Investigation of the pregnancy-induced muscle bundle dispersal of the inner myometrium of adult mouse uterus and its relationship to the metrial gland/MLAp.

In the adult uterus of mice, rats and humans, the initially closely packed muscle bundles of the inner myometrium (muscular tissue that encircles the endometrium where the conceptus implants) undergo a pregnancy-induced dispersal that is clinically significant and hypothesized to regulate important pregnancy events. However, where, when and how this dispersal occurs, what its functions are, as well as its spatial relationship to the mouse metrial gland/mesometrial lymphoid aggregate of pregnancy (MG/MLAp), are unknown. The MG/MLAp, is a pregnancy-induced uterine structure required for successful rodent pregnancy located mesometrial to (above) the decidua basalis (pregnancy-modified mesometrial endometrium) and defined by its accumulation of maternal lymphocytes known as uterine Natural Killer (uNK) cells. To begin to understand how mouse inner myometrium dispersal (IMD) occurs, we spatiotemporally described it by observing the distribution of its muscle bundles and measuring their volume fraction (VF), as well as the VF of uNKs and stromal cells of inner myometrium. We discovered that (a) IMD (defined as reduction in VF of inner myometrium muscle bundles) is restricted to the mesometrial half of the uterus, is first evident at Embryonic day (E) 5.5 (early postimplantation) but not at E3.5 (preimplantation), further increases between E6.5 and E7.5 and remains unchanged from E7.5 to E10.5, (b) IMD initiation (observed between E3.5 and E5.5) occurs in the absence of uNKs and is associated with VF increases of pre-existing inner myometrium stromal cells and (c) the IMD observed between E6.5 and E7.5 is not associated with VF increases of uNKs or stromal cells. To get functional clues about IMD, we examined whether stromal cells between the dispersed muscle bundles undergo decidualization (important for correct fetomaternal interactions) and provide evidence that they do by E10.5, based on their production of Desmin (decidualization marker). Lastly, we examined whether mouse MG/MLAp only comprises the dispersed inner myometrium or additionally includes the mesometrial triangle (a triangular-like area mesometrial to the inner myometrium at the mesometrium-uterus attachment site), as is the case in rats. Our data supports that the dispersed inner myometrium is the only tissue that makes up the mouse MG/MLAp. In conclusion, we provide novel cellular and spatiotemporal insights about IMD that will contribute to understanding its mechanism and function and allow more informed inter-species comparisons about this process.

The involvement of interleukin (IL)-15 in regulating the differentiation of granulated metrial gland cells in mouse pregnant uterus.

Previous studies have suggested that granulated metrial gland (GMG) cells are bone marrow-derived lymphoid cells, which differentiate in situ in the mouse pregnant uterus into natural killer (NK)-like cells. Similar to NK cells, GMG cells express an abundant level of cytolytic mediators such as perforin. The factor(s) regulating the differentiation of GMG cells remain(s) to be identified, although cytokines previously implicated in the stimulation/activation of NK cells (e.g., IL-2, IL-6, IL-7, and IL-12) can be considered as potential candidates. Recently, IL-15, a novel cytokine, which displays biological activities similar to IL-2, has also been shown to be capable of activating NK cells. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we have demonstrated in the present study that IL-15 and its cognate receptor, but not the other cytokines, are expressed in the mouse pregnant uterus, with a time course concomitant with those of cytolytic mediators in differentiated GMG cells. Moreover, IL-15, though not IL-2, is capable of inducing the expression of perforin and granzymes in pregnant uterine tissues explanted in vitro. Data obtained from in situ hybridization study have suggested that the macrophages present in the pregnant uterus may be responsible for the production of IL-15. These results suggest that IL-15 is involved in regulating the differentiation of GMG cells during mouse pregnancy.

The metrial gland in the rat and its similarities to granular cell tumors.

