The trophotaenial placenta of a viviparous goodeid fish. II. Ultrastructure of trophotaeniae, the embryonic component

Embryos of the viviparous goodeid fish Ameca spendens develop within the ovarian lumen, where they establish a placental association with the maternal organism and undergo a 15,000% increase in embryonic dry weight. The placenta consists of an embryonic component, the trophotaeniae, and a maternal component, the internal ovarian epithelium. Examination with light microscopy and with transmission and scanning electron microscopy reveals that trophotaeniae of A. splendens are extraembryonic membranes consisting of five ribbon-like processes originating from a tube-like mass of tissue that extends outward from the perianal region of developing embryos. There are two sets of lateral processes and a longer single median process. Trophotaeniae possess an outer epithelium that surrounds a highly vascularized core of loose connective tissue. Epithelial cells possess apical microvilli and a pronounced endocytotic apparatus. Cells of the trophotaenial epithelium are either tightly apposed along their lateral margins or separated by enlarged intercellular spaces. Regions of the trophotaenial epithelium possessing enlarged intercellular spaces are distributed in patches. The trophotaenial epithelium is continuous with the embryonic hindgut epithelium and is considered to be derived from it. Comparison of trophotaenial morphology in A. splendens with that reported in Xenotoca eiseni reveals differences in histological organization. The former possess unsheathed trophotaeniae, whereas the latter are sheathed. We postulate that the apposition of trophotaenial epithelium to the internal ovarian epithelium constitutes a placental association equivalent to a noninvasive, epithelioform of an inverted yolk sac placenta. Structural relationships of embryonic and maternal tissues of the trophotaenial placenta are discussed in relation to maternal-embryonic nutrient transfer processes.     

In vivo fluorescence imaging of the functional organization of trophotaenial placental cells of goodeid fishes

Embryos of viviparous goodeid fishes undergo a 10 to 150 × increase in dry weight during gestation. Maternal nutrients are transferred across a trophotaenial placenta comprised of the ovarian lumenal epithelium and the trophotaeniae of the embryo. Trophotaeniae are externalized projections of the embryonic hindgut. Epithelial cells of the ribbon trophotaenia (Ameca splendens) resemble intestinal absorptive cells of suckling mammals and endocytose macromolecules. They possess an apical brush border, endocytotic complex, endosomal–lysosomal system, and apical and basal clusters of mitochondria. Cells of the rosette trophotaenia (Goodea atripinnis) lack an endocytotic apparatus, have small lysosomes, two mitochondrial clusters, and transport small molecules. Organelle-specific fluorescent probes were employed to characterize the functional organization of the two types of trophotaenial cells. In A. splendens, Lucifer Yellow, a membrane-impermeable tracer of vesicular transport, first appears in peripheral vesicles (15–45 sec), then passes into elongated tubular endosomes (1–3 min) and later appears in large central vacuoles (10–15 min). These vacuoles accumulate Acridine Orange, a classical probe for lysosomes, and have been shown to contain lysosomal enzymes. Endosomelysosome fusion was observed. In both A. splendens and G. atripinnis, Rhodamine 123 fluorescence was localized in two clusters of fine spots that corresponded to mitochondria. 4′,6-diaminido-2-phenyl-indole (DAPI) staining of nuclei established the positional relationships of cell organelles with respect to the nuclei. 3,3′-dihexyloxacarbo-cyanine iodide (DiOC₆) revealed the perinuclear distribution of the endoplasmic reticulum. In order to compare in vivo fluorescence of Lucifer Yellow with previous ultrastructural observations, we employed fluorescence photoconversion and electron microscopy. © 1994 Wiley-Liss, Inc.     

