The formation of intercellular bridge coating in the hairy whirligig beetle,Orectochilus villosus

Summary The ultrastructure of the intercellular bridges in the ovaries of Orectochilus villosus has been studied. In young egg chambers the inner filamentous coating of the bridge is thin. Characteristic vesicles are observed in close contact with this coating. The inner coating of the vitellogenic chambers is fully developed although vesicles are only sporadically associated with it. These observations suggest that the vesicles are involved in the formation of the filamentous coating.This work was supported by Government Problem Grant MR II, 1.6.4.     

Alarm substance in Gyrinus aeratus (Coleoptera,Gyrinidae)

Gyrinid beetles are common in ponds and lakes in Sweden, where they aggregate in open areas of the surface. Gyrinid beetles have pygidial glands which produce compounds rendering them unpalatable to fish. This study examines whether the pygidial secretion can be used for alarm purposes in addition to other functions. Experiments showed that gyrinid beetles responded to water prepared with the pygidial compounds by evasive behaviour. The beetles did not respond when the compound was mediated by air. One component of the defense system of gyrinids is how they advertise their presence. The aggregation of dark beetles with their typical motion pattern when alerted is very conspicuous and easily identified by an experienced predator. We suggest that this aspect of gyrinid defence can be characterized as aposematic.     

The formation of intercellular junctions in insect stem cell progeny (cockroach intestinal epithelium)

Summary The stages in the development of intercellular junctions have been followed in the mesenteric caecal cells of the cockroach midgut, where two types of mature cell, the columnar and the secretory, exist. Nests of undifferentiated replacement cells occur at intervals along the basal lamina, consisting of central, dividing cells and peripheral semi-lunar cells; the former act as proliferative stem cells to give rise to either pre-columnar or pre-secretory cells. The semi-lunar cells are pre-columnar and produce an attenuated process which gradually projects up to the luminal surface, producing microvilli and a dense extracellular substance en route. Intercellular gap junctions appear between these maturing columnar cell borders first, while septate junctions differentiate later; these are assembled from two different sets of intramembranous particle which become organized into either plaques or rows in parallel alignment, possibly mediated by actin filaments and microtubules. The pre-secretory cells, which are much fewer in number, remain associated only with the basal lamina and never reach the lumen; they develop into one of three distinct mature secretory cell types which release their secretory product in different ways. Offprint requests to: N.J. Lane     

Ultrastructural changes in nurse and follicle cells during late stages of oogenesis in Drosophila melanogaster

Summary During stages 11 and 12, follicle cells surrounding the nurse cells produce lysosomes which presumably aid in the breakdown of the nurse cells. Accompanying a DNA reduction in nurse cell nuclei are several characteristic morphological changes including the appearance of intranuclear rod-like structures and nuclear granules about 300 Å in diameter. Similarities between structures seen in Drosophila nurse cell nuclei and those seen in other organisms are discussed.This research was supported by U. S. Public Health Service Grants 5TIGM903-3 and 1-F1-GM-33, 385-01 and National Science Foundation grant GB-7457.     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

Heterologous gap junctions between oocytes and follicle cells of an insect,Dysdercus intermedius,and their potential role as ion current pathways

Summary In telotrophic insect ovaries, the oocytes develop in association with two kinds of supporting cells. Each ovary contains five to seven ovarioles. An ovariole consists of a single strand of several oocytes. At the apex of each ovariole is a syncytium of nurse cells (the tropharium), which connects by strands of cytoplasm (the trophic cords) to four or more previtellogenic oocytes. In addition, each oocyte is surrounded by an epithelium of follicle cells, with which it may form gap junctions. To study the temporal and spatial patterns of these associations, Lucifer yellow was microinjected into ovaries of the red cotton bug, Dysdercus intermedius. Freeze-fracture replicas were examined to analyze the distribution of gap junctions between the oocyte and the follicle cells. Dye-coupling between oocytes and follicle cells was detectable early in previtellogenesis and was maintained through late vitellogenesis. It was restricted to the lateral follicle cells. The anterior and posterior follicle cells were not dye-coupled. Freeze-fracture analysis showed microvilli formed by the oocyte during mid-previtellogenesis, and the gap junctions became located at the tips of these. As the microvilli continued to elongate until late vitellogenesis, gap junction particles between them and follicle cell membranes became arranged in long arrays. The morphological findings raise questions about pathways for the intrafollicular phase of the ion currents known to surround the previtellogenic and vitellogenic growth zones of the ovariole.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Expression and cellular localization of the Toucan protein during Drosophila oogenesis

The toucan (toc) gene is required in the germline for somatic cell patterning during Drosophila oogenesis. To better understand the function of toc, we performed a detailed analysis of the distribution of the Toucan protein during oogenesis. Toc expression is restricted to the germline cells and shows a dynamic distribution pattern throughout follicle development. Mislocalization of the Toc protein in mutant follicles in which the microtubule network is altered indicates that microtubules play a role in Toc localization during oogenesis.     