Metrial glands are normal structures located in the mesometrial triangle of the pregnant rat uterus from gestational day (GD) 8 through termination of pregnancy. Metrial glands are composed of a dynamic mixed cell population of granulated metrial gland (GMG) cells, endometrial stromal cells, trophoblasts, blood vessels, and fibroblasts. Collections of similar cells may be seen in association with pseudopregnancy and other hormonal disturbances. Granulated metrial gland cells are the hallmark cell of the metrial gland. They are bone-marrow-derived, perforin-positive, natural killer cells that proliferate in the pregnant uterus. Understanding the normal histogenesis of the metrial gland and recognizing the possible existence of GMG cells and a reactive metrial gland in the nonpregnant state are important when examining any uterine lesion that contains granulated cells. This report demonstrates that the cellular composition, morphology, and immunohistochemical staining profile of normal metrial glands are similar to reported granular cell neoplasms in rats and mice. The possibility of a non-neoplastic lesion involving the metrial gland should be considered when proliferative lesions involving granulated cells are observed in the uterus of mice and rats from nonclinical toxicity studies. Positive immunohistochemical staining for perforin and S100 would assist in the classification of such lesions as a reactive metrial gland or decidual reaction.

Uterine natural killer cells in perforin and beta(2)-microglobulin deficient mice.

Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and perforin on proliferation and differentiation of uNK cells in pregnancy, using beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice and perforin-deficient (P(-/-)) mice. The cell population of uNK cells in the metrial gland of P(-/-) mice was tended to be higher than the control B6 mice. The cell population of uNK cells in the metrial gland of beta(2)m(-/-) mice was significantly increased at Day 12 of pregnancy comparing to B6 and P(-/-) mice. On the other hand, the cell population of uNK cells in the decidua basalis of beta(2)m(-/-) mice was tended to be lower than B6 and P(-/-) mice. These results indicate that beta(2)m may be involved in proliferation of uNK cells in the metrial gland, and that beta(2)m may affect the maturation of uNK cells in the decidua basalis.

Vascular endothelial growth factor production by rat granulated metrial gland cells and their morphological features in normal and pathological conditions.

Granulated metrial gland (GMG) cells are pregnancy-specific cells that may have many functions in successful placentation and pregnancy. In the present study, changes in the rat GMG cell structure, distribution and vascular endothelial growth factor (VEGF) expression during early pregnancy were evaluated by light microscopy. Implantation sites taken from females with spontaneous abortion were also investigated. On Day 7 of pregnancy, GMG cells were distributed through the implantation and interimplantation sites. They formed metrial glands in the mesometrial triangle on Day 9, and were observed in the decidua basalis on Day 14 of pregnancy. Avidin-biotin complex immunohistochemistry revealed that GMG cells showed moderate staining for VEGF at the beginning of pregnancy and intense staining on Days 9 and 10 of pregnancy. They were localised mostly near the newly formed blood vessels. The implantation sites from spontaneously aborting females showed numerous leucocytes in the lumen of mesometrial blood vessels. In spontaneously aborting females, GMG cells showed a distinct morphology, increased in number and volume, their granules were denser and degranulation was observed. These results suggest that rat GMG cells might be a guide for placental angiogenesis and they might share a role with leucocytes in pathological conditions.

[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.

Monoclonal antibodies directed against surface and intracellular antigens of mouse granulated metrial gland cells.

Rat monoclonal antibodies with reactivity directed against mouse GMG cells have been produced. One of the antibodies (GMG-1) reacts with a surface antigen of GMG cells and cross-reacts with T lymphocytes. Another (GMG-2) reacts with an intracellular antigen in GMG cells and with asialo-GM1 positive cells in the spleen. Three antibodies (GMG-3, -4, -5) bind to intracellular antigens in GMG cells. The cross-reactivity of these antibodies is discussed with reference to the lineage relationship of GMG cells to NK cells and T cells and the recent suggestion that NK cells and T cells have a common progenitor cell. It is proposed that GMG cells share this common progenitor cell but are otherwise independent of the NK or T cell lineages.

Granulated metrial gland cells in the non-traumatized regions of the uterus of ovariectomized mice with deciduomata maintained on progesterone.

A study has been made of granulated metrial gland (GMG) cells in the non-traumatized regions of the uterus of ovariectomized mice maintained on progesterone and with deciduomata in part of one uterine horn. GMG cells were found in the non-traumatized region of the uterine horns with deciduomata for at least 8 days after induction of the decidual reaction. GMG cells were not found in the contralateral horns without deciduomata. Activation of endometrial stromal cells is characterized by a high mitotic index and, in mice given colchicine, numerous stromal cells were arrested in metaphase in the non-traumatized region of the uterine horns with deciduomata for at least 3 days after induction of the deciduomata. In uterine horns without deciduomata, stromal metaphases were few in number over the same period. A correlation was therefore found between the presence of GMG cells and large numbers of stromal cells in mitosis. There was also a correlation between the absence of GMG cells and small numbers of stromal cells in mitosis. These results are compatible with the hypothesis that the differentiation and maintenance of GMG cells are under the control of endometrial stromal cells.

