Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.

Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.

Ultrastructure of trophoblast giant cell transformation during the invasive stage of implantation of the mouse embryo.

The fine structure of abembryonic and mural trophoblast cells of mouse embryos was analyzed during the initial stages of invasion of the endometrial stroma by the embryo (days 6-8 of pregnancy). On day 6 of pregnancy, most trophoblastic cells are flat and have spindle-shaped nuclei. A few large, round trophoblastic cells (giant cells) are present at the abembryonic pole. As pregnancy proceeds through days 7 and 8, the area occupied by the trophoblast becomes larger because of an increase in the trophoblastic cell population, growth of giant cells, and rearrangement of the latter cells into a network containing maternal blood. As flat cells transform into giant cells, their content of ribosomes, granular endoplasmic reticulum, Golgi complexes, lysosomelike bodies, and heterophagosomes increases. Reichert's membrane is always lined by cell bodies or by laminar processes of trophoblastic cells that are provided with small pores. Transformation of flat cells into giant cells is associated with an activation of the giant cells and their acquisition of invasive behavior.

Ultrastructural study of the mouse antimesometrial decidua.

The ultrastructure of mouse antimesometrial decidual cells was analyzed during the development of the decidua between days 5 and 8 of pregnancy. The first decidual cells, appearing on the 5th day, are polygonal with rounded nuclei and prominent nucleoli; free ribosomes predominate in the cytoplasm. On the 6th to the 8th days the cytoplasm of these cells is typically that of cells actively engaged in macromolecular synthesis. Large numbers of granular and agranular endoplasmic reticulum cisternae are present in addition to well-developed Golgi complexes, mitochondria and lysosomes. Many bundles of microfilaments and lipid droplets occur during this period. An intense accumulation of autophagosomes and lysosomes with very heterogeneous content was noted on the 7th and especially the 8th days. The presence of these organelles is an indication that involution of this part of the decidua has begun.

Involution of the antimesometrial decidua in the mouse. An ultrastructural study.

Involution of the antimesometrial decidua was analysed by electron microscopy on days 9, 10 and 11 of pregnancy in the mouse. During this period, the width of the antimesometrial decidua decreases considerably. Involution begins in the decidual cells situated closest to the embryo (internal decidua) and proceeds towards the myometrium. The cells of the internal decidua showed signs of deterioration characterized by accumulation of clumps of chromatin in the nuclei and dilation of the perinuclear cisterna and endoplasmic reticulum cisternae. Autophagosomes and heterophagosomes accumulated in the cytoplasm of these cells. Cells particularly strongly affected became spherical and were devoid of their plasma membrane. Some cells near the trophoblast as well as the mature decidual cells situated farther from the embryo showed a normal morphology. The trophoblastic cells established close contact with healthy decidual cells and engulfed fragments of disorganized decidual cells. It is suggested that the death of decidual cells is a type of programmed cell death and that it is not due to a direct lytic action by the trophoblast.

Changes of the Golgi apparatus and lysosomes during decidualization in mice.

The Golgi apparatus of the endometrial stromal cells of pregnant mice increases in size simultaneously with the differentiation of stromal cells into decidual cells. The activity of acid phosphatase in this organelle increases during this stage. On the other hand, the involuting decidual cells show morphological and cytochemical signs of Golgi regression (dilated cisternae, lack of enzymatic activity) together with the finding of numerous, pleomorphic lysosomes that have intense cytochemical label. These results confirm morphological data suggesting that decidual cell death occurs by autophagic degeneration.

Distribution of desmoplakin I/II in endometrial cells of mice in the artificially induced decidua.

The decidual reaction is characterized by the redifferentitation of the endometrial connective tissue into a tissue with epithelioid features formed by decidual cells. An ultrastructural study showed a special type of junction formed between differentiating (predecidual) cells of the mouse from day six of pseudopregnancy onward. These contacts share ultrastructural characteristics of both desmosome and adherens junctions. These junctions have usually been classified as desmosome-like. In the present work, besides the ultrastructural analysis, we investigated with light microscopy the presence of desmoplakins I and II, using streptavidin-biotin immunoperoxidase technique. We found a positive punctate staining around predecidual cells while a scarce reaction was observed in the other regions of the uterus. These results suggest that these junctions belong to the desmosome family.

Distribution and space-time relationship of proteoglycans in the extracellular matrix of the migratory pathway of primordial germ cells in mouse embryos.