Intercellular junctions in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary The intramembrane organization of the occluding junctions in the gill epithelium of the Atlantic hagfish, Myxine glutinosa, was studied by means of freeze-fracture electron microscopy. Mitochondria-rich cells, characterized by assemblies of rod-shaped particles in the luminal plasma membrane and by an extensive intracellular amplification of the basolateral plasma membrane, are singly distributed between the pavement cells in the gill epithelium of this marine and stenohaline cyclostome. The occluding junctions between mitochondria-rich cells and pavement cells do not differ from those between adjacent pavement cells, concerning the number of superimposed strands (median 6, range 4–9) and their geometrical organization. These observations suggest that, in contrast to marine teleosts, the paracellular pathway plays a minor role in transepithelial ion movements in the hagfish gill epithelium. The findings are in agreement with the absence of hypoosmoregulatory mechanisms in hagfish, as have been evolved in various marine vertebrates. In addition, small communicating junctions are demonstrated between pavement cells; they possibly serve for a coordinated synthesis and secretion of mucus by the pavement cells.Dedicated to Prof. H. Leonhardt on the occasion of his 70th birthday     

Ultrastructural changes in the glial cells at neuromuscular synapses of Locusta migratoria occurring after nerve stimulation and subsequent rest

The glial processes ensheathing the motor nerve terminals on the retractor unguis muscle of Locusta migratoria are described. Ultrastructural changes observed after electrical nerve stimulation (20 Hz, 7 min) without or with subsequent rest (2 min, 1 h) are analysed morphometrically. Immediately after stimulation both the average terminal circumference (+ 23%) and its proportion covered by glial processes (+ 16%) are significantly increased. The mean number of Schwann cell processes per micron of terminal circumference (without stimulation: 0.86 +/- 0.04) is also affected: Immediately after stimulation it is increased by about 15% and after 2 min of rest even by 36%. The periaxonal cleft (without stimulation: 16.5 nm +/- 0.36) becomes wider immediately after stimulation by about 19%, an effect which is almost reversed after 1 h of rest. It is suggested that these changes are a consequence of the enlargement of the nerve terminal's surface upon massive exocytotic activity and that they are possibly mediated by mechanical attachment between glial and terminal plasma membranes.     

Ultrastructure of the hepatopancreas and associated tissues of the chelicerate arthropod,Limulus polyphemus

Summary The ultrastructure of the hepatopancreas and associated tissues of Limulus is described. All hepatopancreatic tubules contain L- and D-cells. L-cells exhibit apical pinocytosis and contain many different types of inclusions, including lipid containing calcium spherules of unique structure. They also produce a complex secretory product released by apocrine secretion. D-cells contain much rough endoplasmic reticulum, and show other evidence of active protein synthesis. They produce a single type of secretory granule released by merocrine activity. The appearance of L- and D-cells in tubules of various sizes and in fed and starved animals is described. Both cell types may produce digestive enzymes and absorb and transport nutrients to the hemolymph, but neither serves as a major storage site. Storage is an important function of the intertubular R-cells. The appearance of R-cells in fed and starved animals is described. A previously undescribed hemocyte is reported, as are apparent gap junctions between L- and R-cells. A structural and functional comparison of these glands with those of other arthropods is presented.Supported by NSF Grant No. GB-16607 and by the University of Minnesota Graduate School.     

An ultrastructural study of relationships between the ovarian haemal system,follicle cells,and primary oocytes in the sea star,Asterias rubens

Summary The genital haemal sinus, present throughout the gonad wall of sea stars, is supposed to be the site of ultimate accumulation of nutrients for the germinal epithelium. Early vitellogenic pear-shaped oocytes are attached to this sinus by stalk-like processes. The ultrastructure of this association and of the oocyte-follicle cell complex is described with emphasis on mechanisms involved in oocyte nutrition.The genital haemal sinus, and sometimes portions of the surrounding genital coelomic sinus, contain a fine granular ground substance and amoeboid cells. Material similar to the haemal ground substance also fills vacuities in the inner basal laminae of the haemal sinus and intervenes between this layer and adjacent germinal and follicle cells in the ovarian lumen.Vitellogenesis is first detectable as numerous vacuoles accumulate within the oocyte-stalk near the haemal sinus; they contain flocculent material and often fuse with adjacent lysosome-like vacuoles. As vitellogenesis proceeds, oocytes develop complex and tenuous connections with the haemal sinus. These consist of a network of pseudopodia that interdigitate with thin sheet-like extensions of follicle cells. These cells are attached to the oolemma by microfilamentous processes and contain regularly arranged concentrations of glycogen granules and well developed rough endoplasmic reticulum.It is concluded, (1) that follicle cells provide each oocyte with a compartmentalized microenvironment within the ovarian lumen, (2) that such compartments are intimately associated with the nutrient laden haemal sinus, and (3) that nutritive and vitellogenic substances, derived extragonadally and stored temporarily in the ovarian wall, can pass through the oocyte-stalk.     