Differentiation of granulated metrial gland cells in ovariectomized mice given ovarian hormones.

A study has been made of the differentiation of granulated metrial gland (GMG) cells in ovariectomized mice given progesterone and oestradiol at a time equivalent to the first 6 days of pregnancy. By varying the hormone regime, it was shown that progesterone is the ovarian hormone likely to be involved in stimulating GMG cell differentiation in the early pregnant uterus. Progesterone, however, failed to maintain, or allow further differentiation, of GMG cells during stages equivalent to the early post-implantation period, suggesting that the progesterone is not directly stimulating this cell population. The hypothesis that the differentiation and maintenance of GMG cells is under the control of activated stromal cells in the preimplantation period is supported by these results.

Granulated metrial gland cells in the mouse placenta.

A study has been made of granulated metrial gland (GMG) cells and adjacent layer 1 cytotrophoblast in the labyrinthine placenta of the mouse. In those cellular associations in which both cell types appeared healthy with the light microscope, examination with the electron microscope identified features which may form part of the early stages in the interaction which can occur between GMG cells and some layer 1 trophoblast. These included microvillous processes from each cell forming an interdigitating network, a polarization of mitochondria in a GMG cell with interdigitating microvilli, platelets, and some GMG cells which appeared to form a specialized cell junction with a small lymphocyte. The latter observation indicates that a tripartite functional relationship may exist between lymphocytes, GMG cells and layer 1 labyrinthine trophoblast. In a quantitative analysis of GMG cells in the maternal blood spaces of placentae no significant differences in the numbers of GMG cells present in these spaces or involved in an interaction with layer 1 labyrinthine trophoblast were detected between inbred and outbred pregnancies. These results suggest that histocompatibility antigens are not significant factors in determining the frequency of interactions between GMG cells and trophoblast.

A study on the relationship between parity and differentiation of granulated metrial gland cells.

Granulated metrial gland (GMG) cells are a feature consistently associated with rodent pregnancy. Little information is available, however, on the life history of GMG cells in pregnancies other than first pregnancy. To determine if the frequency of GMG cells in the mesometrial triangle of mice is altered by parity, a study of histological sections from pregnant uteri of several females of genotype C.B-17 scid/scid.bg/bg being retired from a breeding program was undertaken. Pregnancies in this study ranged from first to 13th; day 12.5 of gestation was used for all analyses. The frequency of GMG cells in a constant, measured area of the metrial gland on day 12.5 of pregnancy was not significantly altered between first and second pregnancies. A small but statistically significant decline in GMG cell frequency was observed in two females who had given birth to 10 or more litters. However, neither the size of individual day 12.5 GMG cells, nor their granularity changed with parity. The surface area of the placenta in sections was not altered by the decrease in GMG cells. These data suggest that the differentiation of GMG cells from their non-granulated progenitors is a tightly regulated process, that GMG cell progenitors may be fully mature in first pregnancy, and that depletion of GMG cell progenitors is not induced by advanced parity or age.

The killing of rat placental cells by rat and mouse granulated metrial gland cells in vitro.

Small round cells which migrated from explant cultures of rat metrial gland were identified as granulated metrial gland (GMG) cells. They contained large amounts of glycoprotein and displayed the leucocyte common antigen. Other cells which migrated from the explants were probably derived from the fibroblast-like stromal cells of the metrial gland. The asialo-GM1 antigen was found on rat GMG cells in culture and in cryostat sections of rat metrial gland. The rat GMG cells in culture exhibited locomotion and, when co-cultured with placental cells, made numerous contacts with the placental cells. A small number of these contacts (less than 1 per cent) were followed rapidly by the death of the placental cell. Mouse GMG cells which had migrated from explant cultures of mouse metrial gland were also co-cultured with rat placental cells. The migratory activity of the mouse GMG cells also involved numerous contacts being made with rat placental cells and a small number (less than 1 per cent) of these contacts were cytotoxic for the rat placental cells. The observations support previous suggestions that GMG cells are a type of killer cell. The cytotoxic activity of rat and mouse GMG cells against co-cultured rat placental cells is discussed in relation to the nature of the target molecule involved.