In this paper we present an in situ ultrastructural cytochemical study on the distribution and spatial-temporal expression of proteoglycans (PGs) in the extracellular matrix of the migratory pathway of mouse primordial germ cells (PGCs) during the different phases of migration, by the use of the cationic dye ruthenium hexammine trichloride (RHT). Embryos of 9, 10, 11 and 12 days of development were used. The treatment with RHT revealed PGs as electron dense layers, granules, and filaments. Whereas granules prevailed in the extracellular spaces of the migratory route during the whole migratory process, the amount of filamentous structures increased during the migration phase of PGCs. At the end of the migratory process the surface of the PGCs lost its reaction by RHT. There were differences in the size of the granules of PGs at the initial migratory period (9-day-old embryos) as compared with the other days of gestation. There was a strong reaction for PGs in the extracellular spaces, expressed as a meshwork of granules interconnected by filaments, as well as reaction on the basement membranes during the peak of the PGCs migration in 10-day-old embryos. These results support the hypothesis that these molecules may have an important role in the migration of PGCs, although the precise mechanism involved in this process is not yet clear.

Ultrastructural cytochemical study of proteoglycans in the endometrium of pregnant mice using cationic dyes.

Decidualization in rodents is accompanied by remarkable modifications of both fibrillar and non-fibrillar components of the endometrial extracellular matrix. Biochemical studies have shown that the levels of synthesis of hyaluronic acid and sulfated glycosaminoglycans change during decidualization in rodents. As the rodent decidua has regions containing cells in different stages of decidual transformation, we decided to analyse, by an ultrastructural cytochemical technique, the distribution of proteoglycans (PGs) in each region of the decidua of mice on different days of pregnancy. Endometria of mice on days 4, 5 and 7 of pregnancy were processed for electron microscopy in the presence of safranin O, a cationic dye which preserves most of the tissue PGs. The endometrium of non-pregnant mice was used as control. We observed evident differences in the arrangement and distribution of the network of PGs between non-pregnant and 4-day pregnant endometria, as well as between different regions of pregnant endometria. The possible relationship between these modifications and cell transformation that occurs during decidualization is discussed.

The local origin of decidual cells in pregnant mice.

In order to evaluate the participation of extrauterine cells in the formation of mouse antimesometrial decidua, [3H]-thymidine was administered ip on days 1, 5 and 6 of pregnancy and the animals were killed 1 h afterwards. A second group of mice received four ip injections of [3H]-thymidine at 6-h intervals on the 1st day of pregnancy and were killed on the 2nd, 5th or 6th day of pregnancy. A third group of virgin mice in estrus received [3H]-thymidine ip four times at 6-h intervals and was killed 96 h after the first injection. Radioautographs of the uteri showed that few endometrial stromal cells were labelled on the 1st and 2nd day of pregnancy. Although many decidual cells incorporated thymidine on the 5th and 6th day of pregnancy in pulse-labelled animals, only few labelled decidual cells were found on the 5th and 6th day of pregnancy in animals that received several injections of thymidine on the 1st and 2nd day of pregnancy. These results indicate that the antimesometrial decidual cells that develop at the beginning of pregnancy are mostly of local origin. The short-term migration of extraneous cells into the uterus to participate in decidualization is not supported by these data.

Trophoblast invasion during implantation of the mouse embryo.

The interaction between trophoblastic and maternal cells was analysed by electron microscopy on days 6, 7 and 8 of pregnancy, in the mouse. Special emphasis was given to phagocytic activity and invasiveness by the trophoblast. On the sixth day of pregnancy, the trophoblast cells are in contact with the epithelial cells of the implantation crypt, with the basal lamina of the crypt, and with cells of the antimesometrial decidua. On the seventh and eighth days of pregnancy, the trophoblastic cells interact with those of the antimesometrial decidua. The giant trophoblastic cells engulf epithelial cells, maternal blood cells and decidual cells although the pattern of phagocytosis of these structures differs. Both whole epithelial cells and blood cells were ingested. The epithelial cells were deteriorated whereas the blood cells had a normal morphology; the decidual cells were ingested following a severe process of deterioration. Processes of trophoblastic cells interposed between the epithelium of the implantation crypt and its basal lamina seem to contribute to the displacement of the epithelial cells. The invasion of the endometrium by the trophoblast begins with the penetration by processes of trophoblastic cells between the decidual cells. The contact between the surface of both cell types may be very close: adherens type junctions and focal contacts are formed between trophoblastic cells and healthy decidual cells. Trophoblastic cells ingest deteriorated or fragmented cells and gradually occupy the spaces left by the latter.