Trophotaeniae,embryonic adaptations,in the viviparous ophidioid fish,Oligopus Longhursti: A study of museum specimens

Prepartum embryos obtained from old museum specimens of the ovo-viviparous fish, Oligopus longhursti, possess external intestinal appendages. They are structurally identical to the trophotaeniae described by Turner ('37) and Mendoza ('37) in goodeid fishes. This is the first report of trophotaeniae in the viviparous ophidioids. Two developmental Stages, A and B, were observed. A is a tailbud stage, 2.0-2.25 mm in length, and B is a finfold embryo, 3.0-3.25 mm in length (Wourms and Bayne, '73). Trophotaeniae occur in the form of a single median anterior process and a pair of median posterior processes. They originate from a conspicuous peduncle formed around the anus. The processes of stage A are 1.5-2.0 mm long, 0.05 mm in diameter at their base and 0.04 mm at their tip. The stage B processes are 2.75-3.00 mm long, 0.075 mm in diameter at their base and 0.050 mm at their tip. Serial sections show that the surface epithelium of the trophotaeniae is continuous with and identical to the surface epithelium of the trophotaeniae is continuous with and identical to the surface epithelium of the embryonic gut. Examination both by transmission and scanning electron microscopy confirms that the apical surface of the trophotaenial epithelium and intestinal epithelium are covered with microvilli. Trophotaeniae are considered to function in the uptake of nutrients since they are structurally identical to intestinal epithelial cells. We suggest that maternal nutrients absorbed by trophotaeniae rather than yolk reserves are the principal source of embryonic metabolites. Trophotaeniae may afford a selective advantage since their existence in O. longhursti maximizes the number of large size embryos which a female can produce at one time. Occurrence of trophotaeniae in ophidioid, goodeid and zoarcid embryos is a remarkable example of convergent evolution.     

Endocytosis at 0° C, 5° C,and 10° C in trophotaenial absorptive cells of goodeid embryos (Teleostei)

Summary The absorptive epithelium of the trophotaeniae of goodeid embryos is involved in the micropinocytotic uptake of protein macromolecules from the ovarian embryotrophe. Incubations of viable Xenoophorus captivus embryos in vitro with horseradish peroxidase (HRP) and/or cationized ferritin (CF) allows the tracing of the fluid-phase and receptor-mediated pathways, respectively. Effects of lowered temperature on both these endocytotic mechanisms have been investigated. At 10° C, trophotaenial absorptive cells (TACs) have a strong capacity to ingest marker proteins from double tracer media. Surface-bound ligands (CF) and solutes (HRP), taken up in primary pinocytic vesicles, are rapidly channelled to the endosomal compartment. Part of the ingested CF is segregated into dense apical tubules and small vesicles indicating that membrane recycling and transcytosis continue at 10° C. Adsorptive endocytosis of CF at 5° C proceeds at a decreased rate. After incubation periods of 30 min and 1 h, tracer molecules can be found in vesicular, tubular and vacuolar compartments of the apical endocytic zone. At 0° C, no uptake of ligand worth mentioning could be ascertained. Fluid-phase endocytosis, on the other hand, is observable at this temperature. Enzyme reaction product accumulates in flattened vacuoles rather than typical voluminous endosomes. After prolonged exposure to HRP, the epithelial junctional complex becomes leaky and the marker protein penetrates the intercellular space and the lateral lamellar membrane invaginations of TACs.Supported by the Deutsche Forschungsgemeinschaft     

Ultrastructure and protein uptake of the embryonic trophotaeniae of four species of goodeid fishes (Teleostei: Atheriniformes)

