytoskeleton in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg (Diptera: Calliphoridae)

We have examined ovariole morphology and their development in Calliphora erythrocephala Mg. (Diptera: Calliphoridae). It has been shown that several regions can be distinguished in C. erythrocephala germarium. In these regions, cyst morphogenesis subsequently continues until the separation of the formed egg chamber. The germarium has variously branching fusomes. Two egg chambers develop in the C. erythrocephala vitellarium. The distribution of actin filaments and the formation of ring canals was studied in C. erythrocephala nurse cells. The diameter of the ring canal increases in the germarium zone from 1.5 μm to 14.7 μm during the third and fourth studies of oogenesis. A scheme is presented that illustrates the irregular stretching of actin rollers, which are formed during middle and late oogenesis around ring canals, which joins the proximal and neighboring parts and dramatically increases the size of nurse cells.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

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.     

Nest construction and larval behaviour of Bubas bison (L.) and Bubas bubalus (01.) (Coleoptera, Scarabaeidae)

Abstract.

• 1 Female beetles working alone or in cooperation with a male excavated vertical, tunnel-shaped brood chambers. Each chamber was filled with dung to form a cylindrical brood mass which contained two eggs, one near each pole.
• 2 To examine the possible relationship with other Onitini (which lay either one or several eggs per brood mass) factors that influence the two-egg programme were studied. Brood masses with only a single egg were formed if excavation was resumed prematurely. Conversely, when excavation was suppressed several oviposition programmes fused to produce a multi-egg brood mass.
• 3 The larvae repaired their chambers in the typical Scarabaeine manner by building a self-supporting wall formed from their own excrement. This behaviour also prevented direct contact and fighting between adjacent larvae in the same brood mass, and it allowed the larvae to survive inside artificial brood balls. Similar behaviour was observed in larvae of Onthophagus taunts and Ontho-phagus vacca (which develop in one-egg brood masses). The evolution of nesting habits that involve multi-egg brood masses or free-standing brood balls may depend on the pre-existence of this larval repair behaviour.

     

Vertical transmission of cyanobacterial symbionts in the marine sponge Chondrilla australiensis (Demospongiae)

The cyanobacterial symbionts of the marine sponge Chondrilla australiensis (Demospongiae) were examined using fluorescent microscopy and Transmission Electron Microscopy. Unicellular cyanobacteria with ultrastructure resembling Aphanocapsa feldmannii occur in the cortex and bacterial symbionts are located throughout the mesohyl. In C. australiensis, the developing eggs are distributed throughout the mesohyl and are surrounded by nurse cells attached to them by thin filaments. The nurse cells form cytoplasmic bridges with the eggs, apparently releasing their contents into the egg cytoplasm. The presence of cyanobacterial and bacterial symbionts inside developing eggs and nurse cells in 25% of female Chondrilla australiensiswas established using Transmission Electron Microscopy, suggesting that these symbionts are sometimes passed on to the next generation of sponges via the eggs.     

Dicephalic — ADrosophila mutant affecting polarity in follicle organization and embryonic patterning

Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.     

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     

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.     

Morphology of skin incubation in Pipa carvalhoi (Anura: Pipidae)

South American Pipidae show a unique reproductive mode, in which the fertilized eggs develop in temporarily formed brood chambers of the dorsal skin after eggs have been deposited on the back of the female. We studied the skin incubation of Pipa carvalhoi using light microscopy and scanning electron microscopy. The skin consists of a stratified epithelium with a one‐layered stratum corneum, and the dermis. The dermis of the dorsal skin of nonreproductive and reproductive females lacks a distinct stratum compactum, which is typical for most anuran skins. The entire dermis shows irregularly arranged collagen bundles like a stratum spongiosum. Before egg laying, the skin swells, primarily by thickening and further by loosening of the middle zone of the dermis. In the epidermis, large furrows develop that are the prospective sites of egg nidation. The epidermis, which forms a brood chamber around the developing egg becomes bi‐layered and very thin and lacks a stratum corneum. Further, the dermis loosens and becomes heavily vascularized. Egg carrying females do not have mature oocytes in their ovaries indicating a slow down or interruption of egg maturation during this period. Similarities with the brood pouch of marsupial frogs are discussed. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Effect of gamma-irradiation on oogenesis of Drosophila mutants defective for reparation and meiotic recombination

Effects of mutations rad201, mei-9, and mei-41 on cell sensitivity to gamma-radiation in Drosophila oogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201G1 increased the frequency of DLs and egg chamber degeneration, mei-41D5 affected only the DL frequency, and mei-9a, in addition to enhancing the chamber degeneration frequency, promoted radiation "rescue" of some oocytes from the DL induction.     

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.