Coculture system that mimics in vivo attachment processes in bovine trophoblast cells

The establishment of pregnancy requires bidirectional communication between the developing conceptus and the uterine endometrium. The aim of this study was to establish an in vitro coculture system with bovine trophoblast cells and uterine epithelial cells (EECs) that mimics the in vivo attachment process. We previously reported that expression of interferon tau (IFNT), a major secretory product from the trophectoderm, decreases with changes in chromatin structure when the conceptus successfully attaches to the uterine epithelium. Thus, IFNT is a good marker to assess whether attachment has successfully occurred. In this study, bovine trophoblast CT-1 cells were cultured to generate spheroids, which were then placed on type I collagen-coated plates (monoculture) or bovine EECs (coculture) with or without uterine flushings collected from Day 15 cyclic or Days 15, 17, or 19 pregnant animals. In the coculture but not the monoculture, addition of uterine flushings from Day 15 or 17 pregnant animals resulted in decreased IFNT and CDX2 mRNA expression in CT-1 spheroids, accompanied with changes in histone modifications. In monocultured CT-1 spheroids, integrin subunit ITGA8 and ITGB3 mRNAs were minimally expressed but were induced in cocultured CT-1 spheroids with or without uterine flushings. Expression of CDH2, another marker for bovine conceptus attachment to the uterine epithelium, was also induced in the cocultured CT-1 spheroids. These results suggest that this in vitro coculture system could be used to isolate processes essential for conceptus attachment to uterine EECs.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Summary Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytotic vacuoles (pinosomes) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes, which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytic vacuoles ( pinosomes ) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes , which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Uptake of exogenous protein tracer in cells of the rat renal papilla

In order to study the phagocytic potential of different cell types of the rat renal papilla with special emphasis on interstitial cells, horseradish peroxidase (HRP) (8 mg/100 g body weight) was injected intravenously into adult rats. The distribution of peroxidase was studied in animals perfusion-fixed 60 and 180 min after injection and was found to be similar after both time intervals. The epithelial cells of the collecting ducts took up the largest amounts of the tracer. HRP was mainly located in large lysosome-like bodies in the basal part of the cytoplasm, suggesting peritubular uptake from the interstitial space. However, small amounts of the tracer were also seen in apical vesicles close to the luminal plasma membrane. The interstitial cells of peroxidase-injected animals were ultrastructurally altered and had large irregular invaginations of the cell membrane. The cells had taken up only small amounts of the tracer which were located in small round lysosome-like bodies. Thus, the interstitial cells displays no macrophage characteristics, either in the native state or when challenged with an extracellular protein.     

Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Summary Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium.Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15–30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.     

Uptake of exogenous protein tracer in cells of the rat renal papilla

Summary In order to study the phagocytic potential of different cell types of the rat renal papilla with special emphasis on interstitial cells, horseradish peroxidase (HRP) (8 mg/100 g body weight) was injected intravenously into adult rats. The distribution of peroxidase was studied in animals perfusion-fixed 60 and 180 min after injection and was found to be similar after both time intervals. The epithelial cells of the collecting ducts took up the largest amounts of the tracer. HRP was mainly located in large lysosome-like bodies in the basal part of the cytoplasm, suggesting peritubular uptake from the interstitial space. However, small amounts of the tracer were also seen in apical vesicles close to the luminal plasma membrane. The interstitial cells of peroxidase-injected animals were ultrastructurally altered and had large irregular invaginations of the cell membrane. The cells had taken up only small amounts of the tracer which were located in small round lysosome-like bodies. Thus, the interstitial cells displays no macrophage characteristics, either in the native state or when challenged with an extracellular protein.Supported by Karolinska Institutet and the Swedish Medical Research Council (proj. no. 05937)     

Uptake of horseradish peroxidase by bone cells during endochondral bone development