Embryos of most species within the viviparous teleost family Goodeidae develop characteristics perianal processes that are considered to be derivatives of the embryonic hindgut. These processes, termed trophotaeniae, are covered with an epithelium that is continuous with the absorptive epithelium lining the hindgut. Gestation is intraovarian, and trophotaeniae mediate the uptake of maternally provided nutrients into the embryo from the ovarian fluid. Ultrastructural examination of the trophotaeniae of four goodeid species reveals substantial diversity in the organization of the epithelium within the family. The trophotaeniae of Alloophorus robustus, Zoogoneticus quitzeoensis, and Ilyodon furcidens have morphological features associated with the endocytosis of macromolecules and can be shown to endocytose the exogenous protein tracer horseradish peroxidase (HRP) rapidly. The trophotaenial epithelia of these species differ from one another with respect to other morphological features such as cell height, organization of the brush border, and the complexity of the intercellular spaces. The trophotaeniae of Goodea atripinnis lack an endocytotic apparatus and do not endocytose HRP. However, the overall organization of G. atripinnis trophotaenial cells suggests a function as a transporting epithelium. The cells have a dense brush border, numerous mitochondria, and many mitochondria that are enveloped by lamellar sheets of intracellular membrane. Post-fixation with osmium and potassium ferrocyanide reveals a marked difference in the complexity of the subepithelial connective tissue. Alloophorus robustus and Z. quitzeoensis exhibit an extremely electron-dense ground substance containing many acellular components. Goodea atripinnis exhibits an electron-lucid ground substance with few acellular components. © 1994 Wiley-Liss, Inc.     

Embryonic growth and trophotaenial development in goodeid fishes (Teleostei: Atheriniformes)

Embryonic growth and trophotaenial development are examined in two species of goodeid fish, Ameca splendens and Goodea atripinnis. During gestation of A. splendens, embryonic dry mass may increase from 0.21 mg at the onset of development to 31.70 mg at term. In G. atripinnis, embryonic dry mass ranges from 0.25 mg at the onset of development to 3.15 mg at term. Increase in mass is primarily due to the uptake of maternally derived nutrients by trophotaeniae, externalized embryonic gut derivatives. Trophotaenial development in both species is divisible into five phases. During the first phase, the anus is formed. The second phase involves dilation of the anus, enlargement of the perianal lips, differentiation of the hindgut absorptive epithelium, and formation of the trophotaenial peduncle. The third phase is characterized by a further marked hypertrophy and lateral expansion of the perianal lips that results in the formation of short trophotaenial processes. During the fourth phase, there is continued outward expansion of the inner mucosal surface of the trophotaenial peduncle that results in its eversion and lobulation. Placental function is established by this phase. Axial elongation and dichotomous branching of trophotaenial processes occurs during the fifth phase. Development of rosette and ribbon trophotaeniae differ in the degree of axial elongation during the fifth and final phase.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The endodermal trophotaenial epithelium in goodeid embryos acts as a placental exchange site. Fine structural and cytochemical data indicate that the trophotaenial absorptive cells are endocytotically highly active. To test their micropinocytotic capacity and characterize the cellular mechanisms involved in membrane, solute and ligand movements, living embryos of Xenoophorus captivus were incubated in saline media containing horseradish peroxidase (HRP) and/or cationized ferritin (CF) in vitro, and the uptake of these tracer proteins examined by both time sequence analysis and pulse-chase procedures. In some embryos, the effects of prolonged exposure to CF injected into the ovarian cavity, was also investigated.Labelling of the free cell surface was detectable with CF only, but interiorization of both probes was quick from all incubation media. Adsorptive pinocytosis of CF and fluid-phase uptake of HRP sequentially labelled pinocytic vesicles, endosomes, and lysosome-like bodies. In addition, CF-molecules were sequestered within apical tubules and small vesicles. HRP was largely excluded from both organelles and ended up in the lysosomal compartment. For CF, two alternative pathways were indicated by the pulse-chase experiments; transcellular passage and regurgitation of tracer molecules to the apical cell surface. The latter procedure involves membrane and receptor recycling, in which apical tubules are thought to mediate.In double-tracer experiments, using an 81 excess of HRP, external labelling with CF was light or lacking after 1–3 min, and the initial uptake-phase produced pinocytic vesicles and endosomes that mainly contained HRP-reaction product. Prolonged incubation, however, resulted in densely CF-labelled plasmalemmal invaginations and pinocytic vesicles that predominantly carried ferritin granules. After 60 min, the vacuoles of the endosomal compartment contained either high concentrations of HRP-reaction product, both tracers side by side, or virtually exclusively CF.     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

