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.     

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.     

The follicular placenta of the viviparous fish,Heterandria formosa. I. Ultrastructure and development of the embryonic absorptive surface

Embryos of the poeciliid Heterandria formosa develop to term in the ovarian follicle in which they establish a placental association with the follicle wall (follicular placenta) and undergo a 3,900% increase in embryonic dry weight. This study does not confirm the belief that the embryonic component of the follicular placenta is formed only by the surfaces of the pericardial and yolk sacs; early in development the entire embryonic surface functions in absorption. The pericardial sac expands to form a hood-like structure that covers the head of the embryo and together with the yolk sac is extensively vascularized by a portal plexus derived from the vitelline circulation. The hood-like pericardial sac is considered to be a pericardial amnion-serosa. Scanning and transmission electron microscopy reveal that during the early and middle phases of development (Tavolga's stages 10–18 for Xiphophorus maculatus) the entire embryo is covered by a bilaminar epithelium whose apical surface is characterized by numerous, elongate microvilli and coated pits and vesicles. Electron-lucent vesicles in the apical cytoplasm appear to be endosomes while a heterogeneous group of dense-staining vesicles display many features characteristic of lysosomes. As in the larvae of other teleosts, cells resembling chloride cells are also present in the surface epithelium. Endothelial cells of the portal plexus lie directly beneath the surface epithelium of the pericardial and yolk sacs and possess numerous transcytotic vesicles. The microvillous surface epithelium becomes restricted to the pericardial and yolk sacs late in development when elsewhere on the embryo the non-absorptive epidermis differentiates. We postulate that before the definitive epidermis differentiates, the entire embryonic surface constitutes the embryonic component of the follicular placenta. The absorptive surface epithelium appears to be the principle embryonic adaptation for maternal-embryonic nutrient uptake in H. formosa, suggesting that a change in the normal differentiation of the surface epithelium was of primary importance to the acquisition of matrotrophy in this species. In other species of viviparous poeciliid fishes in which there is little or no transfer of maternal nutrients, the embryonic surface epithelium is of the non-absorptive type.     

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.     

Scavenger receptors facilitate protein transport in the trophotaenial placenta of the goodeid fish,Ameca splendens (Teleostei: Atheriniformes)

In the goodeid placental analogue, trophotaeniae provide extraembryonic gut-derived exchange surfaces. Ameca splendens embryos possess endocytosing trophotaeniae that are capable of absorbing a dazzling array of proteinaceous substances. The iron core protein, native ferritin (NF), and several radioiodinated proteinaceous substances were used to study ligand and binding site pathways in the trophotaenial absorptive cells (TACs). Time sequence analysis of NF trafficking indicated an exclusively lysosomal pathway. Binding to TACs of NF was completely inhibitable by proteins containing multiple lysine residues such as apoferritin, bovine serum albumin (BSA), human transferrin (HTf), fetuin, hemoglobin, myoglobin, cytochrome c, ubiquitin, parvalbumin as well as the random copolymers, poly(Glu,Lys,Tyr)6:3:1 and poly(D-Glu,D-Lys)6:4. Peptide hormones and pepsin that contains only one lysine residue did not produce inhibitory effects. Radiolabels such as (125)I-BSA, (125)I-HTf and (125)I-poly(Glu,Lys,Tyr) bound to trophotaeniae in a specific saturable manner. Any two proteins were shown to hinder one another in getting hold of a binding site. Concentration-dependent (125)I-BSA binding and Scatchard analysis of the data revealed both low- and medium-affinity binding with apparent dissociation constants, K(d)s, of 3.4 x 10(-5) M and 2 x 10(-7) M, respectively. Binding of NF and radioiodinated proteins was inhibited in the presence of a large excess of L-Lys, D-Lys, and several dipeptides containing Lys. Both Ca(2+)-depletion and low pH dramatically reduced the TACs' capacity to bind proteins. The effects of acidotropic agents included a reversible loss of surface protein binding sites, tremendous vacuolation, and the arrest of lysosomal degradation. Collectively, present results demonstrate that TACs bind and absorb multiple proteinaceous substances through a mechanism satisfying the criteria of receptor-mediated endocytosis. It is concluded that scavenger protein binding sites are used to ingest proteins for lysosomal degradation, helping to meet the embryos' amino acid requirement.     

Ultrastructure of the full-term shark yolk sac placenta. III. The maternal attachment site

During mid- and late gestation, the uterus of sandbar sharks possesses specialized sites for exchange of metabolites between the mother and fetus. Attachment sites are highly vascular, rugose elevations of the maternal uterine lining that interdigitate with the fetal placenta. The maternal epithelium remains intact and there is no erosion. The attachment site consists of a simple, low columnar juxtaluminal epithelium underlain by an extensive vascular network. Juxtaluminal epithelial cells possess branched microvilli, saccular invaginations of the apical surface, and coated pits. They contain numerous coated vesicles, lipid-like inclusions, a prominent rough endoplasmic reticulum, and many free ribosomes. Tight junctions join the luminal aspect of adjacent cells. Lateral cell boundaries are highly folded and interdigitated. Capillaries are closely apposed to the basal cell surfaces. The endothelium is pinocytotically active. Comparison with the uterine epithelium of non-placental sharks, mammalian epitheliochorial placentae, and selected transporting epithelia reveals that the structure of the maternal shark placenta is consistent with its putative multiple functions, viz: (1) nutrient transfer; (2) transport of macromolecules, e.g., immunoglobulins; (3) respiration; and (4) osmotic and ionic regulation.     