Summary To investigate the mechanisms whereby bone cells absorb organic bone-matrix components during endochondral bone development, rat humeri were examined, employing horseradish peroxidase as a soluble protein tracer.Intravenously-injected peroxidase filled the osteoid layer and penetrated into the osteocyte lacunae and canaliculi, but did not enter the mineralized bone matrix. Whereas osteocytes rarely took up exogenous peroxidase, osteoblasts and osteoclasts actively endocytosed peroxidase in pinocytotic coated vesicles, tubular structures, and vacuoles. They also formed endocytotic vacuoles containing peroxidase in the Golgi area. The Golgi apparatus and dense bodies of these bone cells were, however, free of reaction products. Osteoclast ruffled borders were responsible for peroxidase absorption. In the osteoblast, osteocyte and osteoclast, endogenous peroxidatic reaction was detected only in mitochondria and not in other membrane-bounded vesicles and bodies. These results strongly suggest that both osteoblasts and osteoclasts participate in the resorption of bone-matrix organic components during bone remodelling.     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

Endocytosis in flight-stimulated adipokinetic cells ofLocusta migratoria

An ultrastructural morphometric study was made of the influence of flight activity on endocytosis in the adipokinetic cells ofLocusta migratoria. The endocytotic pathway was revealed by the endocytotic tracer horseradish peroxidase. Endocytosis appeared to be stimulated by flight, as indicated by an increase in the number of tracer-containing endocytotic pits and various intracellular endocytotic and lysosomal organelles. This stimulatory effect continued for at least 10 min after cessation of flight. An increase in the numbers of both tracer-labelled endocytotic pits and endosomal tubules was detected in the cell bodies of flight-stimulated adipokinetic cells. Endosomal tubules are supposed to be involved in recycling membrane material to the plasma membrane soon after it has been endocytosed. It is concluded that the increase in endocytosis in the flight-stimulated cell bodies serves to enlarge the uptake of nutritional and/or regulatory substances. An increase in number of tracer-labelled endocytotic pits was also observed in the cell processes of flight-stimulated adipokinetic cells, which was, however, not accompanied by an increase in number of labelled endosomal tubules, the latter being practically absent in the processes. This indicates that the increase in endocytotic activity in the cell processes is a form of adaptive endocytosis that compensates for membrane material added to the plasma membrane during flight-induced exocytotic release of adipokinetic hormones.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The distribution of the tracer substance horseradish peroxidase (HRP, Mw 40,000) in the neuro-intermediate lobe of the lizard, Anolis carolinensis, was studied at various time intervals (13 min to 24 h) after vascular injection. HRP rapidly entered the extracellular lumen of the neural lobe, but did not penetrate into the third ventricle. The tracer was found in micropinocytotic vesicles (MPVs) of ependymal cells within 13 min after injection. The number of cellular inclusions containing HRP increased during the period of observation (24 h). The tracer was sparsely taken up by aminergic and peptidergic nerve terminals of the external layer. After transection of the hypophysial stalk, numerous dense, labelled droplets were found in the peptidergic terminals, and the number of labelled inclusions in ependymal cells increased.MPVs were frequently found in extensions of stellate cells of the intermediate lobe, and endocytotic vacuoles (EVs) developed especially in the perikaryon. HRP was also found in large cisternae of the secretory cells, appearing predominantly towards the perivascular septum (PVS). These cisternae were found to communicate with the extracellular lumen, probably representing a system of the extracellular space extending into the secretory cell. After transection of the hypophysial stalk, there was an increase in the number of small EVs in secretory cells of the intermediate lobe.The results are discussed in terms of MSH-release regulation and possible participation of the extracellular lumen, glial and stellate cells in the transport of regulating factors and secretory material.Supported by grants from the Swedish Natural Science Research Council (to Dr. Patrick Meurling) and the Royal Physiographic Society of LundThe author is indebted to Mrs. Lena Sandell and Miss Inger Norling for excellent technical assistance and photographic aid; and to Dr. Rolf Libelius and Dr. Ingmar Lundquist for generous advice and stimulating discussions concerning the tracer technique     