The effect of exposure to leupeptin (25 micrograms/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a double-labelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptin-treated yolk sacs were labelled with Con-A Fer at 4 degrees C and then incubated with HRP for 5, 15 or 60 min at 37 degrees C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptin-treated cells did not exhibit any labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

The trophotaenial placenta of a viviparous goodeid fish. I. Ultrastructure of the internal ovarian epithelium, the maternal component

Embryos of goodeid fishes develop to term within the ovarian lumen, where they undergo considerable increase in weight due to transfer of maternal nutrients across a trophotaenial placenta. The placenta consists of an embryonic component, the trophotaeniae, and a maternal component, the ovarian lining. The latter was examined by transmission electron microscopy, scanning electron microscopy, and light microscopy in both gravid and nongravid ovaries of the viviparous goodeid fish, Ameca splendens. The single median ovary of A. splendens is a hollow structure whose lumen is divided into lateral chambers by a highly folded longitudinal ovarian septum. Germinal tissue occurs within folds of the ovarian lining that extend into each of the two lateral chambers. Matrotrophic embryonic development takes place within ovarian chambers. During gestation, the lining of the ovarian lumen is in direct apposition to body surfaces and trophotaenial epithelia of developing embryos. The ovarian lining consists of a simple cuboidal epithelium, termed the internal ovarian epithelium (IOE), overlying a well-vascularized bed of connective tissue. Cells of the IOE are apically convex. Well-developed granular and agranular endoplasmic reticula and numerous large membrane-bound vesicles with electron-dense content occupy the apical cytoplasm of IOE cells. Two functional states of the same cell type are distinguished within the IOE. Phase I cells contain few, if any, large apically situated vesicles; Phase II cells contain many. Secretory products of the IOE are presumed to be an important source of nutrients for embryonic development. Structural and functional relationships of the IOE to the trophotaenial epithelium of developing embryos are discussed in relation to maternal-embryonic nutrient transfer processes.     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

Summary The effect of exposure to leupeptin (25 g/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a doublelabelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptintreated yolk sacs were labelled with Con-A Fer at 4°C and then incubated with HRP for 5, 15 or 60 min at 37°C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptintreated cells did not exhibit any labelling. These findings indicate that, after leupeptin treatment, both endocytotic activity and membrane recycling decrease, and that fusions of the apical vacuolar system with giant lysosomes are retarded or inhibited.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Ultrastructure of embryonic anal processes in Girardinichthys viviparus (Cyprinodontiformes,Osteichthyes)

Scanning and transmission electron microscopy were used to examine the morphology of the perianal processes (trophotaeniae) of goodeid embryos (Girardinichthys viviparus) at two stages of gestation. The epithelial surface of trophotaeniae is composed of two cell types, one of which shows distinct features associated with absorptive activity. Such cells are characterized by microvilli, abundant mitochondria, and an agranular tubulolamellar network. Micropinocytosis at the apical surface is relatively rare. The brush border membranes contain high levels of alkaline phosphatase. The cells of the second type are the minor component of the trophotaenial epithelium. Their surface is distinct, due to the presence of microridges rather than microvilli. The reticulate arrangement of the cells gives rise to intercellular spaces which occasionally are very large. These interstices are populated with leukocytes. The histological appearance of these sections indicates that this tissue is involved in gas exchange. Embryos at very early stages of development possess similar epithelia which are differentiated to a lesser extent. The connective tissue in some parts of the processes shows structural modifications. It is densely packed with numerous leukocytes occupying the spaces between the cytyoplasmic ramifications of the stroma cells. Possible roles of the trophotaeniae in absorption, respiration, excretion, and the acquisition of immunity are discussed, and it is concluded that the perianal processes of the Goodeidae are more than just trophic embryonic structures.     

