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.     

The trophotaenial placenta of a viviparous goodeid fish. III: Protein uptake by trophotaeniae, the embryonic component

Protein uptake and degradation by trophotaenial cells of the viviparous goodeid fish Ameca splendens were studied colorimetrically and ultrastructurally using horseradish peroxidase (HRP) as a tracer and acid (ACPase) and alkaline (ALPase) phosphatase cytochemistry. Trophotaeniae are ribbon-like external projections of the embryonic gut that are equivalent to greatly hypertrophied intestinal villi. During gestation within the ovarian lumen, trophotaeniae are directly apposed to the internal ovarian epithelium (IOE) where they establish a placental association between the developing embryo and maternal organism. Trophotaenial absorptive cells possess an ALPase reactive brush border, an endocytotic apparatus, and ACPase reactive standing lysosomes. Ultrastructural studies of protein uptake indicate that cells of the trophotaenial epithelium take up HRP by micropinocytosis and degrade it within lysosomes. Initially (from 1.5-10 min), HRP is taken up in vitro at 22 degrees C at the apical cell surface and passes via endocytotic vesicles into an apical canalicular system. From 1.5 to 10 min exposure, HRP passes passes from the apical canalicular system to a series of small collecting vesicles. After 10 min, HRP is detected within large ACPase reactive supranuclear lysosomes. Three hours after an initial 1 h exposure to HRP, most peroxidase activity within supranuclear lysosomes is no longer detected. Presence of Golgi complexes, residual bodies, and secretory granules in the infranuclear cytoplasm suggest that products of protein uptake and hydrolysis are discharged across basal and lateral cell surfaces and into the trophotaenial circulation. Trophotaeniae of embryos incubated in vitro in HRP-saline take up HRP at an initial rate of 13.5 ng HRP/mg trophotaenial protein/min. The system becomes saturated after 3 h. Trophotaeniae incubated at 4 degrees C show little or no uptake. In trophotaeniae continuously pulsed with HRP for 1 h, then incubated in HRP-free saline, levels of absorbed peroxidase declined at a rate of 0.5 ng/mg trophotaenial protein/min. HRP does not appear to enter the embryo via extra-trophotaenial routes. These findings are consistent with the putative role of trophotaeniae as the embryonic component of the functional placenta of goodeid fishes. Trophotaenial uptake of maternal nutrients accounts for a massive (15,000%) increase in embryonic dry weight during gestation.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

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)     

An examination of surface epithelium structures of the embryo across the genus Poeciliopsis (Poeciliidae)

In viviparous, teleost fish, with postfertilization maternal nutrient provisioning, embryonic structures that facilitate maternal‐fetal nutrient transfer are predicted to be present. For the family Poeciliidae, only a handful of morphological studies have explored these embryonic specializations. Here, we present a comparative morphological study in the viviparous poeciliid genus, Poeciliopsis . Using microscopy techniques, we examine the embryonic surface epidermis of Poeciliopsis species that vary in their level of postfertilization maternal nutrient provisioning and placentation across two phylogenetic clades and three independent evolutionary origins of placentation. We focus on surface features of the embryo that may facilitate maternal‐fetal nutrient transfer. Specifically, we studied cell apical‐surface morphology associated with the superficial epithelium that covers the body and sac (yolk and pericardial) of embryos at different developmental stages. Scanning electron microscopy revealed common surface epithelial cells across species, including pavement cells with apical‐surface microridges or microvilli and presumed ionocytes and/or mucus‐secreting cells. For three species, in the mid‐stage embryos, the surface of the body and sac were covered in microvillus epithelium. The remaining species did not display microvillus epithelium at any of the stages examined. Instead, their epithelium of the body and sac were composed of cells with apical‐surface microridges. For all species, in the late stage embryos, the surface of the body proper was composed of apical‐surface microridges in a “fingerprint‐like arrangement.” Despite the differences in the surface epithelium of embryos across Poeciliopsis species and embryonic developmental stages, this variation was not associated with the level of postfertilization maternal nutrient provisioning. We discuss these results in light of previous morphological studies of matrotrophic, teleost fish, phylogenetic relationships of Poeciliopsis species, and our earlier comparative microscopy work on the maternal tissue of the Poeciliopsis placenta.     

A scanning electron microscope study of ectodermal differentiations in the caudal mantle epithelium of embryos and juveniles of Loligo vulgaris,Loligo forbesi and Sepia officinalis

Summary

The mantle epithelium of embryos and early juveniles of the squids Loligo vulgaris and Loligo forbesi and the cuttlefish Sepia officinalis was studied using scanning electron microscopy. In embryos of L. vulgaris and L. forbesi, previously undescribed epidermal structures were found. They are missing in S. officinalis embryos. These so-called “extruding structures” are located near Hoyle's organ and first appear at stage XIII of Naef. At the same embryonic stage, Hoyle's organ starts to differentiate and “uniform-type” ciliated cells become visible in the epidermis of both L. vulgaris and L. forbesi. Directly after hatching the epidermis of the species examined starts to slough off and finally the extruding structures, Hoyle's organ and both types of ciliated cells of the mantle epithelium disappear. The function of the extruding structures remains obscure.     