Cytological observations of the ovarian epithelium in mammals during the reproductive cycle

We have studied the ovarian epithelium at various stages of the reproductive cycle in a number of mammalian species utilizing light microscopy, scanning microscopy, the freeze-fracture technique, transmission microscopy and by employing specialized tracers that use lanthanum and horseradish peroxidase. We found that the epithelial cells are joined by incomplete tight junctions, gap junctions, and desmosomes. The cytoplasmic matrix contains a large irregularly shaped nucleus, few microtubules, microfilaments, mitochondria, endoplasmic reticulum and a host of coated and non-coated vesicles of varying diameters. The saccules comprising the large Golgi complex and its companion vesicles are associated with a basal body-centriole complex: some of these saccules and affiliated vesicles are acid phosphatase positive. Surface modifications of ovarian epithelial cells include numerous microvilli, some of which have a bulbous tip, and plications of the lateral plasma membrane which are thought to accommodate volume changes of the ovary during follicular development. Many coated and non-coated endocytotic caveolae were found on these cells, particularly in the basal area. These caveolae internalized exogeneously administered horseradish peroxidase. We view the marked endocytotic activity as an efficient transport mechanism for partially removing substances from the interstitium of the ovary and the peritoneum.     

Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

Endocytosis in absorptive cells of cultured human small-intestinal tissue: Horseradish peroxidase,lactoperoxidase, and ferritin as markers

Summary The occurrence of endocytotic mechanisms in human small intestinal absorptive cells was investigated by culturing biopsy specimens in the presence of horseradish peroxidase (HRP), lactoperoxidase (LPO), and ferritin. The results indicate that both HRP and LPO entered the cells by apical endocytosis, after which they were transported via apical vesicles and tubules to the lysosome-like bodies. Ferritin, which showed a distinct affinity for the cell-coat glycoproteins, was not interiorized by the absorptive cells.These findings suggest that although human absorptive cells have an endocytotic mechanism, possibly fluid-phase endocytosis, cell-coat glycoproteins are not taken up by the cells, as indicated by the absence of ferritin in the apical vesicles and tubules, as well as the lysosome-like bodies. These findings provide indirect support for our hypothesis that the lysosome-like bodies have a function in the regulation of cell-coat glycoprotein transport via a crinophagic mechanism (fusion of apical vesicles and tubules with lysosome-like bodies) rather than via an exocytotic-endocytotic mechanism.     

Kinetic analysis of the triggered exocytosis/endocytosis secretory cycle in cultured bovine adrenal medullary cells

Cultured bovine adrenal medullary cells are an excellent preparation for quantitative analysis of the secretory exocytosis/endocytosis cycle. In this paper we examine the kinetics of endocytosis after stimulation of secretion. Membrane retrieval was monitored by uptake of the fluid phase marker horseradish peroxidase. Horseradish peroxidase was found to be suitable because it can be washed off completely, assayed quantitatively, and its uptake increases linearly with concentration. If this marker is present during stimulation, the rate of uptake is initially slower than catecholamine secretion but faster at a later time, suggesting that the formation of endocytotic vesicles follows exocytosis. To monitor the time-dependent concentration of secretory vesicle-plasma membrane fusion product (omega-profiles), secretion was halted at various time intervals after stimulation and the excess membrane allowed to transform into endocytotic vesicles in the presence of horseradish peroxidase. By adding horseradish peroxidase at various times after inhibition of secretion, the time course of membrane retrieval could be measured directly. All our results are consistent with a two-step kinetic model in which exocytosis and membrane retrieval are consecutive events. The estimated volumes of the compartments involved are roughly equal. The rate of endocytosis is strongly temperature-dependent but unaffected by extracellular calcium in the range of 10(-8)-2.5 X 10(-3) M, suggesting that calcium is not required at the site of endocytotic membrane fusion. Membrane retrieval is also unaffected by Lanthanum (1 mM) but is slowed by hypertonic media.     