Endocytotic pathways across the human gall-bladder mucosa: permeability studies using horseradish peroxidase

Summary Human gall-bladder epithelium obtained straight from the operating theatre was incubated in an Ussing chamber with the fluid phase marker, horseradish peroxidase (HRP), for up to 60 min. When the marker was presented on the apical surface, within 30 min it had moved readily across the apical cytoplasm in transport vesicles to receptosomes and into the lateral intercellular space, extending across the basement membrane into the lamina propria. When HRP was presented at the basal aspect, within 30 min it had moved through the lamina propria, across the basement membrane and into the lateral intercellular space. By 60 min, only small amounts had been taken up by the epithelial cells and transported to receptosomes. These data indicate a rapid transmucosal endocytotic pathway for blood-or bile-borne macromolecules.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macrophages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Aminopeptidases function as endocytic receptors in the trophotaenial placenta of the goodeid fish,Ameca splendens (Teleostei: Atheriniformes)

Viviparity in goodeid teleosts is characterized by the elaboration of trophotaeniae, extraembryonic proctodaeal appendages facilitating maternal-embryonic nutrient transfer. The trophotaenial absorptive cells (TACs) express aminopeptidases (APs) such as APA, APN, gamma-glutamyltransferase (gamma-GT), dipeptidyl aminopeptidase (DAP) IV, and neutral endopeptidase (NEP) as inferred from the results of cleavage experiments with, respectively, Glu-alpha-(4M beta NA), Ala-(4M beta NA), Glu-gamma-(4M beta NA), Gly-Pro-(4M beta NA), and Gl-(Ala)(3)-(4M beta NA). Enzyme reaction product was localized to the apical and basolateral plasma membrane as well as to some intracellular compartments. In the accompanying report (Schindler, 2003) evidence is presented that the trophotaeniae of Ameca splendens embryos randomly, yet specifically, bind and ingest proteins as well as certain copolymers of amino acids. Present results demonstrate that endocytosis is significantly inhibitable by unspecific proteinase inhibitors, such as diisopropylphosphorofluoride, phenylmethanesulfonylfluoride, antipain, 1.10-phenanthroline, and dithiothreitol. The specific microbial AP inhibitors amastatin, bestatin, and phosphoramidon suppressed protein binding to TACs more effectively when added in combination than did either agent alone. Moreover, in the presence of 4M beta NA assay substrates of APs the capability of TACs to bind proteins was significantly reduced. Conversely, the rate at which 4M beta NA substrates were cleaved by trophotaenial APs was modified in the presence of proteins. Depending on protein concentrations the AP-catalyzed reactions either decreased or increased in velocity. Analysis of the enzyme kinetics by methods of linear transformation suggests that proteins bind to APs competitively, thereby adopting the role of enzyme inhibitors. On the other hand, protein binding to APs appears to be a signal to translocate enzymes from an internal pool to the surface membrane. In the presence of primaquine, the rate of AP-catalyzed cleavage of 4M beta NA substrates was significantly reduced. That can be put down to the fact that weak bases disrupt the recycling of endocytosed membrane constituents. In conclusion, there is evidence that APs in the trophotaenial placenta of A. splendens function as scavenger receptors mediating in the delivery of embryotrophic proteins for lysosomal degradation.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The trophotaeniae and abdominal epidermis of Xenoophorus captivus embryos were studied by light, scanning and transmission electron microscopy, freeze-fracture replication, and histochemical techniques for unspecific phosphatases. The trophotaenial epithelium is continuous with both the intestinal mucosa and the epidermis, and contains structural elements similar to both. The predominant component is a simple brush-border epithelium consisting of cuboid cells showing signs of endocytotic activity at their apical surfaces. These are the absorptive elements of the trophotaeniae, and phosphatase ultracytochemistry demonstrates the presence of alkaline phosphatase on the external leaflet of their exposed plasma membranes. Enormously dilated intercellular spaces and large gaps occur in this epithelial covering.Beneath this absorptive epithelium lies an incomplete layer of dense squamous cells that appear to be derived from the stratified epithelium covering trophotaenial areas free of brush border epithelium and the abdominal wall. The exposed cell surfaces of this component are modified to form an elaborate pattern of microplicae which can be seen by scanning EM where gaps appear in the overlying absorptive epithelium. The stratified epithelium of the abdominal wall is underlain with collagen fibrils and an intricate network of capillaries, and is considered to be a site of cutaneous respiration. This cutaneous gas-exchange pathway averages 2–4 m in thickness. Chloride cells are constituents of the stratified epithelium of the trophotaenial base and abdominal wall.The involvement of the endodermal component of the trophotaenial epithelium in the transfer of nutrients and possibly antibodies, and the role of the abdominal epidermis and ectodermal trophotaenial epithelium in gas exchange and osmoregulation, are discussed.     