Follicular placenta and embryonic growth of the viviparous four-eyed fish (Anableps)

In the four-eyed fish, Anableps (Atheriniformes, Anablepidae), eggs are fertilized and embryos develop to term within the ovarian follicles. Development is highly matrotrophic. During gestation, the largest term embryo of A. anableps examined had grown to a total length of 51 mm and attained a dry weight of 149 mg. The postfertilization weight increase is 298,000%. The largest term embryo of A. dowi examined had grown to a total length of 77 mm and attained a dry weight of 910 mg. The postfertilization weight increase is 843,000%. Embryonic weight increases result from nutrient transfer across the follicular placenta. This structure is formed by apposition of the maternal follicular epithelium to absorptive surface cells of the embryo's pericardial trophoderm. The latter, a ventral ramification of the pericardial somatopleure, replaces the yolk sac during early gestation. The external surface of the pericardial trophoderm develops hemispherical projections, termed vascular bulbs. Within each bulb, the vascular plexus of the trophoderm expands to form a blood sinus. Cells of the external surface of the bulbs possess microplicae. Microvilli are absent. During middle to late gestation, the juxtaembryonic follicular epithelium differentiates into two regions. One region consists of shallow, pitlike depressions within which vascular bulbs interdigitate in a “ball and socket” arrangement. Follicular pits are formed by the curvilinear distortion of the apical surfaces of follicle cells. The second region in contact with the dorsal and lateral surfaces of the embryo, is comprised of villous extensions of the hypertrophied follicular epithelium. In both regions, follicle cells appear to constitute a transporting rather than a secretory epithlium. In terms of percentage of weight increase, the follicular placenta of Anableps appears to be the most efficient adaptation for maternal-embryonic nutrient transfer in teleost fishes and closely approaches the efficiency (1.2 × 10⁶%) of oophagy and embryonic cannibalism in lamnoid sharks.     

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig,Hemimerus talpoides

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended “postfertilization” activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.     

The fine structure of an absorptive epithelium in a viviparous teleost

Embryos of the viviparous teleost, “Characodon” eiseni, have unusual anal processes that function only during gestation and are lost shortly after birth. This study was undertaken to determine if the fine structure of the process epithelium supports the assumption that these cells have an absorptive function. The process epithelium is a single layer of columnar cells. At peak activity intercellular spaces become very large and isolate individual cells which simultaneously lose much of their cell mass. The cells are characterized by microvilli on their free surface, much pinocytic activity and by the formation of at least four different kinds of vesicles. There is much evidence that these vesicles fuse together. A distinctive characteristic of these cells is a system of tubules and flattened cisternae that somewhat resemble the endoplasmic reticulum yet they differ from it in several respects. It is suggested these profiles aid in fragmenting the cell at periods of peak absorptive activity, thereby increasing the cell surface. Possible mechanisms of food absorption are considered. The fine structure of these cells supports the contention that these embryonic processes serve as absorptive organs during gestation.     

Myeloid bodies in the pigment epithelium of a teleost embryo, the viviparous Poecilia reticulata

Appearance of myeloid bodies (MB) in the retinal pigment epithelium (RPE) precedes photoreceptor outer segment development in Poecilia reticulata embryos reared under a 12 hrs LD cycle, in constant darkness (DD) and constant light (LL). When first formed, MB are predominantly continuous with rough endoplasmic reticulum (RER). The same is observed in the peripheral growth zone of the developed eye, whereas in differentiated parts, MB are continuous with the smooth endoplasmic reticulum (SER). At onset of photomechanical movements, wavy MB predominate in light-adapted LD embryos, are exclusively present in LL and are located in the RPE processes. SER abounds. Straight MB predominate in dark-adapted LD embryos, are exclusively present in DD and contain electrondense material between lamellae. Diurnal appearance of electrondense material may be coupled with transfer of retinol, mediated by various transport proteins.     

Ultrastructure of the full-term shark yolk sac placenta. II. The smooth, proximal segment

The smooth, proximal portion of the yolk sac placenta of the sandbar shark, Carcharhinus plumbeus is comprised of: (1) An outermost epithelial ectoderm; (2) an intervening collagenous stroma; and (3) an inner mesothelium. The surface epithelium may be one to three cell layers thick. The surface epithelium comprises two cell types. A cuboidal cell that has a dome-like apical surface covered with microvilli and an ovoid nucleus predominate. These cells contain lipid inclusions, many cytoplasmic filaments, and are joined by desmosomes. The second cell type has a convoluted nucleus and a flattened cell apex with microvilli, cilia, and paddle cilia. Golgi complexes and elements of the endoplasmic reticulum are relatively uncommon in the cytoplasm of both cell types. Microplicae also occur on the surface of some cells. The smooth, proximal portion of the placenta is sparsely vascularized. The innermost cellular elements of the surface epithelium rest on a prominent basal lamina. A collagenous zone separates the epithelial basal lamina from the basal lamina of the mesothelium. The mesothelial cells are squamous with a fusiform nucleus, many pinocytotic pits and vesicles, and a large number of cytoplasmic filaments. The endoplasmic reticulum, except for occasional patches of the rough type, and the Golgi complex are poorly developed. Ultrastructural tracer studies show that this portion of the placenta does not absorb horseradish peroxidase (HRP) and trypan blue.     

