Increase of interstitial collagen in the mouse endometrium during decidualization.

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [alpha1(V)](3), was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.

Small leucine-rich proteoglycans (SLRPs) in uterine tissues during pregnancy in mice.

Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.

Granulated decidual cells in the mouse deciduoma: a putative source of decidual prolactin in mice.

Decidual cells are endometrial fibroblasts that redifferentiate during pregnancy in several species of mammals. In this work, we describe a subpopulation of resident decidual cells in the mouse endometrium that are joined by intercellular junctions and have cytoplasmic granules. Decidualization was induced in pseudopregnant mice on the 4th day of pseudopregnancy by injection of 30 microl of arachis oil into the uterine lumen. The uteri were collected on day 8 of pseudopregnancy (at 4 p.m., 8 p.m. and 11 p.m.) and on day 9 (at 8 a.m.). The tissues were fixed for light and electron microscopy. During day 8 of pseudopregnancy, granulated cells were present at the antimesometrial pole of the endometrium; they were concentrated at the periphery of the antimesometrial decidua and disappeared on day 9 of pseudopregnancy. The cytoplasm of the granulated decidual cells had acidophilic granules that stained also with periodic acid-Schiff (PAS). These granules stained with anti-rat prolactin antibody in both light and electron microscope immunocytochemical preparations. Vacuoles of various sizes were always present in the granulated cells. A PAS-positive and prolactin-stained material was often deposited at the periphery of the vacuoles. Our results indicate that the granulated decidual cells are the source of decidual prolactin which accumulates in cytoplasmic granules. These granulated cells therefore form a transient gland in the mouse antimesometrial endometrium (granulated decidual gland).

Ultrastructural cytochemical characterization of collagen-associated proteoglycans in the endometrium of mice.

The decidual reaction in mice is characterized by the transformation of a specific population of endometrial fibroblasts into epithelioid cells, known as decidual cells. An important feature of decidualization in mice is a remarkable modification of the endometrial extracellular matrix. The present work is an ultrastructural cytochemical study of matrix with the purpose of analyzing the arrangement of collagen-associated proteoglycans (PGs) at various regions of nulliparous endometrium and of the antimesometrial decidua of mice using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinase ABC, chondroitinase AC, and hyaluronidase. The staining with cuprolinic blue showed PGs as rods and granules of several sizes. Rods measuring 40-60 nm in length (named F2-rods) were apposed to thin collagen fibrils whereas granules were associated with thick collagen fibrils, particularly in the region occupied by mature decidual cells on the 7th day of pregnancy. The amount of granules was higher than that of F2-rods. Both F2-rods and granules were affected by chondroitinase ABC or AC treatment, indicating that they were PGs containing chondroitin sulfate and dermatan sulfate chains. However, the granules associated with thick collagen fibrils were more resistant to chondroitinase AC treatment than F2-rods, indicating the presence of dermatan sulfate chains that contain both L-iduronic and D-glucuronic acid sugar residues. We suggest that the differences of the nature and amount of PGs may be associated with the changes of the thickness of collagen fibrils observed during decidualization of the endometrium in the mouse.

Organization of desmin-containing intermediate filaments during differentiation of mouse decidual cells.

Decidualization of the mouse endometrium consists of a redifferentiation of the endometrial stromal fibroblasts. During decidualization these fibroblasts undergo growth, change of shape, multinucleation, and establishment of intercellular junctions. One feature of rodent decidual cells is the accumulation of intermediate filaments. In spite of the fact that fibroblasts normally have vimentin intermediate filaments, they acquire a large amount of desmin intermediate filaments while they undergo decidualization. The light and electron microscope immunocytochemical results of the present work show that during the initial stages of decidual transformation the desmin intermediate filaments accumulate around the nuclei, often forming caps around the nuclear envelope. As the decidual cells grow, the filaments form bundles and nets that radiate from the nuclei toward the cell surface. During the final stages of differentiation, on day 8 of pregnancy, staining of differentiated decidual cells decreases and most filaments accumulate under the cell surface. A role for intermediate filaments is suggested for decidualization of mouse endometrial cells.

Light and electron microscopic analysis of controlled injury to follicular unit grafts.

BACKGROUND: Careful manipulation of hair units is essential for a good yield of transplanted hair. OBJECTIVE: To analyze the morphology of dissected follicular units submitted to crushing, stretching, bending, and drying. METHODS: Follicular units were either crushed, bent, stretched with forceps, or left drying on surgical gloves for 3 minutes. The specimens were fixed and prepared for observation with light microscopy or transmission and scanning electron microscopy. RESULTS: No alterations were detected in follicular units that had been crushed, bent, or stretched. Major damage occurred in samples that were left to dry on gloves. CONCLUSION: Letting the follicular unit dry appears to be the worst mishandling to which the follicular units may be subjected during routine hair transplantation.

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.

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice.

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest levels of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice.

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice.

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.

Death and replacement of uterine epithelial cells during oil-induced deciduoma development in the mouse.