Granulated metrial gland cells.

Granulated metrial gland (GMG) cells take their name from the metrial gland. The metrial gland is formed during pregnancy in many rodents with the appearance of GMG cells in the mesometrium at each implantation site. This paper reviews knowledge about GMG cells in rats and mice: the species most extensively studied. Granulated metrial gland cells are characterised by their cytoplasmic granules which contain glycoproteins and hydrolytic enzymes. The cytoplasm of some GMG cells contains extensive deposits of glycogen and moderate amounts of rough endoplasmic reticulum and Golgi bodies are usually present. Some GMG cells are binucleate and at certain stages of pregnancy many undergo mitosis. A few GMG cells are present in the endometrium (in mice) before implantation but in rats and mice during the week following implantation their numbers rapidly increase. During the 2nd week of gestation GMG cells are a prominent cell population in the decidua basalis and they appear in the circular layer of the myometrium and within the mesometrial triangle. By the beginning of the 3rd week of gestation they are present in the metrial gland in large numbers but they disappear and are relatively scarce at parturition. Rat and mouse GMG cells are readily distinguished by differences in the ultrastructure of their electron-dense granules. These differences have made it possible to show that GMG cells differentiate from bone marrow cell precursors by studying GMG cells in radiation-induced chimeric mice. The disappearance of GMG cells from the decidua basalis and metrial gland as pregnancy proceeds is accounted for by their death in situ and by their migration into blood vessels. Some GMG cells probably become trapped in lung capillary beds but the GMG cells in the maternal blood spaces of the placental labyrinth appear to interact with some layer 1 trophoblast cells and degeneration of the trophoblast and GMG cells occurs. Other cell types present in the uterus are described and their relationships to GMG cells considered. A close morphological relationship exists between cells in the decidua basalis and GMG cells and between fibroblast-like stromal cells in the metrial gland and GMG cells. Although initially GMG cells are closely packed between smooth muscle cells at the base of the mesometrium, the organisation of muscle cells in this region is disrupted with the formation of the metrial gland. Macrophages are considered, particularly in relationship to endocytotic activity of cells in the uterus, and it is argued that "it is not appropriate simply to dismiss GMG cells as macrophages".(ABSTRACT TRUNCATED AT 400 WORDS)

Granulated metrial gland cells in 'minor' species.

Granulated metrial gland cells, also known as uterine natural killer cells or large granular lymphocytes, are pregnancy associated leucocytes of granular phenotype. They are well characterised in mice and humans in terms of their structure, origin and distribution although the function of these cells has yet to be determined. In this review, granulated metrial gland cells in 'minor' species of rodents, insectivores, primates and species with epitheliochorial placentae are described. Emphasis is given to the comparative structure and distribution of granulated metrial gland cells in these minor species and to their possible functional association with trophoblast. Comparative studies of granulated metrial gland cells in minor species complements other approaches such as can be provided using mutant mice.

Evaluation of the murine metrial gland for immunological function.

The metrial gland (MG) is a transient uterine structure associated with rodent pregnancy. The gland is a complex structure consisting of stromal and vascular elements, as well as a population of histologically distinctive, large, granulated metrial gland (GMG) cells. The functions of the MG and of the GMG cells, as well as their relationship to the success of pregnancy, are unknown. Based upon morphological and morphometric studies it has been proposed that the MG might be involved in the immunology of pregnancy and that GMG cells could be immunocompetent. Explant cultures of MG have therefore been evaluated for immunological function. Lytic activity against the NK sensitive target cell line YAC and mitogen responsiveness could not be detected. MG tissue and medium conditioned by overnight culture of MG tissue (MG-CM) suppressed the response of murine spleen cells to Con A. MG-CM also reduced the lytic activity of splenic NK cells against YAC target cells. However, uptake of [3H]thymidine was elevated when YAC cells were cultured in MG-CM. The response of embryonic and uterine cells to growth in MG-CM was complex. MG-CM had little effect on isotope incorporation by decidual cells recovered at 6.5 days or by embryonic cells recovered from 12.5 day embryos. However, thymidine incorporation was less in MG-CM than in control medium for 12.5 day placental cells, 6.5 day embryonic sac, 6.5 day ectoplacental cone and 3.5 day blastocysts. Cytotoxicity and cytostasis accounted for reduced uptake of isotope in cultures of 3.5 day blastocysts and 6.5 day embryonic tissues. Loss of viability could not be detected in any other assays. Both YAC cells and unstimulated splenocytes showed altered morphology and improved viability when cultured in MG-CM. This study suggests that the only immunological role the MG might have during normal pregnancy is that of non-specific intra-uterine suppression. Alternatively, differential regulation of cell proliferation might be a function of the MG, within the pregnant uterus. The latter mechanism could also account for the apparent observation of non-specific immunosuppression.