Collagen distribution in the mouse endometrium during decidualization.

The distribution of collagen and reticular fibers was studied in the endometrium of virgin and pregnant mice. The collagen and reticular fibers were examined in picrosirius-stained sections observed in a polarizing microscope and in silver-impregnated sections. Picrosirius-stained sections of animals in estrus, diestrus and on the 2nd day of pregnancy had fine greenish fibers distributed irregularly in the endometrium and thicker red fibers concentrated near the myometrium. Argyrophyl fibers in virgin mice were scarce and irregularly distributed. On the 4th day of pregnancy very few fibers were observed in the endometrium. On the 5th, 6th, and 7th days of pregnancy long greenish fibers were found surrounding decidual cells. A network of argyrophyl fibers was observed in the silver-impregnated decidua. It is suggested that new fibers are produced by decidual cells.

Implantation and decidualization in rodents.

This article reviews the main events of embryo-implantation and decidualization in rodents. In common laboratory rodents the embryo attaches to the uterine epithelial lining, usually on days 4 to 6 of pregnancy. A progressive degree of proximity between trophoblast and epithelium occurs until the epithelial cells undergo apoptosis and detach from the basement membrane. During the attachment stage, the spindle-shaped connective tissue cells that underlie the epithelium next to the embryos transform into polyhedral and closely packed decidual cells. Following the epithelial detachment and the breaching of the basement membrane the embryo is thus in direct contact with decidual cells. These cells accumulate organelles associated with synthesis of macro-molecules, intermediate filaments, and eventually lipid droplets and glycogen. Another remarkable feature of decidual cells is the establishment of gap and adherens intercellular junctions. Differentiation of fibroblasts into decidual cells advances antimesometrially and mesometrially, creating in the endometrium several regions of cells with different morphology. The whole phenomenon of decidualization which is normally triggered by the embryo can be artificially induced in pseudo-pregnant or hormonally-prepared animals with the use of diverse stimuli. The uterine epithelium is probably responsible for the transduction of the initial stimulus. Prostaglandins have been shown to be important in the induction of decidualization. More recently other substances such as leukotrienes, platelet-activating factor (PAF), and transforming growth factor (TGF) have been thought to play a role in induction. Much evidence points to prostaglandin production by the decidual cells. New proteins such as a luteotropic factor, desmin, and other molecules were shown to be produced after rat stromal cells undergo decidual transformation. The extracellular matrix of the mouse decidua contains very thick collagen fibrils. Mouse decidual cells are also very active in phagocytosing the thick fibrils, contributing to the remodeling and involution of the decidua that accompanies embryonic growth. Radioautographic data indicates that mouse decidual cells produce and secrete collagen and sulfated proteoglycans.

Growth of mouse embryos implanted in the subcutaneous tissue of recipient mice.

Eight-days-old mouse embryos were transferred to the subcutaneous tissue of the dorsal skin of host mice. A high rate of embryos developed into hemorrhagic (HN) or nonhemorrhagic nodules (NHN). The latter had trophoblastic cells as well as embryoblastic derivatives whereas HN contained almost only trophoblastic cells. At least two kinds of trophoblastic cells were present in NHN: small cells and large cells similar to trophoblastic giant cells. In HN most trophoblastic cells arranged themselves into a network whose meshes contained host blood. Although embryoblastic derivatives such as endoderm and Reichert's membrane were apposed to host connective tissue cells, trophoblastic cells were always surrounded by collagen fibrils or by a layer of an amorphous material.

Volume changes during mitosis in pancreatic acinar cells of the rat.

The volume of interphasic and mitotic pancreatic acinar cells of 20-day-old rats was estimated by analysis of drawings from Araldite-embedded serial thick sections. Assuming the average interphase cell volume to be 100%, the volume of prophase cells was 145%, reaching a volume of 180% during anaphase. Postmitotic acinar cell volume was 74%.

Invasiveness of mouse trophoblastic cells in connective tissue.

Ectoplacental cones from 8-day-old mouse embryos were grafted into the dorsal subcutaneous tissue of host mice. The grafts were collected between the 3rd and 8th days after transfer and processed for morphological analysis by light and electron microscopy. Approximately 60% of the grafts formed hemorrhagic nodules in which only invasive, giant trophoblastic cells developed. These cells shared many morphological features with the trophoblastic cells involved in normal implantation. At the periphery of the nodules, trophoblastic giant cells were frequently seen growing toward the host connective tissue. The association between the trophoblast and fibrilar components of the extracellular matrix was examined. No direct association with collagen fibrils was noted; however, many areas of the surface of invasive cells were in close proximity with microfibrils of the extracellular matrix. Since only the invasive trophoblast cells exhibited such an association, a direct comparison was made with the process of trophoblast migration within the connective tissue.

