Library

Notes: Summary In the eggs ofPimpla turionellae, which are characterized by a long germ anlage (“long-germ egg” type), the cleavage nuclei primarily populate the anterior part and only later appear in the posterior of the egg lumen during the intravitelline cleavage. Gastrulation and segmentation also start within this anterior region. Time-lapse motion pictures served to observe and to check quantitatively even slow movements during cleavage and blastogenesis. In motion diagrams made by means of microkymographic technics the flow within the ooplasm along the longer axis of the egg has been timed. Shortly before the first cleavage in thestrictly unfertilized male eggs a short-time“unipolar flow” sets in from a primary initial region at 90% of their length. Thus a pillar of “central plasm” between both of the poles becomes shifted towards the posterior, while its outer coating layer of “marginal-plasm” is displaced forwards by the same distance. In eggs from fertilized females two successive flows of the same “unipolar” type have been observed. At the end of the third cleavage the energids, heretofore loosely grouped together, become distributed within the central plasm to form a “nuclear column”. At the same time a fluently pulsatory “bipolar flow” sets in, within asecondary initial region at 80% of the egg length. Comparable to two mirror-image fountains, parts of the central plasm are carried towards the front pole and to the rear pole of the egg, respectively, while the marginal plasm, together with the oolemma, flows in opposite directions at times. With each pulsation the moving areas of the bipolar flow are shifted more and more towards the egg poles. The occurrence of bipolar flow pulsations, amounting to five, is correlated with the nuclear divisions in a still unknown way. In the rhythm of the bipolar flow, the energids become dispersed within the central plasm with a certain spatial lagging. After the bipolar flow has come to a halt, four further cleavages are indicated by faint local pulsations of the ooplasm. The cleavage nuclei move to the egg surface and pole cells become separatedtied off During blastoderm formation another four faint pulsations are observed, especially within the central ooplasm, all of them clearly synchronized with superficial cleavages. Occurring in mitotic waves, these cleavages indicate a third initial region, with the individual position varying between 10 and 28% of the egg length. Furthermore the technics of time-lapse motion pictures permit a local and temporal determination of extravitelline pole space formation, of a ring-shaped contracted region of slightly thickening periplasm within the secondary initial region, and the dislocation of the oosome towards the egg surface, which results from the activity of the posterior fountain during the phase of bipolar flow. Invagination and segmentation of the embryo become distinct within the secondary initial region, thus identifying this region as a differentiation centre. The correlation of plasm flow and nuclear divisions is discussed as well as the correlation of the initial regions to the different patterns of egg architecture in the longgerm egg type. The correlation between bipolar pulsations and the development of the metameric pattern including the function of the oosomal region is also discussed. The ooplasmic movements as known from egg types other thanPimpla are compared to the above observations.

Notes: Summary Time-lapse photomicrographs of eggs from the Ichneumonid waspPimpla and from the gall midgeWachtliella reveal that during fixation the ooplasm performs excessive streaming movements when conventional fluids such as Bouin's or Dubosq-Brasil's are used. InWachtliella, intravitelline cleavage may continue for two mitotic cycles during fixation. The cytoplasm of individual egg regions may become displaced up to 45% of the egg's length inPimpla, and up to 14% of the egg's length inWachtliella. Therefore fixed preparations may grossly misrepresent the distribution of egg components in the living state. The changes can be traced back by microkymographic registration of movements during fixation. Microkymograms for this purpose were obtained by projecting a time-lapse film through a slit onto photosensitive material mounted on a rotating drum. The shifting of egg components and the mitotic activity of nuclei fail to occur when the egg is treated with a buffered solution of OsO4 (1%, 1 min). Subsequent fixation with glutaraldehyde results in satisfactory preservation of microtubules.

Notes: Abstract Explanted oocytes and eggs of different developmental stages from the Hymenopteron Pimpla were fused in pairs as “parabiotic tandems”. Interactions within the tandem were analysed by time lapse films. Except for the exchange of nuclei, no joint development was observed, and each partner followed its own time pattern. The rapid cell cycles of the cleavage energids switched over to longer cycles according to the local developmental stage of the different egg regions, although all of the nuclei were still contained in a single plasmodium. In a second experimental series, nuclei in newly deposited eggs were X-rayed and replaced by cleavage energids from later stages injected into the wrong (=posterior) egg pole. Even injected blastoderm nuclei immediately took up mitotic activities and underwent rapid cell cycles characteristic of early cleavage. Normal embryos could be formed, although the nuclei had populated the egg in a reverse direction.

