Dynamics of annulate lamellae in Drosophila syncytial embryos

The mitotic events in eukaryotic cells are controlled by a family of evolutionary conserved cyclin-dependent kinases (cdk) that phosphorylate cell proteins, which results in the structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that changes in cdk1 activity controlling the assembly and disassembly of nuclear pore complexes also affect the structure of cytoplasmic pores in annulate lamellae. Here, we report a comparative electron microscopic analysis of the dynamics of these organelles during mitosis throughout the development of a Drosophila syncytial embryo. We presume that the distribution of annulate lamellae containing mature cytoplasmic pores across the cytoplasm reflects local reductions in the mitotic kinase cdk1 activity during the development of Drosophila syncytial embryos.     

Fine structure of the pore-annulus complex in the nuclear envelope and annulate lamellae of germ cells

Summary Octagonal symmetry in the pore margin has been demonstratedin situ in annulate lamellae and the nuclear envelope of germ cells. The annular material is located to variable extent within the pore and also extends beyond the pore margin; in the latter case it may be continuous with extra-pore annular material of some adjacent pores. In thin sections of fixed material, the annular material of both the nuclear envelope and annulate lamellae appears to be composed of a matrix within which are embedded thin filaments and small granules, the disposition and interrelationship of which are described and discussed. The so-called intra-annular granule is described as consisting of a number of smaller units (similar to the granular component of the annular material) which become aggregated in the center of some pores in both the nuclear envelope and annulate lamellae. The possible significance of intra-annular granules is discussed in terms of binding and movement of macromolecules.This investigation was supported by research grants (HD-00699, GM-09229) and a Career Development Award from the National Institutes of Health, U. S. Public Health Service. The author acknowledges the skillful technical assistance of Mrs.Robert Decker.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

The formation of “accessory nuclei” in the developing oöcytes of the parasitoid hymenopterans Ophion luteus (L.) and Apanteles glomeratus (L.)

Summary The appearance and subsequent distribution of accessory nuclei in the developing oöcytes of the Ichneumonoid Ophion luteus and Braconid Apanteles glomeratus is described. They are produced by a folding of annulate lamellae produced from the nuclear envelope. The nucleus and accessory nuclei give rise to other annulate lamellae by two distinct modes of budding. These organelles are involved in membrane formation.     

Relationships between annulate lamellae and filament bundles in oocytes of the zebrafish,Brachydanio rerio

Summary Bundles of filaments have been observed in the vitellogenic oocyte of the zebrafish, Brachydanio rerio; and these filaments illustrate a close spatial and structural relationship to annulate lamellae. The filaments range from 6–8 nm in diameter, and the annulate lamellae may cap both rounded ends of the bundle as well as extend parallel to the surface of the filament bundles. The ends of the filaments can be observed to exhibit an apparent termination in close relation to pore margins of the annulate lamellae, the membrane of the interpore regions of the annulate lamellae, as well as many nearby polyribosomes. The possible functional significance of this unique relationship is discussed in reference to a recent hypothesis regarding the function of annulate lamellae.     

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

Summary Ultrastructural 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.     

Effects of lithium chloride on embryos ofOphryotrocha labronica LaGreca and Bacci

Summary Cleavage embryos of the polychaeteOphryotrocha labronica were exposed to LiCl solutions of various strength for up to 4 days. High concentrations strongly inhibited development and led to endodermal protrusion, whereas moderate concentrations permitted development of larvae, although with characteristic disturbances as a consequence. Ultrastructural analyses of embryos exposed to moderate concentrations demonstrated manifest changes already during the first 24 hours, viz. of lipid droplets and yolk granules, the latter being ruptured and the contents dispersed to an extent far surpassing that at normal development. The premature widespread splitting of yolk granules by LiCl was confirmed by quantitative measurements on embryos which during oogenesis had their yolk granule DNA selectively labelled with³H-thymidine.The skilful technical assistance of Miss Britt Jönsson, Mrs. Siv Nilsson and Mrs. Annagreta Petersen is gratefully acknowledged. This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.     

INCREASED NUMBERS OF ANNULATE LAMELLAE IN MYOCARDIUM OF CHICK EMBRYOS INCUBATED AT ABNORMAL TEMPERATURES

Annulate lamellae have been observed in the myocardium of 18-day-old chick embryos maintained at the normal temperature of 100°F and at 90°F during the last week of incubation. An increased number of annulate lamellae was observed in heart muscle of embryos incubated at 90°F. This is probably caused by a persistent production of these organelles, since annulate lamellae are present in greater frequency than in 11-day-old embryos incubated at 100°F. In the hypertrophic hearts of 18-day-old embryos incubated at 90°F, the annulate lamellae were associated with a net increase of protein content and an elevated concentration of myocardial glycogen. It is suggested that the increased number of annulate lamellae is a sequela of reduced environmental temperature during incubation.     