Granulated metrial gland cells: hypotheses concerning possible functions during murine gestation.

Granulated metrial gland (GMG) cells are morphologically distinctive lymphoid cells found in the murine uterus only during gestation. The life history of GMG cells suggests that they have important roles during mammalian gestation but these roles have been difficult to define. Genetic and immunologic data suggest that GMG cells may be a specialized subset of natural killer (NK) lymphocytes. This has directed research on GMG cell functions towards questions of effector cell-target cell interactions. A broader range of potential functions is discussed and shifts in functional roles played by GMG cells are proposed over the course of gestation.

Receptors for IgG2b on cells of the mouse metrial gland.

Single cells prepared from metrial glands of mice killed at days 10, 13 and 17 of pregnancy were assayed for the expression of Fc gamma receptors in a standard rosetting assay using sheep red blood cells sensitised with a mouse monoclonal IgG2b antibody. Rosettes, indicating Fc gamma receptors, were found on both granulated metrial gland (GMG) cells and non-GMG cells, comprising mainly stromal cells, from each stage of pregnancy. Some animals were given an intravenous injection of horseradish peroxidase 2 h before they were killed in order to identify endocytic cells. No GMG cells were found to have endocytosed the horseradish peroxidase. Non-GMG cells which showed endocytic activity all expressed Fc gamma receptors but these receptors were also found on some of the non-GMG cells which had not exhibited endocytosis. The finding of Fc gamma receptors on GMG cells provides further evidence that these cells may be related to NK cells.

Mouse metrial gland cells do not kill Yac-1 myeloma cells.

Mouse metrial gland cell suspensions, which included granulated metrial gland cells, were assessed for their ability to lyse NK cell target Yac-1 myeloma cells in a 51chromium release cytotoxicity assay. Metrial gland cells did not kill Yac-1 cells even after in vivo stimulation of NK cytotoxicity activity by polyinosilic-cytidilic acid. The precise relationship of granulated metrial gland cells to the NK cell lineage remains to be clarified.

A monoclonal antibody, 4H12, recognizes a surface antigen found on granulated metrial gland cells in the murine decidua.

A monoclonal antibody (MAb), designated 4H12, was selected for reactivity to a surface antigen on PYS-2 teratocarcinoma cells. 4H12 was the product of a fusion of lymphoid cells of a non-immunized pregnant C57BL/6 mouse to NS-1 myeloma cells. Initial studies utilizing immunohistochemistry revealed that MAb 4H12 bound to an antigen found on cells in the decidua basalis of 7-, 8- and 10-day pregnant mice. Antigen-positive cells of 11--19-day pregnant mice were also found predominantly in the decidua. A few antigen-positive cells were found in the labyrinth of the placenta and up against Reichert's membrane. Antigen-positive cells were morphologically and spatially distinct, oval to round with large periodic acid Schiff positive granules. Indirect immunofluorescent (IIF) labeling of decidual cultures showed antigen on the surface of cells that were small, oval to round and adherent. The antigen recognized by MAb 4H12 was removed from tissue sections with trypsin and protease and therefore is suggested to be a protein. We conclude that MAb 4H12 recognizes a surface antigen found on cells historically described as granulated metrial gland (GMG) cells. This MAb should greatly facilitate the further analysis of the life history and function of GMG cells during pregnancy.

Leucocyte membrane antigens on mouse granulated metrial gland cells.

An indirect immunofluorescence technique has been used to study the expression of leucocyte membrane antigens on mouse granulated metrial gland (GMG) cells. GMG cells isolated from cultured metrial gland explants and GMG cells in cryostat sections of uterine implantation sites were examined. GMG cells were found to express both the asialo-GM1 antigen and the Thy-1 antigen. GMG cells did not express the Lyt-1, Lyt-2, Mac-1, L3T4 or IgM antigens. These results provide new evidence that GMG cells are a type of NK cell.