Notes: Abstract Oocytes explanted from adult ovaries of the arrhenotokous Hymenopteron Pimpla turionellae remain in an inactive state, because development has not been initiated by mechanical deformation during natural oviposition. However, they could be induced to enter development by injecting cleavage energids into the posterior pole. After lag phases of up to 32 h, the implanted nuclei initiated a normal cleavage process, except that the polarity of its progress was reversed. In other oocytes, the injected energids congregated in a ring-shaped region at the egg surface to form a superficial “nuclear front”, which slowly advanced towards the anterior egg pole, thereby successively stimulating portions of the quiescent ooplasm to take part in development. Up to 41 rapid cell cycles started from that front, each of them with an anaphase wave running backwards into the region already peripherally occupied by nuclei. Thus, the blastoderm was formed extremely metachronously and by rapid obviously biphasic cell cycles, which never occur at the egg surface during normal cleavage. A germ band, however, was only formed under the following conditions: (1) that cleavage did not follow the nuclear front mode, and (2) that ooplasm from the donor's posterior pole was co-injected with the graft nuclei. We conclude that embryonic differentiation requires some of the events which had been omitted in eggs where development failed, especially the exponential increase of the cell cycle length, and the activity of some posterior factor(s) during egg activation.

Notes: Summary The control of nuclear division and migration was studied in time-lapse films of the multinucleate egg cell of a gall midge by experimental alterations of the mitotic pattern. During each cleavage cycle, a wave of randomly oriented saltations of yolk particles (WROS) is seen to travel through the ooplasm. This wave proved to be an indispensable prerequisite for the accompanying anaphase wave and for the activation of the nuclear migration cytasters: WROS cycles can occur autonomously without cleavage nuclei being present, but there is no anaphase without a WROS passing the dividing nucleus. WROSs and mitotic waves can be inverted, and the WROS cycles and the cleavage cycles can be desynchronized by temperature grandients or by locally impaired gas exchange. If a nucleus is not ready for anaphase when met by a WROS, it will only divide in the course of the next WROS. WROSs thus indicate autonomous anaphase-triggering waves governing the cleavage divisions. Rhythmic ooplasmic movements continue even if the WROSs as well as the nuclear divisions are inhibited by colchinine. The characteristics of the WROSs support the hypothesis that each of them is the visible effect of a wave of calcium release (similar to that established in vertebrate eggs) which acts locally on the microtubular system and may continue even if the WROSs are suppressed. The correlations between a possible calcium release, WROS activity, microtubule disassembly and nuclear cycle are discussed.