Reproduction,placentation, and embryonic development of the Atlantic sharpnose shark,Rhizoprionodon terraenovae

The Atlantic sharpnose shark Rhizoprionodon terraenovae (Richardson) is a small carcharhinid that is a common year-round resident along the southeast coast of the United States. It is viviparous and its embryos develop an epithelio-vitelline placenta. Females enter shallow water to give birth in late May and early June. Mating occurs shortly after parturition, and four to seven eggs are ovulated. Fertilized eggs attain the blastoderm stage in early June to early July. Separate compartments for each egg are formed in the uterus when the embryos reach 3–30 mm. Embryos depend on yolk for the first 8 weeks of development. When embryos reach 72 mm their yolk supply is nearly depleted and they shift to matrotrophic nutrition. When the embryos reach 40–55 mm, placental development begins with the vascularization of the yolk sac where it contacts the uterine wall. Implantation occurs at an age of 8–10 weeks by which time the embryos reach 70–85 mm. The expanding yolk sac engulfs the maternal placental villi, and its surface interdigitates with the villi to form the placenta. The rest of the lumenal surface of the uterus is covered by non-placental villi that appear shortly after implantation. Histotrophe production by the non-placental villi begins just after their formation. The placenta grows continuously during gestation. The egg envelope is present throughout gestation, separating maternal and fetal tissues. Embryos develop numerous appendiculae on the umbilical cord. Young sharks are born at 290–320 mm after a gestation period of 11 to 12 months. © 1993 Wiley-Liss, Inc.     

Proliferation of Oogonia and folliculogenesis in the viviparous teleost Ilyodon whitei (Goodeidae)

Oogonial proliferation in fishes is an essential reproductive strategy to generate new ovarian follicles and is the basis for unlimited oogenesis. The reproductive cycle in viviparous teleosts, besides oogenesis, involves development of embryos inside the ovary, that is, intraovarian gestation. Oogonia are located in the germinal epithelium of the ovary. The germinal epithelium is the surface of ovarian lamellae and, therefore, borders the ovarian lumen. However, activity and seasonality of the germinal epithelium have not been described in any viviparous teleost species regarding oogonial proliferation and folliculogenesis. The goal of this study is to identify the histological features of oogonial proliferation and folliculogenesis during the reproductive cycle of the viviparous goodeid Ilyodon whitei. Ovaries during nongestation and early and late gestation were analyzed. Oogonial proliferation and folliculogenesis in I. whitei, where intraovarian gestation follows the maturation and fertilization of oocytes, do not correspond to the late oogenesis, as was observed in oviparous species, but correspond to late gestation. This observation offers an example of ovarian physiology correlated with viviparous reproduction and provides elements for understanding the regulation of the initiation of processes that ultimately result in the origin of the next generation. These processes include oogonia proliferation and development of the next batch of germ cells into the complex process of intraovarian gestation. J. Morphol. 275:1004–1015, 2014. © 2014 Wiley Periodicals, Inc.     

Structure and function of placental exchange surfaces in goodeid fishes (Teleostei: Atheriniformes)

The species of the family Goodeidae have evolved reproductive strategies involving intraovarian gestation, early evacuation of nearly yolk‐exhausted embryos from the ovigerous tissue into the ovarian cavity, placental matrotrophy during intraluminal gestation, and the birth of highly developed fry. The inner ovarian lining becomes hypervascularized during gestational periods and functions as the maternal component of the placental association. Embryotrophic liquid is secreted by the inner ovarian epithelium into the ovarian cavity. Comparative electrophoretic analyses of embryotrophe and maternal blood serum provide evidence for the transfer of maternal serum proteins into the embryotrophe. Trophotaeniae, proctodaeal processes of the embryos, provide a surface for nutrient absorption. Endocytic activity was demonstrated by ingestion of unspecific tracer proteins in various species. Moreover, the trophotaenial absorptive cells (TACs) in Ameca splendens ingest various proteins or random copolymers conjugated to colloidal gold as well as radioiodinated proteins in a way that satisfies the criteria of receptor‐mediated endocytosis. Several aminopeptidases (APs) on the surface of TACs were identified as protein binding sites as evidenced by inhibition of binding and uptake of marker proteins in the presence of AP substrates or AP inhibitors. Morphological adaptations of the embryonic circulatory system pertaining to nutrient and gas exchange were characterized. The embryonic epidermis comprises two layers of squamous cells closely underlain by a dense capillary net. Efficient gas exchange is facilitated by a thin embryotrophe‐blood barrier of both the embryonic skin and the intraovarian lining. J. Morphol. 276:991–1003, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

Follicular placenta of the viviparous fish,Heterandria formosa: II. Ultrastructure and development of the follicular epithelium