Morphological studies of the goldfish hindgut mucosa in organ culture

Summary The morphology of the absorptive cells of the goldfish hindgut mucosa, and their capability for horseradish peroxidase (HRP) uptake, were investigated by electron microscopy after a 24-h organ culture. The columnar appearance and the fine structure of the absorptive cells were well preserved for 24 h at room temperature and 37° C with 5% CO₂ in air, in all the media used in this study. Mitoses were frequently observed in the epithelium at the bottom of cultured mucosal folds, and re-epithelization was also observed in many explants.Some structural changes were, however, noted in the cultured absorptive cells, as compared with the non-cultured absorptive cells; the deep invaginations of the surface membrane between the microvilli decreased in number; supranuclear giant vacuoles were reduced in size or almost disappeared; the distributional pattern of mitochondria in the absorptive cells was altered.The HRP uptake experiments showed that the absorptive cells cultured for 24 h could still take up HRP by endocytosis and transport it, indicating that the absorptive cells maintained their capability of macromolecule uptake and transport after 24 h of culture. In addition, HRP experiments, in which reaction product was detected within numerous cytoplasmic tubules (CT), various vacuoles and CT-vacuole complexes, suggested a close relationship between CT and vacuolar system in the apical cytoplasm during endocytotic events in the absorptive cells.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

Summary The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4°C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5 60 min at 20° and 37°C in Con-A-free serum resulted in a temperature-dependent internalization of membranebound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37°C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constitutents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Membrane differentiations of an absorptive epithelium covering embryonic trophotaeniae in a goodeid teleost

Summary The trophotaenial absorptive cells (TACs) in goodeid embryos facilitate nutrient absorption during prolonged periods of intraovarian gestation. In a study of membrane differentiations associated with solute and ligand transfer in the trophotaeniae of Xenotoca eiseni, embryos were incubated in vivo with cationized ferritin (CF) prior to freeze-cleaving. This exposure to high concentrations of an adsorptive ligand was meant to induce swelling of the endosomal compartment. Macromolecular trafficking in TACs occurs via an apical endocytic complex consisting of plasma membrane invaginations, a large population of small vesicles, uniformly thick apical tubules, and endosomes. Freeze-fracture replicas showed that the microvillar plasma membrane P-face of TACs was studded with intramembrane particles (IMPs) at a fairly high density, whereas that of the cell surface proper contained a distinctly lower density and the tubulovesicular endocytic pits contained almost no IMPs. The majority of small vesicles and apical tubules in a near surface position displayed P-fracture faces with only a few odd IMPs, indicating that membrane, shuttling between the apical plasma membrane and intracellular sorting organelles, obviously does not carry along many large-sized integral membrane proteins. The distended endosomal compartment had many P-face-associated particles primarily clustered into patches. Specializations of the lateral plasma membrane included 4–8 tight junctional strands, relatively large complements of gap junction proteins, and numerous plaques of desmosomal membrane particles. A system of lamellar cisternae underlay the lateral cell surface that was in continuity with the intraepithelial space by numerous tubular canals, giving rise to an intracellular amplification of the basolateral plasma membrane. Their outward openings appeared as tiny pits on the cytoplasmic faces of freeze-cleaved cell membrane. The density of IMPs on the P-faces of the surface plasma membrane was apparently lower than that on its invaginated lamellar complex. Hence, it is concluded that the mobility of integral membrane proteins in the plane of the membrane may be hampered in movement across the surface pores.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.