Notes: Summary The experimental results ofGeyer-Duszynska (1959), speaking in favour of three ooplasmic factors localized in the pole plasm, in the basophilic oosome material contained therein, as well as in the periplasm of the posterior egg pole ofWachtliella persicariae, suggested to investigate for further factor regions with other technical means. Since ooplasmic factor regions may be indicated by initial regions of morphogenetic development, kinematics were used for in vivo analysis of early embryonic development by means of time-lapse motion pictures. Electron microscopic investigations added to the micro-morphological aspects of plasmic systems within the egg for a better understanding of nature and effectivity of ooplasmic factors. Cleavage nuclei do not move exclusively by means of their spindle activity during anaphase movement. The nuclear envelope of cleavage energides consists of either two unit membranes with pores or of many tube-like as well as membranous elements. The appearence of a complex multi-layered nuclear envelope coincides with the moving phase of energides, an observation which is discussed in relation to the possibility of active nuclear movement. During late preblastoderm the entoplasm contains horse-shoe-shaped and multilobed vitellophagues with dense karyoplasm. With the blastoderm formed, the nuclei may become pycnotic, their membranes fragmenting at the same time. These fragments probably are piled up to form annulated membranes. The pole plasm does not show specific structures apart from the oosome material, contained therein. It is free of yolk material and nearly exclusively consists of ground plasm. The basophilic oosome material within the pole plasm is not surrounded by any membranes. It consists of numerous ribosome-like units and is restricted to the plasm of the future pole cells. The micro structure of the oosome material is preserved at least till the germ band has reached its maximal length. The cell membranes develop by invagination of the oolemma which penetrates into the egg interior. While pole cells and blastoderm cells become tied off, the ground plasm possibly participates in the growing-in process of the cell membranes by developing fibrous differentiations at the terminal extensions of oolemma folds. There is no clear cut limitation between periplasm and entoplasm. The periplasm which is without yolk material, appears rich in ground plasm and does not contain specific ultra structures. During the process of cleavage external ooplasmic regions of the egg are shifted in rhythmical pulsation parallel to its longer axis by a maximum of about 6 % of the entire egg length. Topographic statements of certain areas concerning any anlage therefore are bound to suffer from an adequate lack of exactness. Since comparable shifting processes within the egg plasm probably are common in insects other thanWachtliella, they should be considered as a certain source of error. At 60+−3 % of egg length as measured from its posterior pole, there exists a cleavage centre, an initial region of intravitelline cleavage and of repeated mitotic waves. Adjacent to the middle of the egg follows an initial region of germ band formation (differentiation centre). By their electron microscopic appearance, both developmental centres are not characterized by specific ultra structures. The factor region at the posterior pole exclusively represents an initial region of cell wall formation during superficial cleavage. Other than any experimental marking procedure the technique of time-lapse motion pictures permits to evaluate quantitatively and without artificial interfering the shifting of presumptive segment material during morphogenetic movements of the germ band. The embryonic material of the blastoderm at the egg equator is used for building up the first abdominal segment. The prothoracal and mesothoracal material at about 60% of the egg length stays in site when the germ band becomes extended lengthwise. Closely behind the differentiation centre there is a region of maximal extension as well as of shortening of the germ band. No proliferous growth of segments (segment formation zone) has been found.

Notes: Summary In the eggs ofWachtliella persicariae the cleavage nuclei move relative to the surrounding ooplasm. This ‘active’ migration is caused by an organelle whose ultrastructure was studied throughout the mitotic cycle. It consists of a greatly enlarged polar cytaster derived from the mitotic apparatus, linked to the nucleus by 100 Å filaments. The microtubules of the cytaster were found only during periods of active nuclear migration, i.e., from the onset of anaphase to the early prophase of the next mitotic cycle. They are always solitary and follow the course of the astral rays, which are known to temporarily adhere to peripheral structures of the egg cell and to exert tractive forces. In contrast to the cytaster microtubules, the microtubules in the spindle are bundled and persist from early metaphase through late telophase. During ontogenesis the first migration cytaster is built up between 3 and 12 min after oviposition near the anterior egg pole, in the vicinity of the sperm nucleus. In non-inseminated eggs time lapse films show a migration cytaster to develop autonomously in a region free from nuclei, but it does not follow the normal path of the male pronucleus. In several cases the female pronucleus, which remains without a cytaster of its own, was observed to move to the cytaster generated in the absence of the male pronucleus. Whether or not it is adhering to a nucleus, the cytaster divides into two at the correct time, i.e, corresponding to the first cleavage division in fertilized eggs. In some non-inseminated eggs this type of ‘pseudocleavage’ has been observed to occur repeatedly, giving rise to an increasing number of anucleate cytasters.

