Guinea-pig Yolk Sac Research Articles

Development and morphology of the inverted yolk sac in the guinea pig (Cavia porcellus)

Although the guinea pig is an important animal model for human placentation, aspects of fetal nutrition are not fully understood, especially in regard to the yolk sac that is regarded to be essential for early development of the embryo. We investigated differentiation by means of histology, histochemistry, immunohistochemistry, and transmission electron microscopy. Data suggest that the guinea pig's yolk sac was not sufficiently developed to facilitate substantial fetal nutrition in early pregnancy. On Day 12, it was a flat, inverted, but avascular structure. This was followed by differentiation to form the typical, highly villous and vascularized condition of advanced gestation. Finally, the yolk sac degenerated toward term. We suggest that the guinea pig and other caviomorphs rely predominantly on hemotrophic nutrition via the placenta even in very early pregnancy. In contrast to the general pattern of mammals, histiotrophic nutrition via yolk sac routes seems to be most essential during mid-gestation.

Ontogeny of Expression of Insulin-like Growth Factor (IGF) and IGF Binding Protein mRNAs in the Guinea-pig Placenta and Uterus

To determine the temporal and spatial distribution of insulin-like growth factor (IGF) and its family of binding proteins (IGFBPs), guinea-pig yolk sac and chorioallantoic placentae were collected at 15, 20, 25, 29, 44–45, 55 and 65–66 days of gestation. Messenger RNAs for IGF I, IGF II and IGFBP 1–6 were identified in tissue sections by in situ hybridization, using35S-cRNA probes. Epithelial and mesenchymal cell types were identified by immunohistochemistry for cytokeratin and vimentin, respectively. At 15 days of gestation, IGF-II mRNA was expressed in ectoplacental mesoderm, cytotrophoblasts and syncytiotrophoblast, and IGFBP-5 mRNA was detected in the syncytiotrophoblast. In the mid-gestation placenta, IGFBP-5 mRNA was expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth. Near term, when expansion of the labyrinth was complete, IGFBP-5 mRNA was coexpressed with IGF-II mRNA in the marginal and interlobular syncytium. These observations suggest that interaction between IGF-II and IGFBP-5 plays a role in the vascularization of the placenta by fetal vessels. IGF-II mRNA was not expressed in the maternal tissues at any gestational age. IGFBP-2, -3 and -5 mRNAs were expressed in the endometrial stroma at 7–12 days of gestation but, following establishment of the placenta, IGFBP mRNAs were more abundant in the myometrium than in the decidua. IGF-II mRNA was detected in trophoblasts invading the walls of maternal vessels, and the endothelium of the preplacental vessels expressed IGFBP-4 mRNA, while IGFBP-2 and IGFBP-5 mRNAs were present in the tunica media of mesometrial arteries that had not been invaded by trophoblast. These findings suggest that IGF-II produced by the trophoblast acts in an autocrine and/or paracrine fashion to promote trophoblast invasion and that this process is modulated by interaction with IGFBPs present in maternal tissues.

The artificially perfused guinea-pig yolk sac placenta: Transfer and uptake of water, glucose and amino acids

The development of a new technique to investigate maternal fetal transfer across the near term guinea pig yolk sac placenta by in-situ perfusion of the yolk sac vessels is described. The maternal fetal transfer of labeled water, d- and l-glucose, O-methyl- d-glucose (oMDG), d- and l-alanine, d- and l-aspartate, L-lactate and alphaamino- isobutyric acid (AIBA) was investigated after injection of these substances into the maternal circulation. After 15 min of perfusion at 0.5 ml/min the water clearance was 132 ± 12 μ/ min ( SEM, n = 30). The clearances for d- or l-glucose were less than 1.2,μl/min. The activity of label in the venous yolk sac perfusate of all other substances was not different from background activity when 14C-label was used. The clearance of 3H- l-alanine approached the clearance value of water. The total uptake (as defined for single-injection double tracer dilution experiments) from the perfusate of d-glucose, oMDG, alanine and aspartate in comparison to l-glucose was also studied. Mean d-glucose uptake was 11.2 ± 1.9 per cent ( n = 8), it was significantly reduced to 4.9 ± 2 per cent ( n = 5) by cytochalasin B (1 × 10 −4 mmol/l), and by increasing concentrations of d-glucose (1 to 20 mmol/l, n = 4). The uptake of oMDG was 8.8 ± 1.5 per cent ( n = 8). l-alanine uptake was 25 ± 3.4 per cent, d-alanine uptake was 8.3 ± 1.5 per cent ( n = 12). Both uptake values were decreased significantly by 10 mmol/l l-alanine, but unaffected by [Na +] (< 15 mequ/l). There was no uptake of AIBA. The uptakes of l-aspartate were 34.9 ± 3.7 per cent and oft-aspartate 40.4 ± 4.8 per cent ( n = 11). Both uptake values were significantly and reversibly reduced by 1 mmol/l l-aspartate and d-aspartate, and by low [Na +] (< 15 mequ/l). It is concluded that water can move by diffusion from maternal circulation into the yolk sac capillaries in considerable amounts whereas the contribution of the yolk sac placenta to fetal nutrition with d-glucose, l-alanine and l-aspartate is negligible. The membranes of yolk sac cells contain specific transport systems for d-glucose, d-/l-alanine and d-/l-aspartate transfer. The function of the vitelline placenta in the near-term guinea-pig is comparable more to the gut than to the chorio-allantoic placenta.