Immunohistochemical localisation of ecdysteroids in the follicular epithelium of locust oocytes

Summary Frozen sections of growing terminal follicles of the locust ovary were incubated with an ecdysteroid-specific rabbit antibody and the bound antibody visualised by the use of FITC-labelled goat-anti-rabbit antiserum. A bright fluorescence was seen in the cytoplasm of the follicle cells in terminal follicles with a length between 4.0 and 6.0mm with a maximum intensity at 5.5mm, indicating the presence of ecdysteroids in these cells in this particular developmental stage.     

Presence of a cytotomy factor in the karyoplasm ofAcipenser oocytes at the onset of the period of rapid growth

Summary Experiments with oocyte enucleation and transplantation of germinal vesicles show that already at the beginning of the period of rapid growth, the oocyte karyoplasm contains the substances necessary for the appearance in the cytoplasm of the ability to divide.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Ultrastructural analysis of the developing follicle during early vitellogenesis in tilapia,Oreochromis niloticus,with special reference to the steroid-producing cells

The development and distribution of steroid producing cells (SPCs) in the ovary of tilapia have been studied by light and electron microscopy. At 40–50 d after hatching, these cells are seen only in the vicinity of blood vessels; there are no SPCs in the interstitial region, nor in the thecal layer enclosing young oocytes at the peri-nucleolus stage. By 70–80 d after hatching, the number of SPCs in the area near blood vessels has increased, and the capillaries have spread among the developing peri-nucleolar stage oocytes, and into the ovarian tunica. Clusters of SPCs have also migrated into the interstitial region and into the tunica along with these capillaries. In the ovary 100 d after hatching, some SPCs can be found in the thecal layer enclosing vitellogenic oocytes. Moreover, masses of SPCs can now be observed infiltrating the thecal layer of the oocyte. Serum testosterone (T) and estradiol-17 (E2) levels at 40–70 d after hatching, are low (T, 0.75–1.10 ng/ml, E2, 0.36–1.08 ng/ml), but at 100 d, plasma E2, but not T, is elevated (T, 1.95 ng/ml, E2, 4.65 ng/ml). These results suggest that SPCs appearing in the vicinity of blood vessels move into the interstitial region between oocytes, and finally enclose the oocytes at an early vitellogenic stage. It is interesting to note that the enclosure of oocytes by SPCs coincides with significant increases in E2 production.     

Ultrastructural,fluorescence microscopic and microfluorimetric study of the innervation of the axial complex in the sea urchin,Sphaerechinus granularis (Lam.)

Summary In juxtaposition with the contractile epithelia of the axial complex of the sea urchin, Sphaerechinus granularis, several types of nerve fibers with different vesicle populations were determined. Nerve terminals, filled with clear vesicles and dense core vesicles, form synaptoid neuromuscular junctions. Close to the somatocoelic epithelia of the axial and terminal sinus septa, numerous axon profiles form a nerve plexus. Among the epithelial cells covering the plexus, two types of nerve cells can be distinguished which presumably produce neurosecretory and aminergic granules, respectively. Monoamine fluorescence (formaldehyde-induced fluorescence, Falck-Hillarp technique) was analyzed microspectrofluorimetrically. The emission spectrum of the fluorophores occurring in the present material shows a maximum at 475 nm and is characteristic of catecholamines; the excitation maximum at 380 nm after formaldehyde treatment is typical of catecholamines at low pH only. Since the peak ratio (370:320 nm) does not change after HCl-vapor treatment, the fluorophores are likely to be indicative of dopamine.     

The olfactory epithelium of the lantern fish,Tarletonbeania crenularis (myctophidae)

Summary The permeability of the septate junction of the nephrocytes of Helix pomatia from the basal lamina to lumen has been studied with two electron opaque tracers, horse-radish peroxidase and lanthanum. Ultrathin sections of tissues from animals treated with these materials showed that the septa offer a considerable restraint to the movement of horse-radish peroxidase, but that the lanthanum permeates the septate region of the junction. Tangential sections of lanthanum-infiltrated junctions showed that the septa are corrugated sheets. The infiltration by lanthanum shows that in some areas there are discontinuities in these sheets which may allow the passive movement of solutes from the intercellular space to the kidney lumen.