Notes: Summary Kinematics and ultrastructure of centrifuged and untreated eggs fromWachtliella persicariae were investigated for the micromorphological properties of ooplasmic factor regions and their role in early developmental processes by means of time-lapse motion pictures and electron microscopic analysis (see part I). After centrifugation the eggs show up to five different layers, among them a pole of fatty yolk with lipid droplets, a region of clear plasm (rich in ground plasm) which itself may become subdivided into a centripetal region with nuclei and endoplasmic reticulum, followed by a centrifugal part with mitochondria and ribosomes, another region containing orange clods of proteid yolk and finally a cup of glycogen. Displacement of pole plasm from the posterior pole always is accompanied by dislocation of the basophilic oosome material contained therein. At sufficient r.p.m. both of them enter the centripetal area of clear plasm. Structures of “di-polar density” type are orientated by centrifugation. The initial phase till the centrifuge reaches its final r.p.m. may act decicively upon the site of certain egg components after centrifugation as upon the nuclei, and thus may essentially influence the experimental results. In case centrifugation coincides with certain dividing phases of energides, the nuclear envelope becomes fragmented. The fragments then may appear piled up to form annulated membranes which have been recognized as pathological structures in centrifuged eggs. Besides lamellar cytosomes are often found. In centrifuged as well as in untreated eggs the nuclear envelope either consists of two layers as usual or may be of the complex multi-layered type (see part I). As for the movement of nuclei, the possible role of the complex nuclear envelope is not yet clear. The pigment halo of cleavage nuclei does not play an active part in nuclear migration. In centrifuged eggs yolk nuclei are of the usual type i.e. either roundish, horse-shoe-shaped or multi-lobed. They mostly appear in parts of the entoplasm which are poor in yolk. A surrounding rich in yolk does not seem to be essential for transforming normal cleavage nuclei into vitellophagues. For their changing into the multi-lobed type yolk nuclei must be surrounded by a sufficient amount of ground plasm. Pole cells have been found in the posterior pole region only. Their formation requires an abundant amount of ground plasm, the presence of cleavage energides, as well as pole plasm and oosome material, if not either of the two latter systems. Since in centrifuged eggs pole plasm and basophilic oosome material are always shifted together into the region of clear plasm, contrary to the opinion of other authors (p. 42; part I, p. 124) the technique of centrifugation does not permit any decision as to which of both ooplasmic systems controls the karyotic differentiation of the germ line or the formation of pole cells, respectively, or whether both systems are essential to promote these processes. The oolemma may also become invaginated to form cell membranes when no nuclei are present (“pseudoblastoderm”), the formation occurring in regions with sufficient amounts of ground plasm only. For that reason the formation of pole cells is restricted to the posterior pole rich in ground plasm, whereas blastoderm cells exclusively occur in the area of preblastoderm plasm. The ground plasm plays a decisive part in the dynamics of cell membrane formation. As for blastoderm cells, the nuclei seem to be necessary only to control their regular shape. In contradiction to the opinion of other authors (p. 42, part I, p. 124), the periplasm of young eggs cannot range among the essential prerequisites of blastoderm formation. During centrifugation it does not stay at the surface of the egg poles where nevertheless a blastoderm may be formed. Yet blastoderm formation is only possible if, in spite of the compact condition of polar yolk material, the egg poles become covered with preblastoderm plasm from the region of clear plasm, rich in ground plasm, and thus replacing sufficient amounts of periplasm and ground plasm shifted by centrifugation.

Notes: Summary To affirm the hypothesis that during the early cleavage inWachtliella persicariae L., central and marginal ooplasm do not only differ by their kinematic aspects (Wolf, 1969; Wolf and Krause, 1971) but also physiologically, the radial symmetry of the ooplasm has been destroyed experimentally. For this purpose a device has been developed permitting a local ultrasonic treatment of insect eggs limited to well defined regions of various sizes (Fig. 1). The changes in the egg architecture can be observed directly while the treatment is going on and their effects on embryogenesis are analyzed by time-lapse motion pictures. Cleavage nuclei which have been transferred prematurely to the egg surface during low-intensity ultrasonic radiation of the whole egg length (Fig. 2 a), move back into the central ooplasm after treatment; the marginal plasm becomes populated only later at the same time as in untreated eggs. Local high-intensity ultrasonic treatment, too (Fig. 2 c), obviously does not cause any damage to the nuclei, while the ooplasm within the treated area becomes completely mixed up. After exposure the untreated region may become partially coated by a sheath of mixed up ooplasm. The populating of the egg surface by the nuclei is restricted to those sites where the untreated marginal ooplasm remains at the surface. These are the only areas for the later formation of preblastoderm and blastoderm. For the normal distribution of the cleavage energides a certain ooplasmic component sensitive to ultrasonic treatment seems to be responsible, the ultrastructure of which will be subjected to further investigations.