Embryos of the viviparous poeciliid fish, Heterandria formosa, develop to term in the ovarian follicle where they undergo a 3,900% increase in embryonic dry weight. Maternal-embryonic nutrient transfer occurs across a follicular placenta that is formed by close apposition of the embryonic surface (i.e., the entire body surface during early gestation and the pericardial amnionserosa during mid-late gestation) to the follicular epithelium. To complement our recent study of the embryonic component of the follicular placenta, we now describe the development and fine structure of the maternal component of the follicular placenta. Transmission electron microscopy reveals that the ultrastructure of the egg envelope and the follicular epithelium that invests vitellogenic oocytes is typical of that described for teleosts. The egg envelope is a dense matrix, penetrated by microvilli of the oocyte. The follicular epithelium consists of a single layer of cuboidal cells that lack apical microvilli, basal surface specializations, and junctional complexes. Follicle cells investing the youngest embryonic stage examined (Tavolga's and Rugh's stage 5–7 for Xiphophorus maculatus) also lack apical microvilli and basal specializations, but possess junctional complexes. In contrast, follicle cells that invest embryos at stage 10 and later display ultrastructural features characteristic of transporting epithelial cells. Apical microvilli and surface invaginations are present. The basal surface is extensively folded. Apical and basal coated pits are present. The cytoplasm contains a rough endoplasmic reticulum, Golgi complexes, and dense staining vesicles that appear to be lysosomes. The presence of numerous apically located electron-lucent vesicles that appear to be derived from the apical surface further suggests that these follicle cells may absorb and process follicular fluid. The egg envelope, which remains intact throughout gestation and lacks perforations, becomes progressively thinner and less dense as gestation proceeds. We postulate that these ultrastructural features, which are not present in the follicles of the lecithotrophic poeciliid, Poecilia reticulata, are specializations for maternal-embryonic nutrient transfer and that the egg envelope, follicular epithelium, and underlying capillary network form the maternal component of the follicular placenta. © 1994 Wiley-Liss, Inc.     

Fine structure at the basal surface of intestinal epithelium in the midgut region of the balanidae,with special reference to “Neural-like” processes

Observations on fine structure at the basal end of the intestinal epithelium in the midgut region of Balanus balanoides and Balanus improvisus reveal complex interrelationships among several tissues. Numerous elongate cell processes extend towards the intestinal epithelium penetrating between layers of intestinal muscle through blood spaces and into the basal lamina underlying the epithelium. Two types of morphological relationships occur between cell processes and the basal end of the intestinal epithelial cell: 1. The cell process may penetrate the basal lamina and lie closely apposed to the epithelium. 2. The cell process may give rise to narrow, medially-directed, finger-like extensions (projections). The narrow projections penetrate the basal lamina and, in addition, terminate as dilated bulbs within inpocketings of the epithelium. In some respects the cell processes are suggestive of neural tissue.     

Ultrastructure of the epidermis and digestive tract inSebastes embryos,with special reference to the uptake of exogenous nutrients

Synopsis Ultrastructural features of the epidermis and rectum were studied inSebastes schlegeli andS. melanops during the late stages of embryonic development, to confirm uptake of maternal substances. Ruthenium red (RR) and horseradish peroxidase (HRP) were used at fixation and in live embryos, respectively. Epidermal tissue of embryos after developmental stage 24 comprised two squamous cell layers. The outer, thinner cells and their intercellular spaces were easily infiltrated with RR, but the inner cells had no RR deposition. The HRP was not incorporated into the epidermis except in a few outer cells, which had well-developed microvillous projections of cytoplasm. Sacciform cells, chloride cells, and mucous cells distributed in the inner layer but protruding to the epidermal surface had no intracellular RR and HRP depositions. The rectal cells of embryos at about developmental stage 28 had many globular inclusions containing electron-dense substances. The rectal cells were found to take up and digest HRP actively. It is suggested that the embryonic epidermis is structurally loose and takes up low weight molecules, while rectal cells, after the opening of the mouth, actively ingest exogenous, high weight molecules.     

Morpho‐histological and ultra architectural changes during early development of endangered golden mahseer Tor putitora

Ultrastructural and histological changes in the embryonic and larval surface during ontogenesis of the endangered golden mahseer Tor putitora is studied here for the first time. Embryonic development was completed 91–92 h after fertilization at an ambient temperature of 23° ± 1° C (mean ± s.d. ). The gastrula stage was characterized by presence of the Kupffer's vesicle, notochord, ectoderm and endoderm cells. Primordial germ cells were clearly identifiable from c. 55 h post‐fertilization at the organogenesis stage. Mean total length of newly hatched larvae was 7·0 ± 0·5 mm. Scanning electron microscopy of newly hatched larvae demonstrated vitelline arteries, microridged epithelial cells and mucous gland openings over much of the body surface. Eye, oral cavity, pharyngeal arches, heart, intestinal loop, prosencephalon, cephalic vesicle and nasal epithelium were clearly distinguished in 3 day old hatched individuals. In 6 day old individuals, caudal‐fin rays and internal organs were evident. The dorsal fin became prominent at this stage and larvae began swimming at the surface. The reserved yolk material was totally absorbed 8–11 days after hatching and larvae began feeding exogenously. Tor putitora exhibited a longer early developmental period than other cyprinids reared at similar temperatures.