BACKGROUND: A decidual cell reaction can be induced in rodent endometrium by an intrauterine injection of oil. The epithelial lining is thought to be instrumental to transduce intralumenal stimuli for decidualization. One of the consequences of oil injection is the death of uterine epithelial cells. No information is available on the effect that sustained contact with oil has on the epithelium. METHODS: A decidual cell reaction was induced in 4-day pseudopregnant mice by injection of 30 microliters of arachis oil into the uterine lumen. Samples from the uteri were collected 24, 48, and 72 h after the injection and prepared for transmission electron microscopy. RESULTS: Twenty-four hours after the oil injection, some of the initial modifications of epithelial cell surfaces were very similar to those induced by the contact with the blastocyst during normal pregnancy. Uterine epithelial cells internalized injected oil and many cells were seen in various stages of degeneration. At 48 h, many epithelial cells were detached from the basal lamina. At 72 h, the uterine lining was re-established by flattened cells. CONCLUSIONS: The contact of oil with the uterine epithelium of pseudo pregnant mice induces epithelial cell death in the antimesometrial region of the uterine crypt. There is, however, replacement of epithelial lining by epithelial cells, which probably migrate from the mesometrial region of the crypt. The prolonged presence of oil within the uterine lumen seems to induce cycles of epithelial cell death and replacement.

Cloning of a thymic stromal cell capable of protecting thymocytes from apoptosis.

A clone of thymic stromal cells, namely 2BH4, was established by primary culture, cellular transfection and limiting dilution. Morphological analysis by transmission electron microscopy revealed that these cells grow as multilayers, producing a well-defined basement membrane to which they attach and frequently form structures similar to hemidesmosomes. The adjoining cells are connected by intercellular junctions, as tight junctions, intermediate junctions, and desmosome-like junctions, as well as interdigitations. Their cytoplasm contains microtubules, strands of actin filaments, and scarce intermediate filaments. Fluorescence microscopy revealed that 2BH4 cells stain with anti-cytokeratin antibodies, the majority of them giving a faint reaction. In addition, they express Thy-1.1, LFA-1, ICAM-1, and the gp23 epithelial antigen, and synthesize laminin. They have a doubling time of 16 hr and are able to bind thymocytes. Thymocytes cultured in the presence of 2BH4 cells are partially protected from both spontaneous and PMA- or dexamethasone-induced apoptosis. This protection is conferred neither by soluble factors normally produced by the 2BH4 cells nor by the sole contact with fixed 2BH4 cells. Rather, thymocytes must interact with metabolically active 2BH4 cells in order to receive the protective signal(s).

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.

Biosynthesis of glycosaminoglycans in the endometrium during the initial stages of pregnancy of the mouse.

Significant changes in the synthesis of glycosaminoglycans occur during the transformation of stromal cells of the endometrium into decidual cells which takes place during the initial stages of pregnancy in mice. Hyaluronic acid, which is practically absent in the endometrium of virgin mice, increases dramatically on the fifth day of pregnancy, reaching its maximal concentration on day 6 followed by a 50% decrease on day 7. Changes in hyaluronic acid concentration also occur in pseudopregnant mice indicating that they are not related to the presence of the embryo in the uterus. The absolute concentration of the sulfated glycosaminoglycans, e.g., heparan sulfate, dermatan sulfate and chondroitin sulfate in the decidua did not change significantly. There was, however, a striking decrease of their biosynthesis in pregnant and pseudopregnant mice when compared to virgin mice, as shown by the use of radioactive inorganic sulfate as a precursor for the study of in vivo synthesis. A radioautographical analysis confirmed that the highest incorporation of radioactive sulfate was observed in virgin endometria when compared to pregnant ones. These studies also have shown a characteristic pattern of labeling in different regions of the endometrium that repeats itself during the different days of pregnancy.

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.

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.

Histochemical demonstration of phospholipid containing choline in the cytoplasm of murine decidual cells.

The localization of lipids in the endometrium of virgin and 6- to 9-day-pregnant mice was detected by histochemical methods. Total lipids (as shown by staining with Sudan black) and phospholipids containing choline (PCC) were detected. Sections subjected to reactions that have been proposed for the demonstration of vitamins E and D and cholesterol gave negative results. In the virgin animals, lipids were found in epithelial cells but not in the endometrial storma. On the other hand in the pregnant animals, the endometrial stroma contained both Sudan-black-stained lipids and PCC. Maximal staining was reached on day 8 of pregnancy. The staining was more conspicuous in the more differentiated decidual cells adjacent to the embryos than in the less differentiated predicidual cells. The nondecidualized stroma, situated peripherally near the myometrium did not stain for lipids. In the cytoplasm of decidual cells the reaction for PCC was observed in the form of granules, which were often arranged in groups surrounding the nuclei. We suggest that decidual cells store PCC to be mobilized as a precursor for mediators of decidualization, such as prostaglandins, that would act as paracrine inducers of the decidual reaction.

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.

Dissociation of F-actin induced by hydrostatic pressure.

F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits.