Dieter Zissler

Oogenesis in the honeybee Apis mellifera: cytological observations on the formation and differentiation of previtellogenic ovarian follicles

SummaryOogenesis is known to be important for embryonic pattern formation. For this reason we have studied the early differentiation of the honeybee ovariole histologically, ultrastructurally, and by staining F-actin with rhodaminyl-phalloidin. At the anterior tip of the ovariole, stem cells are lined up in a single file; they are organelle-poor but contain characteristic electrondense bodies with lysosomal properties. The presence of these bodies in cystocytes as well as prefollicle cells indicates that both cell types may be derived from the apical stem cells. During later stages of oogenesis, the follicle cells differentiate cytologically in different regions of the follicle. The organization of the intercellular bridges between cystocytes derived from a single cystoblast has been studied in detail. The polyfusomes in the intercellular bridges of cystocyte clusters stain with rhodaminyl-phalloidin and hence contain F-actin. Later, when the polyfusomes begin to desintegrate, F-actin rings form which line the rims of the intercellular bridges. Actin might be recruited from conspicuous F-actin stores which were detected in the germ-line cells. The F-actin rings are dissembled some time before the onset of vitellogenesis when the nurse chamber has grown to a length of about 200 μm. At the basal side of the follicle cells (close to the basement membrane facing the haemocdele) parallel microfilament bundles encircle the ovariole. The microfilament bundles which are oriented mostly perpendicular to the long axis of the ovariole were first observed around the zone where the cystocyte divisions occur; after this phase the micro-filament bundles become organized differently in the follicle cells associated with the nurse cells and in the follicular epithelium of the oocyte.

The Cytoplasmic Architecture of the Insect Egg Cell

Investigations on pterygote insect oogenesis have yielded a multitude of data on the architecture and development of early and late oocytes (for reviews see King, R. C., 1970; Telfer and Smith, 1970; Mahowald, 1972; Anderson, 1974; Telfer, 1975; see also Miya et al., 1969; Truckenbrodt, 1970; Buning, 1972). Oogenesis has also been studied in some Apterygota (Cone and Scalzi, 1967; Cantacuzene and Martoja, 1972; Palevody, 1972, 1973; Matsuzaki, 1973; Bilinsky, 1976, 1977, 1979; Klag, 1977, 1978). Yet the end product of oogenesis was usually exempted from investigation; the newly laid insect egg thus remains largely unknown with respect to ultrastructure. This may mainly be due to technical reasons: the prevalence of yolk and the extremely resistant egg covers of insect eggs provide considerable handicaps for fixation and sectioning. Descriptions of the egg shell are therefore much more frequent than publications dealing with the egg cell.

A morphogenetic determinant in the anterior pole of an insect egg (Smittia spec., Chironomidae, Diptera): Localization by combined centrifugation and ultraviolet irradiation

Abstract In chironomid midges, the development of the head and thorax in the embryo requires the function of cytoplasmic determinants localized near the anterior pole of the egg. Experimental inactivation of these determinants causes a dramatic switch in the developmental program of the embryo. Instead of the normal segment pattern, the aberrant pattern “double abdomen” is formed. Head, thorax, and anterior abdominal segments are then replaced by an additional set of posterior abdominal segments jointed with reversed polarity to the original abdomen. To determine the cellular fraction which contains the effective targets for uv induction of double abdomens, Smittia eggs were centrifuged prior to uv irradiation. Accumulation of proteid spheres or lipid droplets in the irradiated anterior pole region caused a considerable decrease in the double abdomen yield. Removal of these components from the target area enhanced double abdomen formation. The maximum yield of double abdomens was obtained after uv irradiation of a cytoplasmic layer in which organelles larger than ribosomes could not be detected. The results of these and other experiments, suggest that ribosomes, ribosomal subunits, or other ribonucleoprotein particles act as effective targets for the uv induction of double abdomens in Smittia eggs.

The cytoplasmic architecture of the egg cell ofSmittia spec. (Diptera, Chironomidae)

Summary1.The egg of the Chironomid midgeSmittia spec. has been studied by light and electron microscopy. The present paper describes the fine structure of the anterior and posterior pole regions before pole cell formation. These regions were selected because of their functional involvement in body pattern determination and pole cell formation.2.In the anterior cytoplasm (region I), 3 subregions can be recognized. A thin outer layer (Ia) which resembles the more equatorial periplasm (region II) but contains fewer organelles, covers a layer rich in mitochondria (Ib). This in turn borders a central cone of cytoplasm (Ic) which protrudes into the anterior face of the yolk endoplasm and frequently contains a cytaster-like structure but no chromatin.3.The posterior cytoplasm (region III) includes a germ plasm or oosome similar to the type found in other lower dipterans. It is lens-shaped and contains a 3-dimensional network of electron-dense material. This material is probably granular, but may appear fibrous due to the spatial arrangement of the granules.4.A series of organelles of multivesicular or lysosome-like appearance is described. These may be involved in the formation or utilization of proteid yolk.5.Special structures or organelles restricted to the anterior pole region were not found. This might indicate that the role of this region in the switch from head formation to tail formation after UV irradiation could be due rather to differences in quantity or arrangement of ubiquitous structures than to qualitative differences between this and other egg regions. However, qualitative singularities cannot be excluded. They are obvious in the posterior pole region which contains the oosome.Zusammenfassung1.Das Ei der ZuckmückeSmittia wurde licht-und elektronenmikroskopisch untersucht. Die vorliegende Arbeit beschreibt den Bau von Vorder-und Hinterpolregion vor Bildung der Polzellen. Diese Regionen wurden wegen ihrer Bedeutung für die Bildung des Segmentmusters und der Polzellen ausgewählt.2.Das vordere Zytoplasma (Region I) kann in 3 Subregionen gegliedert werden. Eine dünne Außenschicht (Ia), die dem äquatorialen Periplasma (Region II) ähnelt, aber weniger Organellen enthält, bedeckt eine Schicht mit vielen Mitochondrien (Ib). An diese schließt sich ein zentraler Zytoplasmazapfen an (Ic), der von vorn in das Dotter-Endoplasmasystem hineinragt und häufig einen Cytaster ohne Chromatin enthält.3.Das hintere Zytoplasma (Region III) enthält ein Keimplasma oder Oosom des Typs, der von anderen niederen Dipteren bekannt ist. Es ist linsenförmig und wird von einem 3-di-mensionalen Netzwerk aus elektronendichter Substanz durchzogen. Dieses Material ist vermutlich granulär, kann aber infolge der Anordnung der Grana fibrillär erscheinen.4.Es wird eine Serie von multivesiculären und lysosomen-artigen Organellen beschrieben, die möglicherweise mit dem Aufoder Abbau des Proteiddotters verknüpft sind.5.Besondere, auf den Vorderpolbereich beschränkte Strukturen oder Organellen wurden nicht gefunden. Dies könnte bedeuten, daß die Rolle dieser Region bei der UV-induzierten Umsteuerung von Kopf zu Schwanzbildung eher auf Unterschieden in Menge oder Anordnung normaler Zellstrukturen beruht, als auf qualitativen Unterschieden zwischen dem Vorderpolbereich und den übrigen Eiregionen. Qualitative Besonderheiten des Vorderpolbereichs sind jedoch nicht auszuschließen. Der Hinterpolbereich enthält als solche Besonderheit das Oosom.

Switch in pattern formation after puncturing the anterior pole of Smittia eggs (Chironomidae, Diptera)☆

Abstract The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region.

Mitochondria and polarity in the egg ofSmittia spec. (Diptera, Chironomidae): UV irradiation, respiration measurements, ATP determination and application of inhibitors

