The structure and differentiation of granulated metrial gland cells of the pregnant mouse uterus

Summary A study was made with the light and electron microscopes of the granulated metrial gland cells of the decidua basalis of the pregnant mouse uterus, up to day 11 of pregnancy. The granulated metrial gland cells are large, up to 50 in diameter, mono- or binucleate and the glycogen rich cytoplasm typically contains many large glycoprotein granules which may be up to 5 in diameter. Morphological evidence is described in support of a lymphocyte-like cell being the precursor to the granulated metrial gland cell. This differentiation sequence is similar to that already proposed in the rat but differences between the ultrastructure of the mature metrial gland cells of rats and mice were noted.     

Immuno-electron-microscopic localization of enkephalin in the secretory granules of C cells in the chicken ultimobranchial glands

Interstitial cells in the pineal gland of the rat were characterized immunocytochemically using the monoclonal antibodies MRC OX-42 and ED1 for macrophages/microglia, and MRC OX-6, which recognizes major histocompatibility complex (MHC) class II antigen. A polyclonal antibody against GFAP was used to identify astrocytes. Cells immunopositive for OX-42 and/or ED1 were distributed throughout the gland; they extended processes primarily along the perivascular spaces and occasionally within the parenchyma of the gland. Ultrastructurally, these OX-42-positive cells were characterized by a nucleus with sparse heterochromatin and cytoplasmic vacuoles/lysosomes. Cells expressing MHC class II antigen had a distribution and morphology similar to OX-42-immunopositive cells, suggesting that pineal macrophages/microglia play a role as antigen-presenting cells. GFAP-positive astrocytes were concentrated at the proximal end of the pineal where the pineal stalk enters the gland. The occurrence of antigenpresenting cells in the circumventricular neuroendocrine gland has important functional implications as these cells may be mediators of neuroimmunomodulatory mechanisms, and involved in certain disease states such as autoimmune pinealitis.     

Immunophenotypic evidence for distinct populations of microglia in the rat hypothalamo-neurohypophysial system

The morphology, distribution and immunophenotype of microglia throughout the adult rat hypothalamo-neurohypophysial system was examined. Four macrophage-associated antibodies (OX-42, F4/80, ED1 and ED2) were used; the expression of major histocompatibility complex antigens was investigated by use of antibodies against OX-6, OX-17 (MHC class II) and OX-18 (MHC class I). Three distinct types of microglia were identified. The first was located in the magnocellular nuclei; these radially branched (ramified) microglia had round cell bodies and long branched processes, and were strongly immunoreactive only for OX-42. The second was located outside the blood-brain barrier in the median eminence, pituitary stalk and neurohypophysis often close to blood vessels; these compact microglia had irregular cell bodies and shorter processes, and were strongly labelled by OX-42 and F4/80, weakly labelled by OX-18, and generally unlabelled by ED1, ED2, OX-6 and OX-17. The third type was found in small numbers throughout the system at the surface of the neurvous tissue or around blood vessels; these perivascular microglia were elongated cells with no branching processes, and were strongly labelled by ED1, ED2, OX-18, OX-6, OX-17 and F4/80 antibodies but showed variable OX-42 immunoreactivity. Cells in a perivascular location were heterogeneous with respect to their immunophenotype. The presence in the normal adult rat hypothalamo-neurohypophysial system of MHC class-II molecules (OX-6 and OX-17) on a sub-set of perivascular microglia suggests that these cells are capable of presenting antigen to T lymphocytes. The microglia, which lie on either side of the blood-brain barrier, are well placed to facilitate interaction between the immune and neuroendocrine systems.     

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on days 13 and 14 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the natural suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector: target system at a ratio of 50 : 1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 60% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 ± 13.4 and 75.0 ± 12.5 µm², respectively) and pseudopregnant (97.5 ± 4.9 and 69.2 ± 3.5 µm², respectively) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 ± 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109 ± 5 µm². The data obtained confirm the known data on a low killer activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 26–34.Original Russian Text Copyright © 2005 by Podporina, Mikhailov.     

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on day 13 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the nuclear suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector : target system at a ratio of 50:1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 30% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 +/- 13.4 and 75.0 +/- 12.5 microm2, respectively) and pseudopregnant (97.5 +/- 4.9 and 69.2 +/- 3.5 microm2) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 +/- 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109.1 +/- 5.2 microm2. The data obtained confirm the known data on a low activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.     

Measurement of natural killer activity and target cell binding by mouse metrial gland cells isolated by enzymic or mechanical methods

Cells of the metrial glands of mice were isolated by enzymic or mechanical dissociation procedures. Morphological observations indicated that up to half of the enzymically dissociated cells and nearly all of the mechanically dissociated cells were granulated metrial gland cells, but the presence of some fibroblast-like stromal cells among the latter population was not ruled out. Moreover, the granulated metrial gland cells had lost a substantial part of their granule content during isolation. Both cell preparations had little or no natural killer (NK) activity, indicating either that granulated metrial gland cells are not NK-like or that their NK activity was impaired by loss of granule-associated lytic substances or by other factors. Enzymically dissociated metrial gland cells did not bind significantly to the NK target cell YAC-1, nor did they develop granules, NK activity, or the ability to bind YAC-1 cells during culture in vitro, either in normal medium or with the addition of indomethacin or lymphokines. Mechanically dissociated metrial gland cells bound avidly to YAC-1 cells but not to P815 cells or adult thymus cells, which are not NK target cells. Since many if not most of the mechanically dissociated metrial gland cells appeared morphologically to be granulated metrial gland cells, their selective binding to an NK target cell suggests that granulated metrial gland cells may be related in some way to NK cells.     

Isolation of serine protease from granulated metrial gland cells of mice and rats with lectin from Dolichos biflorus.

Granulated metrial gland cells were the only cells in the endometria of pregnant mice and rats that reacted histochemically with fluoresceinated lectin (DBA) from Dolichos biflorus. Cell extracts of uteri of pregnant animals, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analysed by lectin overlay blotting, contained DBA-reactive, 40-50 kDa, doublet glycoprotein bands. This glycoprotein was purified on a DBA agarose affinity column. It was identified by amino acid sequencing as a serine protease closely related to granzymes of T lymphocytes. We conclude that this granzyme accounts for the selective reactivity of granulated metrial gland cells with fluoresceinated DBA in histological sections of uteri of pregnant rodents and show that DBA affinity columns can be used for purification of granzyme derived from granulated metrial gland cells.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

妊娠小鼠子宫腺细胞区的碱性磷酸酶与酸性磷酸酶的研究

小鼠子宫系膜三角区在妊娠后出现上皮样细胞群,群内的细胞称颗粒子宫腺细胞(granu-lated metriial gland cells,GMG细胞),该区改称子宫腺细胞区(metriial gland cell area,MGCA).取孕12～19天MGCA,液氮速冻,恒冷箱切片,偶氮偶联法显示碱性磷酸酶(ALP)与酸性磷酸酶(ACP).结果为,ALP主要分布于GMG细胞群间的疏松结缔组织中;GMG细胞为阴性反应.ACP主要位于GMG细胞内,群间结缔组织含量较少.两种酶的活性随胎龄增加而减弱.ALP与ACP的定位与活性变化特性显示它们与GMG细胞功能关系密切.     

Localisation of the MRC OX-2 Glycoprotein on the Surfaces of Neurones

The MRC OX-2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B-lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX-2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double-immunofluorescence staining of cerebellar interneurone cultures with MRC OX-2 antibody and tetanus toxin showed that all tetanus-positive cells (neurones) were MRC OX-2-positive. Glial fibrillary acidic protein-positive astrocytes were not labelled by MRC OX-2 antibody. Thus OX-2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy-1.     

The metrial gland

The information available about the metrial gland of the pregnant rodent uterus with its content of granulated metrial gland (GMG) cells is reviewed. Recent research shows that GMG cells differentiate from bone marrow cells and supports the suggestion that GMG cells are involved in the immunological relationship between mother and foetus. There is probably a complex association between GMG cells and stromal cells of the metrial gland, and it is suggested that the association between GMG cells and the placental labyrinthine cells represents a functional interaction.     

In situ localization and characterization of bone marrow-derived cells in the decidua of normal murine pregnancy.

The study reported here was designed to examine the in situ distribution and characteristics of hemopoietically derived decidual cells during normal pregnancy in mice prenatally reconstituted with bone marrow cells carrying a transgenic marker. Bone marrow cells from a transgenic CD-1 strain (CD-1 beta; carrying 1000 copies of beta-globin genes in tandem) were injected into the yolk sac of Day 17 conventional CD-1 embryos. The pregnant females were allowed to deliver normally, and the female offspring raised to puberty were mated with CD-1 males and then killed on Day 12 of gestation. The extent of chimerism in sections of their spleens, uteri, and other organs was evaluated by in situ hybridization of the sections with a biotinylated cDNA probe specific for the beta-globin genes followed by avidin-biotin-peroxidase staining. Tissue controls were provided by CD-1 beta and CD-1 mice, respectively. Tissues were also processed without the application of the probe or with the application of biotinylated lambda DNA as specificity controls. Reconstituted mice exhibited variable degrees of hemopoietic chimerism as indicated by labeling of their splenic lymphocytes (18-54%; mean 42%) as well as hemopoietic cells in other organs. Variable cellular labeling was also noted in their decidua basalis and metrial glands. Labeled cells in these tissues were identified as typical decidual cells, macrophages, and granulated metrial gland (GMG) cells. Labeling of typical decidual cells varied extensively among implantation sites in the same chimera, the average labeling ranging from 17% to 33% (mean 24%) in various chimeras. Labeling was also noted in GMG cells, lymphocytes, and some decidual cells migrating out of metrial gland explants after 24-h culture. The non-pregnant uterus of a chimeric mouse revealed significant labeling of endometrial stromal cells indicative of their hemopoietic origin. These results revealed a hemopoietic origin of certain typical decidual cells and GMG cells identified in situ during normal murine pregnancy and a hemopoietic origin of certain endometrial stromal cells that may represent precursors of decidual cells. The precise timing of the predecidual stem cell migration from the bone marrow to the uterus remains to be defined.     

Contribution of cytokines on the suppression of lung metastasis

Weekly injection of a protein-bound polysaccharide PSK in mice with Lewis Lung Cancer (LLC) significantly decreased the number of lung metastatic foci concomitant with enhancement of cytostatic activity in the bronchoalveolar lavage (BAL) cells. These effects were more marked when the agent was given intratracheally, inducing a larger number of pulmonary macrophages, lymphocytes and neutrophils concomitant with increases in BAL tumor necrosis factor- (TNF-), mouse inflammatory protein- (MIP-1), mouse inflammatory protein- (MIP-1), interleukin-1 (IL-1) and interleukin-6 (IL-6), but not interleukin-2(IL-2) and interleukin-4 (IL-4). Pre-treatment with anti TNF- antibody reduced these effects. The time course and production of PSK-induced cytokines were similar between the tumor-bearing mice and control mice. BAL neutrophils in mice with LLC showed a tendency toward acceleration of O2- production compared with circulating neutrophils. Pulmonary macrophage phagocytosis was also significantly higher in the LLC mice.These results suggest that enhancement of cytostasis appears to be induced by activation and/or improvement of function in inflammatory and immune cells through cytokines under immunomodulator treatment in lung metastasis, possibly via a TNF--dependent mechanism.     

颗粒子宫腺细胞的分离、纯化与细胞化学研究

胰蛋白酶消化小鼠子宫腺细胞区,制成单细胞悬液,滴片后进行细胞学及细胞化学研究。结果：有多种类型细胞存在于悬液中,其中中等大小细胞（直径２０μｍ左右）大部分为颗粒子宫腺细胞（ＧｒａｎｕｌａｔｅｄＭｅｔｒｉａｌＧｌａｎｄＣｅｌｌｓ,ＧＭＧｃｅｌｌｓ）。ＧＭＧ细胞约占所有细胞的５０％。利用Ｐｅｒｃｏｌｌ非连续性密度梯度离心可以提高ＧＭＧ细胞的纯度,使之达８０％以上。细胞化学研究显示ＧＭＧ细胞含ＡＣＰ,ＮＳＥ,ＬＮＡｓｅ及糖蛋白成份。     

The differentiation of the decidua and the distribution of metrial gland cells in the pregnant mouse uterus

Summary A study was made with the light microscope of the cellular changes involved in the formation of the decidua in the pregnant mouse uterus up to day 11 of pregnancy. The differentiation sequence was similar to that found by previous workers but the morphology and development of the basal zone is described in more detail. In addition, the morphology of glycogen rich cells in an area termed the lateral decidual zone is described. The distribution of granulated metrial gland cells and their precursors is described. These cells are first found in the uterine stroma before the blastocyst has implanted. Later they occur in the decidua and in the circular smooth muscle zone beneath the mesometrial triangle prior to the formation of the metrial gland. Granulated metrial gland cells are also found in the maternal blood spaces of the implantation site.     

Immunocytochemical localization of a growth-associated protein (GAP-43) in rat adrenal gland

We have localized at light and electron-microscopic level the growth-associated protein GAP-43 in adrenal gland using single and double labelling immunocytochemistry. Clusters of GAP-43-immunofluorescent chromaffin cells and many immunofluorescent fibres were observed in the medulla. GAP-43-immunoreactive fibres also formed a plexus under the capsule, crossed the cortex and ramified in the zona reticulata. Double labelled sections showed the coexpression of GAP-43 with a subpopulation of tyrosine hydroxylase-and of dopamine--hydroxylase-immunoreactive chromaffin cells. Dual colour immunofluorescence for GAP-43 and calcitonin gene-related peptide (CGRP) revealed that some of the GAP-43-immunoreactive fibres also express CGRP. Pre-embedding electron microscopy showed GAP-43 immunoreactivity associated with the plasma membranes and cytoplasm of noradrenaline-producing chromaffin cells, and with processes of nonmyelin-forming Schwann cells. Immunoreactive unmyelinated axons and terminals were also observed. The immunostained terminals made symmetrical synaptic contacts with chromaffin cells. Immunoreactive unmyelinated fibres and small terminals were present in the cortex. Our results show that GAP-43 is expressed in noradrenergic chromaffin cells and in various types of nerve fibres that innervate the adrenal. Likely origins for these fibres include preganglionic sympathetic fibres which innervate chromaffin cells, postganglionic sympathetic fibres in the cortex, and CGRP containing sensory fibres.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Migration of granulated metrial gland cells from cultured explants of mouse metrial gland tissue

Summary The migration of granulated metrial gland (GMG) cells from cultured explants of metrial gland tissue obtained from mice killed between days 10 and 16 of pregnancy has been studied. GMG cells migrated from all of the explants but more GMG cells were found around explants obtained from mice at day 10 of pregnancy than around explants obtained at later stages of pregnancy. The number of GMG cells found around each explant reached a peak at days 1, 2 or 3 of culture but only a few GMG cells were found around the explants by day 7 of culture.