Carlo Taddei

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

Abstract 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.

The membrane-bound respiratory chain of Pseudomonas pseudoalcaligenes KF707 cells grown in the presence or absence of potassium tellurite

The respiratory chain of Pseudomonas pseudoalcaligenes KF707 in membranes isolated from cells grown in the presence or absence of the toxic oxyanion tellurite (TeO3(2-)) was examined. Aerobic growth in the absence of tellurite shows an NADH-dependent respiration which is 80% catalysed by the cytochrome (cyt) bc1-containing pathway leading to two terminal membrane-bound cyt c oxidases inhibited by different concentrations of KCN (IC50 0.2 and 1 microM). A third oxidase, catalysing the remaining 20% of the cyanide-resistant respiration and fully inhibited by 2-3 mM KCN, is also present; this latter pathway accounts for 60-70% of the total NADH-dependent respiration in membranes from cells grown in LB medium supplemented with potassium tellurite (35 microg x ml(-1)). Two high-potential b-type haems (E(m,7) +395 and 318 mV) are redox centres of a membrane-bound cyt c oxidase (possibly of the cbb3 type) which shows a 50% decrease of its activity in parallel with a similar decrease of the c-type haem content (mostly soluble cyt c) in membranes from tellurite-grown cells; the latter type of cells specifically contain a cyt b type at +203 mV (pH 9.0) which is likely to be involved in cyanide-resistant respiration. Comparison of the growth curve of KF707 cells in parallel with tellurite uptake showed that intracellular accumulation of tellurium (Te(0)) crystallites starts from the mid-exponential growth phase, whereas tellurite-induced changes of the respiratory chain are already evident during the early stages of growth. These data were interpreted as showing that reduction of tellurite to tellurium and tellurite-dependent modifications of the respiratory chain are unrelated processes in P. pseudoalcaligenes KF707.

Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizard Podarcis muralis

Using immunocytochemistry at light‐ and electron‐microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian α‐keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and β‐layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized β‐ and α‐layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the β‐layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron‐dense areas within the β‐layer, suggesting retention of α‐keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40–41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77–78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed. J. Morphol. 246:179–191, 2000.

Drosophila vitelline membrane cross-linking requires the fs(1)Nasrat, fs(1)polehole and chorion genes activities

Abstract. During the final step of Drosophila vitelline membrane formation, the structural proteins composing this layer become cross-linked by covalent bonds. In the present report, we analyzed the vitelline membrane cross-linking in mutants having defects either in this layer or in the chorionic layers. In the fs(1)Nasrat and fs(1)polehole mutant alleles conferring defects in vitelline membrane formation, disruption of vitelline membrane cross-linking was observed, indicating the involvement of these two genes in the process. On the contrary, in the fs(1)Nasrat and fs(1)polehole alleles showing defects only at the termini of the embryo the vitelline membrane is properly formed, confirming a multifunctional activity of their gene products. Altered vitelline membrane cross-linking was also detected in a mutant of the chorion protein gene Cp36 and in the chorion amplification mutant fs(1)K1214, suggesting a role of the structural components of chorion layers in the process of vitelline membrane hardening.

Structural modifications of the nuclear components during lizard oogenesis in relation to the differentiation of the follicular epithelium.

This paper deals with an electron microscope study of nucleolar ultrastructural modifications that occur in the oocytes of the lizard Podarcis sicula during ovarian follicle differentiation. In small diplotene oocytes around which a monolayered follicular epithelium forms, the nucleolus appears as a fibrillo-granular structure. Afterwards, simultaneously with the beginning of pyriform cell differentiation inside the granulosa, the nucleolus progressively condenses and breaks into fragments, forming dense spherical bodies. In larger follicles, with well differentiated pyriform cells, a typical nucleolus is no longer detectable in the oocyte nucleus. These ultrastructural modifications suggest a possible impairment of the oocyte nucleolus in ribosome organization. A possible involvement of pyriform cells in supplying ribosomes to the growing oocyte is discussed.

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard Podarcis sicula

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.

The germarium of panoistic ovarioles of Bacillus rossius (Insecta Phasmatodea): Larval differentiation

Summary In newly hatched larvae of Bacillus rossius (Insecta Phasmatodea) viewed with light and electron microscopes, ovarioles appear as bag-like structures; during the first instar they initially assume a bell-shaped appearance. Afterwards, they begin to elongate and their vitellarium essentially consists of a row of oocytes with a clear growth gradient. This typical ovariole morphology becomes more evident in the subsequent instars up to the fourth. The germarium appears as a region interposed between growing oocytes of the vitellarium and somatic cells of the terminal filament. Light and electron microscope observations indicate that the structure of the germarium does not markedly change during the different instars: it contains, besides somatic prefollicular cells, germ elements arrested in the “diffuse” stage which precedes diplotene of growing oocytes. Only in newly hatched larvae, squash preparations of the germarium also show germ cells in earlier meiotic prophase (zygo-pachytene). The observati...

The germarium of panoistic ovarioles of Bacillus rossius (Insecta Phasmatodea): Structure and function during imaginal life

Summary In adult females of Bacillus rossius (Insecta Phasmatodea) the germarium, localized at the ovariole tip just below the terminal filament and above the vitellarium, progressively reduces in size and eventually disappears at the end of the ovulatory period. The observations with light and electron microscopes show that in the end-chamber most germ cells are arrested in a post-pachytenic diffuse stage, which just precedes diplotenic oocyte growth. These observations also indicate that the reduction in size of the germarium of ovulating females should probably be ascribed to a progressive and extensive activation of the resting germ cells. The average number of ovulated eggs per ovariole (6.7±0.9) is consistent with this view. However, occasional findings of lepto-zygotenic germ cells in some preovulatory ovarioles of adult females do not completely rule out the persistence of scarce undifferentiated germ elements (oogonia) in the larval germarium at the onset of adult life. Furthermore, the reduction...

Ribosomal bodies and annulate lamellae in the oocytes of the lizardPodarcis sicula

SummaryUltrastructural studies suggest that, in the oocytes of the lizardPodarcis sicula, ribosomal bodies are structurally continuous with annulate lamellae during their organization and disaggregation. This observation may indicate the dynamic transformation of the cytomembranes of one structure into those of the other, and vice versa. Moreover, the presence of annulate lamellae has been detected for the first time in lizard oocytes. The hypothesis is advanced that ribosomal bodies and annulate lamellae, in spite of some different structural characteristics, may play a similar role during the oocyte growth.

Microtubule organization and nucleation in the differentiating ovarian follicle of the lizard Podarcis sicula.

We analyzed the organization of the microtubular cytoskeleton and the distribution of centrosomes at the different stages of differentiation of the ovarian follicle of the lizard Podarcis sicula by examining immunolabeled α‐ and γ‐tubulins using confocal microscopy. We observed that in the follicular epithelium the differentiation of the nurse pyriform cells is accompanied by a reorganization of the microtubules in the oocyte cortex, changing from a reticular to a radial pattern. Furthermore, these cortical microtubules extend in the cytoplasm of the connected follicle cells through intercellular bridges. Radially oriented microtubules were still more marked in the oocyte cortex during the final stages of oogenesis, when the yolk proteins were incorporated by endocytosis. The nucleation centres of the microtubules (centrosomes) were clearly detectable as γ‐tubulin immunolabeled spots in the somatic stromal cells of the germinal bed. A diffuse cytoplasmic immunolabeling together with multiple labeled foci, resembling the desegregation of the centrosomes in early oogenesis of vertebrates and invertebrates, was revealed in the prediplotenic germ cells. In the cytoplasm of growing oocytes, a diffuse labeling of the γ‐tubulin antibody was always detectable. In the growing ovarian follicles, immunolabeled spots were detected in the mono‐layered follicle cells which surrounded the early oocytes. In follicles with a polymorphic follicular epithelium, only the small follicle cells showed labeled spots. A weak and diffuse labeling was observed in the pyriform cells while in the enlarging intermediate cells the centrosomes degenerated like in the early oocytes. Our observations confirm that in P. sicula most of the oocyte growth is supported by the structural and functional integration of the developing oocyte with the pyriform nurse cells and suggest that their fusion with the oocyte results in an acquirement by these somatic cells of characteristics typical of the germ cells. J. Morphol. 2012.