Transfer of 22Na+ across the guinea pig yolk sac placenta perfused in situ

Transfer of 22Na+ across the guinea pig yolk sac placenta perfused in situ

D-glucose uptake and transfer in the artificially perfused guinea-pig yolk sac placenta

Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive functions. NMDA receptors play a crucial role in modulating the activity of these cells. Considering that the amino-acid D-Aspartate has been recently shown to be an endogenous NMDA receptor agonist, the aim of the present study was to examine the effects of D-Aspartate on the functional properties of nigral dopamine neurons. We compared the electrophysiological actions of D-Aspartate in control and D-aspartate oxidase gene (Ddo−/−) knock-out mice that show a concomitant increase in brain D-Aspartate levels, improved synaptic plasticity and cognition. Finally, we analyzed the effects of L-Aspartate, a known dopamine neuron endogenous agonist in control and Ddo−/− mice. We show that D- and L-Aspartate excite dopamine neurons by activating NMDA, AMPA and metabotropic glutamate receptors. Ddo deletion did not alter the intrinsic properties or dopamine sensitivity of dopamine neurons. However, NMDA-induced currents were enhanced and membrane levels of the NMDA receptor GluN1 and GluN2A subunits were increased. Inhibition of excitatory amino-acid transporters caused a marked potentiation of D-Aspartate, but not L-Aspartate currents, in Ddo−/− neurons. This is the first study to show the actions of D-Aspartate on midbrain dopamine neurons, activating not only NMDA but also non-NMDA receptors. Our data suggest that dopamine neurons, under conditions of high D-Aspartate levels, build a protective uptake mechanism to compensate for increased NMDA receptor numbers and cell hyper-excitation, which could prevent the consequent hyper-dopaminergia in target zones that can lead to neuronal degeneration, motor and cognitive alterations.

Uptake and transfer of amino acids in the artificially perfused guinea-pig yolk sac placenta

Uptake and transfer of amino acids in the artificially perfused guinea-pig yolk sac placenta

Effects of monensins in on the receptor-mediated endocytosis of 125I-labelled IgG by guinea-pig yolk sac in vitro

The effects of the carboxylic ionophore, monensin, on the receptor-mediated binding and uptake of 125I-labelled IgG by the guinea-pig yolk sac have been studied in vitro. Exposure of tissue to 10 μm monensin resulted in a rapid inhibition of uptake which correlated with a time- and time-dependent loss of cell-surface receptor activity. Monensin appeared to bring about a change in receptor distribution since the lost activity could be detected after permeabilizing the tissue with saponin. Electron microscopic examination of monensin-treated tissue revealed that the apical plasma membrane of endoderms cell was depleted of coated and uncoated pits and that the apical cytoplasm contained numerous large vacuoles. Dilatation of the Golgi apparatus was also observed. Normal surface receptor activity and ultrastructural features could be largely recovered by removal of monensin. Recovery of receptor activity was unaffected by the presence of cycloheximide. These results are consistent with a model in which IgG receptors are recycled and in which monensin blocks this process by causing receptors to be trapped intracellularly. Ammonium chloride or a combination of valinomycin and carbonyl cyanide p-(trifluoromethoxy)-phenyllhydrazone also brought about a loss of surface IgGreceptors, lending support to the idea that inhibition of recycling was the result of pertubation of an intracellular acidification event and implying that passage through an acidic compartment may be improtant for correct receptor processing.

Sorting and transepithelial transport of adsorbed protein tracers: effects of temperature.

The epithelium of the guinea pig yolk sac is involved in the selective transport of macromolecules to the fetus. We studied the compartments involved in sorting and transepithelial transport of protein tracers and the effect of lowered temperature (18 degrees C) on these events. Explants of yolk sac were incubated with a mixture of cationized ferritin (CF) and horseradish peroxidase (HRP, Sigma type VI). At 4 degrees C, both tracers were bound to the cell surface and binding of an HRP-gold complex was shown to be inhibited by mannan. At 37 degrees C and 18 degrees C, both tracers were taken up into tubules and vesicles in the apical cytoplasm. Usually the tubules contained a mixture of tracers, but they often showed a polarized distribution with CF and HRP at opposite ends. The vesicles also contained mixtures of the tracers, but some contained only one. In addition, there were some irregularly shaped vacuoles composed of saccules that contained either a mixture, HRP alone, or CF alone. These results suggest that these adsorbed ligands are binding to unique microdomains of the endocytic complex. After 20 min at 37 degrees C coated vesicles 100 nm in diameter were located in the apical cytoplasm and coated vesicles of the same size were located at the lateral cell membrane. Usually they contained only HRP or CF, although occasional mixtures were seen. At 18 degrees C, HRP was transported across the cells in 100 nm vesicles. However, transport of CF was completely inhibited at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative hydrolase cytochemistry of the mature guinea-pig and marmoset yolk sac with special reference to proteases