Annulate lamellae in plant cells: Formation during microsporogenesis and pollen development in Canna generalis Bailey

Summary The occurrence of stacked annulate lamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchitecture and relationship to endoplasmic reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamellar cisternae may originate as a degenerative form of endoplasmic reticulum.     

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis

Summary During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 m from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.     

A multilamellate body in the granulated hypophysial cells of the teleost,Hemihaplochromis philander

Summary A multilamellate body (MLB), bearing close resemblance to an array of annulate lamellae, has been observed in several adenohypophysial cell types of the teleost, Hemihaplochromis philander. In longitudinal section, each MLB comprises a ladder-like row of 12–50 sausage-shaped profiles, termed lamellae. A few lamellae in each section show connections with the endoplasmic reticulum. Apposition of paired lamellar membranes at regularly spaced intervals results in a beaded appearance, whereby sites of membrane apposition are probable pore sites. The MLB differs from annulate lamellae in having poorly developed pores and closer packing of lamellae. It is suggested that the MLBs described here, may represent annulate lamellae at a stage of development or break-down when pores are incomplete.Acknowledgement is made to the South African Council for Scientific and Industrial Research for financial aid towards this work. The authors also wish to thank the Natal Parks, Game and Fish Preservation Board for providing specimens     

Annulate lamellae and "yolk nuclei" in oocytes of the dragonfly, Libellula pulchella

Annulated membranes in the form of single and short lamellae are present adjacent to and parallel to the nuclear envelope in oogonia and early oocyte (synaptene) stages of the dragonfly, Libellula pulchella. These solitary and short annulate lamellae are usually continuous with long, part rough- and part smooth-surfaced cisternae which extend into more distal areas of the oogonial ooplasm. These particular annulate lamellae then either disappear or decrease in number to be replaced by a much more extensive system of annulate lamellae in the cortical ooplasm of previtellogenic oocytes. The differentiation of extensive stacks of annulate lamellae is consistently observed to be restricted to large cytoplasmic areas of considerable electron density. These cytoplasmic regions consist of material which stains basophilic and contains RNA but differs structurally from the large number of ribosomes which surround the dense masses. The cytoplasmic dense masses, in terms of their formation and staining reactions, are comparable to the "yolk nuclei" or "Balbiani bodies" described in insect oocytes in earlier studies. The results of the present study thus provide evidence that the appearance of cortical ooplasmic stacks of annulate lamellae in the dragonfly oocyte is specifically limited to cytoplasmic areas of high electron density which contain RNA but which do not have a ribosomal morphology.     

Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components?

Annulate lamellae are cytoplasmic organelles composed of stacked sheets of membrane containing pores that are structurally indistinguishable from nuclear pores. The functions of annulate lamellae are not well understood. Although they may be found in virtually any eucaryotic cell, they occur most commonly in transformed and embryonic tissues. In Drosophila, annulate lamellae are found in the syncytial blastoderm embryo as it is cleaved to form the cellular blastoderm. The cytological events of the cellularization process are well documented, and may be used as temporal landmarks when studying changes in annulate lamellae. By using morphometric techniques to analyze electron micrographs of embryos, we are able to calculate the number of pores per nucleus in nuclear envelopes and annulate lamellae during progressive stages of cellularization. We find that annulate lamellae pores remain at a low level while nuclear envelopes are expanding and acquiring pores in early interphase. Once nuclear envelopes are saturated with pores, however, the number of annulate lamellae pores increases more than 10-fold in 9 min. Over the next 30 min it gradually declines to the initial low level. On the basis of these results, we propose (a) that pore synthesis and assembly continues after nuclear envelopes have been saturated with pores; (b) that these supernumerary pores accumulate transiently in cytoplasmic annulate lamellae; and (c) that because these pores are not needed by the embryo they are subsequently degraded.     

Presence of acid phosphatase activity in heavy bodies of sea urchin eggs

Cytochemical studies were made on the acid phosphatase (Acp-ase) in the heavy bodies of eggs of the sea urchins, Hemicentrotus pulcherrimus and Temnopleurus toreumaticus, from the stage of mature unfertilized eggs to the late gastrula stage. High Acp-ase activity was found in masses composed of ribosome-like granules in the heavy bodies from the early stage of the formation of heavy bodies until the time of their disappearance, whereas the annulate lamellae and the space between the annulate lamellae and the masses packed with ribosome-like granules did not have activity. This activity was inhibited by the addition of 0.01 M NaF to, or the omission of substrate from, the Gomori medium. Using the reaction products of Acp-ase and the masses of ribosome-like granules as markers of heavy bodies, we found small groups of tightly packed ribosome-like granules that were not surrounded by annulate lamellae. The function of Acp-ase in the heavy bodies is still unknown.     