Summary1)The development of the head end in the embryo ofSmittia is determined by cytoplasmic components in the anterior region of the egg. Experimental inactivation or removal of these determinants results in the formation of “double abdomens”. In embryos with this aberrant metameric pattern, head and anterior segments are replaced by an additional set of posterior segments joined in mirror image symmetry to the original abdomen. Several types of experiment were carried out to determine whether mitochondria act as determinants of the anterior pole in the embryo.2)The following observations are compatible with a determinative rôle of mitochondria: mitochondria are more abundant in the anterior tip of theSmittia egg which responds most effectively in experiments inducing double abdomens by local UV irradiation. UV irradiation inflicts visible damage upon mitochondria in the irradiated egg region, and significantly decreases the oxygen consumption of the eggs.3)The following data argue against a determinative rôle of mitochondria: photoreverting treatment after UV irradiation did not cause a significant increase of oxygen uptake. The ATP content of whole eggs and anterior egg halves was significantly increased after UV irradiation of the anterior egg quarter. Localized application of dinitrophenol and ethidium bromide via puncture of the anterior pole, as well as irradiation with intense red light after sensitization of mitochondria with Janus Green B, did not produce double abdomens.4)After UV irradiation during intravitelline cleavage, photoreverting treatment can be delayed for several hours without diminishing its efficiency; the deadline for photoreversal coincides perfectly with the deadline for UV induction, i.e. with blastoderm formation. This result suggests that the effective targets are metabolically inactive from egg-laying until blastoderm-formation.5)The results are considered as evidence that mitochondria and the determinants of anterior pole in theSmittia embryo are not identical despite their apparently similar cellular localization.Zusammenfassung1)In der Embryogenese vonSmittia wird die Entwicklung des Kopfendes durch cytoplasmatische Komponenten in der Vorderpolregion des Eies determiniert. Experimentelle Inaktiveirung oder Entfernung dieser Determinanten bewirkt die Entstehung des aberranten Segmentmusters “Doppelabdomen”. In diesem spiegelbildlich symmetrischen Segmentmuster sind Kopf, Thorax und vordere Abdominalsegmente durch ein zweites Abdominalende mit revertierter Polarität ersetzt. In Versuchen mit verschiedenen methodischen Ansätzen wurde geprüft, ob Mitochondrien als Determinanten des embryonalen Vorderpols fungieren.2)Die folgenden Ergebnisse sind vereinbar mit der Vorstellung, daß mitochondriale Funktionen die Längsachsenpolarität imSmittia Ei determinieren: Mit größter Effizienz lassen sich Doppelabdomina durch Bestrahlung der vordersten Eiregion erzeugen; diese Region zeigt zugleich eine starke Anreicherung von Mitochondrien. Die UV-Bestrahlung verursacht sichtbare Schäden an Mitochondrien der exponierten Eiregion und eine signifikante Abnahme der Atmungsrate.3)Die folgende Ergebnisse stehen im Widerspruch zur Annahme einer Determination der Längsachsenpolarität durch mitochondriale Funktionen: Photoreversion nach UV-Bestrahlung bewirkt keine signifikante Zunahme der Respirationsrate. Der ATP-Gehalt ganzer Eier und vorderer Eihälften wird durch Bestrahlung des vorderen Eiviertels signifikant erhöht. Durch Anstechen der Eier am Vorderpol und anschließende Inkubation in Dinitrophenol oder Ethidiumbromid lassen sich keine Doppelabdomina erzeugen; Bestrahlung mit intensivem Rotlicht nach Sensibilisierung mit Janus Grün B bewirkt ebenfalls keine Bildung von Doppelabdomina.4)Nach UV-Bestrahlung in frühen Entwicklungsstadien kann die photorevertierende Belichtung um mehrere Stunden verzögert werden, ohne den Photoreversionseffekt signifikant zu beeinträchtigen. Die sensiblen Phasen für UV-Induktion und Photoreversionen dauern beide vollkommen übereinstimmend bis zur Blastodermbildung. Demnach scheinen die wirksamen Eikomponenten während dieser Zeit metabolisch inaktiv zu sein.5)Nach diesen Ergebnissen wird es als äußerst unwahrscheinlich angesehen, daß die Vorn-Hinten-Polarität im Ei vonSmittia durch mitochondriale Funktionen determiniert wird.

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

SummaryThe 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.

Fortpflanzung und Entwicklung

Insekten pflanzen sich fast ausschlieslich geschlechtlich fort. Geschlechtliche Fortpflanzung ist an besonders differenzierte Geschlechtszellen, Gonocyten, gebunden. Im Allgemeinen ist die Fortpflanzung der Insekten zweigeschlechtlich, amphigon: Eine weibliche, eine Eizelle (Ovum) wird von einer mannlichen, einer Samenzelle (Spermium, Spermatozoon) befruchtet. Die Befruchtung ist ein Zweistufenprozess: Dem Eindringen des Spermiums in die Eizelle, der Syngamie, folgt die Vereinigung des weiblichen Vorkerns mit dem mannlichen, die Karyogamie (s. 13.2.1.1). Seltener vollzieht sich die Fortpflanzung eingeschlechtlich, indem sich eine Eizelle ohne Befruchtung entwickelt (Parthenogenese, s. 13.1.2.3).