Proteases and gamma-glutamyl transferase linked to plasma membranes, lysosomes and secretion granules were investigated cytochemically together with phosphatases, glycosidases and non-specific esterases in the mature guinea-pig and marmoset yolk sac. Species-independently, the yolk sac epithelial cells showed high activities of lysosomal proteases, glycosidases and non-specific esterases and phosphatases, whereas species-dependent patterns were found for plasma membrane proteases, gamma-glutamyl transferase and phosphatases. Furthermore, the guinea-pig yolk sac epithelium showed marked regional differences and that of both species intercellular differences, which allowed the subdivision of yolk sac epithelial cells in various types. The high activities of all lysosomal enzymes suggests their general importance for the yolk sac function in guinea-pigs and marmosets. The different equipment of the plasma membrane with proteases, gamma-glutamyl transferase and phosphatases indicates a more species-specific physiological role of this part of the yolk sac epithelial cells.

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.

The role of coated vesicles in selective transfer across yolk sac epithelium

Yolk sac epithelial cells nonselectively absorb macromolecules, yet selectively transfer certain proteins to the fetus. In the present study, horseradish peroxidase-conjugated guinea pig IgG (IgG-HRP) and cationized ferritin (CF) were used to follow coated pits and vesicles in guinea pig yolk sac and to establish their role in selective protein transfer. After exposure to IgG-HRP, reaction product was localized in small coated pits forming directly from the cell surface between microvilli or in small coated pits budding from larger saccular surface invaginations. The heaviest reaction product was associated with coated membrane regions. Reaction product was also found in coated vesicles fused with the lateral cell membrane. At 4°C, CF at higher concentrations labeled both microvilli and coated membrane invaginations, but at lower concentration only the glycocalyx of coated pits was labeled. Upon warming to 37°C, CF was internalized and transferred to the lateral cell membrane, also via coated vesicles. The results lend strong support to the involvement of coated pits and vesicles in selective transfer across yolk sac epithelium. The results suggest there may be some relation between the binding of IgG to its receptor in the glycocalyx of the coated pit and the maintenance of the clathrin coat on the cytoplasmic aspect of the membrane. The retention of the clathrin coat may direct the coated vesicle away from the lysosomal system and permit the transmission of intact immunoglobulin.

A freeze-fracture study of the guinea pig yolk sac epithelium.

The yolk sac epithelium functions in endocytic absorption of macromolecules from the uterine lumen and in maintaining permeability barriers between the maternal (uterine) compartment and underlying fetal compartments. In this study, cell membranes and intercellular junctions of the guinea pig yolk sac were examined using the freeze-fracture technique. Intramembranous particle (IMP) distribution and size were examined in microvilli, intermicrovillous membrane, and endocytic pits. IMPs on the P-fracture face were not evenly distributed within these membrane domains and the IMPs were of several sizes. In addition, short filamentous strands sometimes bridged between the cytoplasm and the E-face of endocytic pit membranes. These results indicated that the regions of yolk sac cell membrane involved in endocytosis did not have unusual numbers or distributions of IMPs compared to other membrane regions. The intercellular junctions between endoderm cells consisted of zonulae occludentes, gap junctions, and desmosomes. The zonula occludens usually consisted of four or five interconnected strands or ridges, and were elaborately developed where three cells abutted one another. Gap junctions were infrequent, small, and often associated with the zonula occludens. Desmosomes were observed both as typical macular structures and as more extensive, elongated structures. These results confirm previous thin-section studies of yolk sac indicating the presence of tight junctions. However, the extent of development of tight junctions does not correlate with the regard absence of a potential difference across yolk sac epithelium. The presence of gap junctions does not confirm results of previous freeze-fracture studies of other yolk sacs.

Differentiation of parietal endoderm cells of the guinea pig yolk sac, with particular reference to the development of endoplasmic reticulum

Cytodifferentiation in parietal endoderm cells was studied by light and electron microscopy in yolk sacs of guinea pigs between 22 days of gestation and term (67–70 days). The parietal yolk sac endoderm forms an epithelial covering of the margin of the chorioallantoic placental disc. Early in gestation the epithelium is simple, but during the middle of pregnancy the endoderm proliferates rapidly to become a thick pseudostratified layer. Late in gestation the epithelium is again reduced in thickness. Early in the course of differentiation the endoderm cell cytoplasm contains large numbers of free polyribosomes and scant elements of endoplasmic reticulum (ER). By 28–30 days, granular ER becomes highly developed, consisting of dilated fenestrated cisternae. By 34–38 days, however, agranular ER, in the form of anastomosing tubules, becomes the dominant cytoplasmic feature of these cells. In addition, whorls or lamellae of agranular membranes are present. From 40 days to term there is little change in the distribution of ER. The granular ER appears to be involved in the synthesis of Reichert's membrane. The functional significance of the well developed agranular ER is not known, but the possibilities that it may be involved in steroid or carbohydrate metabolism are discussed.