Growth of mouse ectoplacental cone cells in subcutaneous tissues. Development of placental-like cells.

Ectoplacental cones of mouse embryos collected on day 8 of pregnancy were grafted into the dorsal subcutaneous tissue of host mice. The grafts were collected between days 3 and 8 after transfer and processed for light and electron microscope morphological analysis as well as for cytochemistry of nonspecific alkaline phosphatase. Fragments of normal mouse placentas collected between days 12 and 18 of pregnancy were processed similarly. About 37% of the grafts were nonhemorrhagic nodules formed by different kinds of trophoblastic cells. These cells had many morphological and cytochemical features of cells present in normal mouse placentas. Nonphagocytic giant cells, glycogen cells, as well as cells with a well-developed granular endoplasmic reticulum were similar to cells found in the placenta and were always present in the grafts. Cells showing features intermediate between the above-mentioned cells and those whose cytoplasm was poor in organelles also were found in the grafts. The latter resembled cells of layer 1 of the labyrinth of the placenta. These results suggest that trophoblastic cells of the ectoplacental cones had differentiated into placental cells following their transfer to the subcutaneous tissue.

Remodeling of the mouse endometrial stroma during the preimplantation period.

An ultrastructural cytochemical study of acid phosphatase activity performed in mouse endometrium on the second day of pregnancy showed that stromal cells which were heavily labeled by the cytochemical reaction had disarranged organelles. On the other hand, the cytoplasm of several stromal cells had collagen-containing phagosomes that were also labeled, indicating that the collagen fibrils were being digested by lysosomal enzymes. It is suggested that cell death and phagocytosis of collagen are events of the remodeling of the mouse endometrium that occur prior to decidualization.

Collagen remodeling during decidualization in the mouse.

An ultrastructural study of the features and distribution of collagen fibrils was performed in the endometrium of virgin and pregnant (2nd to 11th day) mice. Collagen-containing structures were observed in the cytoplasm of fibroblasts on the 2nd day of pregnancy. Treatment of tissues with lanthanum nitrate established that these structures were intracytoplasmic. Their association with lysosome-like bodies suggested the occurrence of intracellular digestion of collagen, probably connected with remodeling of the endometrial stroma prior to decidualization. On the 4th day of pregnancy, very few collagen fibrils were present in the intercellular space. From the 6th day of pregnancy onwards, "thick" collagen fibrils were observed between decidual cells. The diameter of these fibrils measured up to 300 nm whereas the fibrils present in the endometrium of virgin mice measured 40-68 nm.

Diameter increase of collagen fibrils of the mouse endometrium during decidualization.

The diameter of collagen fibrils was measured in different regions of the antimesometrial endometrium of mice on days 5, 6, and 7 of pregnancy as well as in the endometrium of virgin mice. The average diameter of fibrils of virgin mice was 39.18 nm (range: 20-80). In the region of fully decidualized cells, the averages and ranges were 45.32 nm (30-170), 89.39 nm (30-270), and 125.88 nm (20-370), respectively, on days 5, 6, and 7 of pregnancy. Thick fibrils larger than 70 nm had irregular profiles. Our results show that the increase in diameter is associated with the decidualization of the mouse endometrium.

Incorporation of 3H-amino acids by endometrial stromal cells during decidualization in the mouse. A radioautographical study.

The incorporation of 3H-amino acids by endometrial stromal cells was analyzed by radioautography during decidualization in the mouse. 3H-proline was administered to 5- and 6-day pregnant mice and to virgin mice in estrus used as controls. 3H-tryptophan was administered to 6-day pregnant mice. The animals were killed 30 min. after the injection of the radioactive label. The quantitative analysis of the radioautograms showed that the concentration of silver grains per unit area due to 3H-proline incorporation was higher in decidual and predecidual cells on the fifth and sixth day of pregnancy than in fibroblasts of virgin mice. Considering the pregnant endometrium by itself, the incorporation of both 3H-proline and 3H-tryptophan was higher in fully differentiated decidual cells and predecidual cells than in fibroblasts of the periphery of the endometrium and involuting decidual cells. These results show a clear correlation between different endometrial regions recognized by morphological criteria and their metabolic activity. The data also show that different cells of the pregnant endometrium have different metabolic activities, independently of the precursor that was used.