An ultrastructural analysis of mitosis and cytokinesis in the zygote of the sea urchin, Arbacia punctulata

Post-fertilization events leading to the cleavage of the zygote of the sea-urchin, Arbacia punctulata were examined with the light and electron microscopes. Prior to prophase of the first cleavage division, endoplasmic reticulum and annulate lamellae become organized around the zygotic nucleus to produce a crescent-shaped structure which is defined as the streak (Harvey, '56). With the advent of prophase the streak undergoes morphogenic events which lead to the formation of the mitotic asters. During this transition there is a loss of annulate lamellae and a concomitant increase in endoplasmic reticulum. Annulate lamellae are not found as a part of the mitotic apparatus and are not again observed within the embryo until the two cell stage. During telophase, karyomeres are formed which consist of chromosomes delimited by a porous bilaminar envelope. Blastomere nuclei are produced following the fusion of the outer laminae, and subsequently by the fusion of the inner laminae of the envelopes encompassing the karyomeres.     

The origin of annulate lamellae in the oocyte of the ascidian,Boltenia villosa stimpson

Summary The formation of the extranuclear annulate lamellae has been revealed to be connected with a process of nuclear emission which is very active during the previtellogenetic stages of the Boltenia oocyte development. This process involves both of the nuclear membranes. At many spots on the surface of the nuclear envelope, the outer membrane pulls away from the inner membrane, thus forming what has been designated as blisters of various sizes and shapes. Masses of nuclear content, apparently not from the nucleolus, are pushed into the blisters. These blisters may become detached from the nuclear envelope and lie free in the cytoplasm. But in many cases, the detachment seems delayed, and in each blister many emission masses are squeezed tightly together and flat one on top of the other. These masses, in sections, may present the appearance of a stack of elongated outlines. The membrane, limiting any two adjacent masses in close contact, develop annuli. It is thus that an annulate lamella is formed. Whether an annulate lamella is formed between a pair of neighboring masses depends on their proximity. So the production of the annulate lamellae is incidental to, but not a necessary part of the process of nuclear emission. After the original outer nuclear membrane forming the blister has disintegrated, the annulate lamellae are left exposed in the cytoplasm.It is clear that, 1. both membranes of an annulate lamella are of inner nuclear membrane origin, 2. they hold between them some of the content of the enlarged perinuclear space resulting from the raising of the outer nuclear membrane when the blister is formed, and 3. the material held between any two lamellae is from the nucleus.The intranuclear annulate lamellae simply arise from the narrow pouches formed by the inner nuclear membrane towards the interior of the nucleus, and on these narrow pouches annuli are developed. So the intranuclear annulate lamellae is also composed of two membranes of an inner nuclear membrane origin holding between them a quantity of the content of the perinuclear space.Supported by Grant GM-11858 of National Institute of Health. The author is indebted to Dr. Richard Cloney of the Department of Zoology, University of Washington, for the use of the electron microscope.     

Intranuclear annulate lamellae in oocytes of the tunicate,Styela partita

Summary Intranuclear annulate lamellae have been observed with the electron microscope in oocytes of the tunicate, Styela partita. Morphological evidence suggests that the annulate lamellae may arise by a specialized fusion process of individual vesicles. Intranuclear vesicles appear to be formed, in time, before differentiated annulate lamellae. It is also suggested that the position and structure of an annulus is in large part determined by the fusion of the vesicles. An annulus may be present as soon as two vesicles have completed their fusion process. Finally, it is again suggested on the basis of morphological evidence that the intranuclear vesicles are derived by the blebbing activity of the inner layer of the nuclear envelope.This investigation was supported by grants (RG-9229, 9230) from the National Institutes of Health, Public Health Service. The electron microscope facilities used were also supported by a grant (GM-05479) from the National Institutes of Health to Professor H. W. Beams.     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

NEGATIVE STAINING AND ADENOSINE TRIPHOSPHATASE ACTIVITY OF ANNULATE LAMELLAE OF NEWT OOCYTES

Semi-isolated annulate lamellae were prepared from single newt oocytes (Triturus alpestris) by a modified Callan-Tomlin technique. Such preparations were examined with the electron microscope, and the negative staining appearance of the annulate lamellae is described. The annulate lamellae can be detected either adhering to the nuclear envelope or being detached from it. Sometimes they are observed to be connected with slender tubular-like structures interpreted as parts of the endoplasmic reticulum. The results obtained from negative staining are combined with those from sections. Especially, the structural data on the annulate lamellae and the nuclear envelope of the very same cell were compared. Evidence is presented that in the oocytes studied the two kinds of porous cisternae, namely annulate lamellae and nuclear envelope, are markedly distinguished in that the annulate lamellae exhibit a much higher pore frequency (generally about twice that found for the corresponding nuclear envelope) and have also a relative pore area occupying as much as 32% to 55% of the cisternal surface (compared with 13% to 22% in the nuclear envelopes). The pore diameter and all other ultrastructural details of the pore complexes, however, are equivalent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various animal and plant cells, the annuli of the annulate lamellae pores reveal also an eightfold symmetry of their subunits in negatively stained as well as in sectioned material. Furthermore, the annulate lamellae are shown to be a site of activity of the Mg-Na-K-stimulated ATPase.     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).