A mini organ culture as a model for studying the gallbladder epithelium of mouse

Summary— A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.     

Studies of hCG binding and endocytosis in rat ovary by ultrastructural immunocytochemistry

Summary Localization of hCG binding sites and the process of endocytosis in pseudopregnant rat ovaries were investigated by indirect electron-microscopic immunocytochemistry. Immature female rats were treated with pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovarian luteinization. Eight days after priming with PMSG-hCG and 1–6 h before sacrifice the animals were given another injection of hCG to bind the receptors. Receptor sites to hCG localized by reaction product were present in most luteal cells, but not in primary follicular cells. The receptor sites were distributed on luteal cell surfaces facing interstitial spaces. Endocytotic pits containing hCG binding sites were rarely seen 1 h after hCG injection. At 2 h, hCG and presumably its receptor were taken up within endocytotic vesicles with the evidence of reaction product coated on the vesicle wall. With time, fusion of endocytotic vesicles with lysosome occurred and the reaction product appeared in phagolysosomes. The reaction product was localized on phagolysosomal inner surface or in free granular form. These findings suggest that hCG and its receptors were internalized through endocytotic pits and endocytotic vesicles and delivered to lysosomes probably for degradation. An additional experiment for localization of acid phosphatase was also performed to delineate the lysosomes and phagolysosomes.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Increased uterine vascular permeability at the time of embryonic attachment in the pig

The temporal relationship between embryonic attachment and endometrial vascular permeability was investigated in the gilt. Light and electron microscopy failed to reveal structural differences between Day 10 cycling and pregnant maternal epithelia, including evidence of blastocyst contact. Chorionic adhesion was preserved at mesometrial regions in 3 of 5 Day 13 pregnant animals and appeared to be related to localized differentiation of the underlying maternal epithelium. In order to study uterine vascular permeability, 44 gilts between Days 11 and 19 of the cycle and pregnancy were injected i.v. with a 0.5% solution of Evans Blue (2.5 ml/kg body weight). Examination of excised uteri under ultraviolet light revealed a well-defined zone of endometrial fluorescence corresponding to extravascular content of the dye. Exclusive to pregnant gilts, this response appeared in conjunction with blastocyst elongation at Day 12, and was consistently confined to areas of embryonic membrane contact thereafter. The changes in endometrial morphology and vascular permeability suggest involvement of some embryonic factor(s) acting in a localized manner. Increased histotrophe production is probably facilitated by the flux of plasma constituents to maternal epithelial cells. Coincidence of increased uterine vascular permeability at the site of attachment with elevated blood flow would enhance transport of nutrients toward the conceptus and allow access of blastocyst-induced products to the maternal circulation.     

Transport of pinocytic vesicles in the eye of a snail,Helix aspersa

The heads of small adult snails, Helix aspersa, were injected with horseradish peroxidase (HRP) for one to five hours before extirpating the eyes and preparing them cytochemically for electron microscopy. There was internalization of tracer by pinocytic vesicles (pinosomes) at the bases of types-I and -II sensory cells, ganglion cells and, in lesser amounts, by pigmented supportive cells. Vesicles and vacuoles filled with HRP were transported in two directions: lensward as far distad as the ends of the cells (retrograde) and brainward down the optic nerve (anterograde). We believe that the numerous reacted vacuoles in the cell somata are formed by fusion of vesicles, tubules and C-shaped organelles filled with tracer; we present evidence that they become secondary lysosomes. Sensory cell type II possesses more HRP-reacted vacuoles distally than the other retinal cells. Other vesicles are also described. There was no uptake of tracer by the distal ends of the retinal cells following injection HRP into the hemolymph. The swelling of the optic nerve, immediately behind the eye, contains more HRP-filled pinosomes and vacuoles than does the nerve below the dilatation. The significance of endocytosis and transport of pinosomes within the eye and down the optic nerve is discussed.