Cyto-epitheliochorial placenta of the viviparous lizard Pseudemoia entrecasteauxii: a new placental morphotype

The structural features of the uterine epithelium of the chorioallantoic placenta and omphalloplacenta in the viviparous Australian skink, Pseudemoia entrecasteauxii, were investigated using SEM and TEM techniques. In particular, the structural characteristics that would allow interpretation of function were analyzed, particularly those of gas exchange in the chorioallantoic placenta and histotrophy in the omphaloplacenta. Pseudemoia entrecasteauxii has a complex placenta consisting of a placentome, paraplacentome, and omphaloplacenta. The paraplacentome has a well-vascularized lamina propria in which projecting uterine capillaries displace the overlying uterine epithelial cells, reducing them to attenuated cytoplasmic extensions. Associated cell nuclei and organelles are lost from this region, to provide a capillary lumen to uterine lumen barrier of 0.5-1.0 microm. Hence, the paraplacentome is likely a prominent site for gaseous exchange via simple diffusion. The omphaloplacenta has a similar cytology to that of the placentome, but the uterine epithelial cells are hypertrophied and the apical plasma membrane actively secretes vesicles into the uterine lumen. The omphaloplacenta shows features that are associated with histotrophic transport of nutrients via vesicle secretion, very similar to that of lipid apocrine secretion. The placentome consists of cuboidal cells in the uterine epithelium, with large centrally located nuclei overlying the well-vascularized lamina propria. Although the placentome has a similar cytological structure to that of the omphaloplacenta, granules or active vesicle secretion were not observed. Thus, the placentome may be associated with histotrophy, but not via apocrine secretion. Squamate placentation is epitheliochorial; however, we propose a new term be used to describe the type of placentation in P. entrecasteauxii: "cyto-epitheliochorial," because of the extreme attenuation of uterine epithelial cells of the paraplacentome.     

Khawkinea quartana, a Colorless Euglenoid Flagellate. I. Ultrastructure

Khawkinea quartana, a naturally occurring colorless homologue of Euglena, was examined with the electron microscope. The organism is biflagellate though only one of the 2 flagella emerges from the anterior reservoir. The pellicular strips covering the body of the organism are supported by microtubules which are continuous in part with microtubules bordering the reservoir. Additional rows of microtubules are found associated with the kinetosomes. An eyespot is located in the wall of the reservoir and, adjacent to it, the contractile vacuole. The nucleus, mitochondria, and Golgi complexes are similar to those described in other euglenoid flagellates. The food reserve is paramylon. The study supports the phylogenetic origin of Khawkinea from pigmented Euglena through the loss of chloroplasts.     

Ultrastructure of the full-term shark yolk sac placenta. I. Morphology and cellular transport at the fetal attachment site

During ontogeny, the yolk sac of some viviparous sharks differentiates into a yolk sac placenta that persists to term. The placenta is non-invasive and non-deciduate. Hematrophic transport is the major route of nutrient transfer from mother to fetus. The placental unit consists of: (1) an umbilical stalk; (2) the smooth, proximal portion of the placenta; (3) the distal, rugose portion; (4) the egg envelope; and (5) the maternal uterine tissues. Exchange of metabolites is effected through the intervening egg envelope. The distal rugose portion of the placenta is the fetal attachment site. It consists of: (1) surface epithelial cells; (2) a collagenous stroma with vitelline capillaries; and (3) an innermost boundary cell layer. The columnar surface epithelial cells are closely apposed to the inner surface of the egg envelope. Wide spaces occur between the lateral margins of adjacent cells. Surface epithelial cells contain an extensive apical canalicular-tubular system and many whorl-like inclusions in their basal cytoplasm. Capillaries of the vitelline circulation are closely situated to these cells. A well-developed collagenous stroma separates the surface epithelium from an innermost boundary cell layer. In vitro exposure of full-term placentae to solutions of trypan blue and horseradish peroxidase (HRP) reveals little uptake by the smooth portion of the placenta but rapid absorption by the surface epithelial cells of the distal, rugose portion. HRP enters these cells by an extensive apical system of smooth-walled membranous anastomosing canaliculi and tubules. Prominent whorl-like inclusions that occupy the basal cytoplasm of the surface cells, adjacent to the pinocytotically active endothelium of the vitelline capillaries, are hypothesized to be yolk proteins that are transferred from the mother to embryo throughout gestation.