Rat Metrial Gland Research Articles

IL1β-NFκβ-Myocardin signaling axis governs trophoblast-directed plasticity of vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) plasticity is fundamental in uterine spiral artery remodeling during placentation in Eutherian mammals. Our previous work showed that the invasion of trophoblast cells into uterine myometrium coincides with a phenotypic change of VSMCs. Here, we elucidate the mechanism by which trophoblast cells confer VSMC plasticity. Analysis of genetic markers on E13.5, E16.5, and E19.5 in the rat metrial gland, the entry point of uterine arteries, revealed that trophoblast invasion is associated with downregulation of MYOCARDIN, α-smooth muscle actin, and calponin1, and concomitant upregulation of Smemb in VSMCs. Myocardin overexpression or knockdown in VSMCs led to upregulation or downregulation of contractile markers, respectively. Co-culture of trophoblast cells with VSMCs decreased MYOCARDIN expression along with compromised expression of contractile markers in VSMCs. However, co-culture of trophoblast cells with VSMCs overexpressing MYOCARDIN inhibited their change in phenotype, whereas, overexpression of transactivation domain deleted MYOCARDIN failed to elicit this response. Furthermore, the co-culture of trophoblast cells with VSMCs led to the activation of NFκβ signaling. Interestingly, despite producing IL-1β, trophoblast cells possess only the decoy receptor, whereas, VSMCs possess the IL-1β signaling receptor. Treatment of VSMCs with exogenous IL-1β led to a decrease in MYOCARDIN and an increase in phosphorylation of NFκβ. The effect of trophoblast cells in the downregulation of MYOCARDIN in VSMCs was reversed by blocking NFκβ translocation to the nucleus. Together, these data highlight that trophoblast cells direct VSMC plasticity, and trophoblast-derived IL-1β is a key player in downregulating MYOCARDIN via the NFκβ signaling pathway.

Molecular regulation of vascular smooth muscle cell phenotype switching by trophoblast cells at the maternal-fetal interface

IntroductionEstablishment of hemochorial placenta is associated with development and remodelling of uterine vasculature at the maternal fetal interface. This results in calibration of high resistance uterine arteries to flaccid low resistance vessels resulting in increased blood flow to the placenta and fetus in humans and rodents. Mechanisms underlying these remodelling events are poorly understood. In this report, we examine regulation of vascular remodelling using vascular smooth muscle cell (VSMC) phenotype switching as a primary parameter. MethodsCellular dynamics was assessed by Immunofluorescence, qRT-PCR, western blotting in timed pregnant rat tissue. In vitro co-culture of trophoblast cells with vascular smooth muscle cells was used to understand regulation mechanism. ResultsAnalysis of cellular dynamics on days 13.5, 16.5 and 19.5 of gestation in the rat metrial gland, the entry point of uterine arteries, revealed that invasion of trophoblast cells preceded disappearance of VSMC α-SMA, a contractile state marker. Co-culture of VSMCs with trophoblast cells in vitro recapitulated trophoblast-induced de-differentiation of VSMCs in vivo. Interestingly, co-culturing with trophoblast cells activated PDGFRβ signalling in VSMCs, an effect mediated by secreted PDGF-BB from trophoblast cells. Trophoblast cells failed to elicit its effect on VSMC de-differentiation upon inhibition of PDGFRβ signalling using a selective inhibitor. Moreover, co-culturing with trophoblast cells also led to substantial increase in Akt activation and a modest increase in Erk phosphorylation in VSMCs and this effect was abolished by PDGFRβ inhibition. DiscussionOur results highlight that trophoblast cells direct VSMC phenotype switching and trophoblast derived PDGF-BB is one of the modulator.

Mode of cell death in the rat metrial gland during peripartum regression.

In the rodent uterus, the metrial gland develops during midpregnancy and undergoes regression prior to parturation. The involution of the gland is reported to be accompanied by the loss of gland cells due to their death in situ. Cell death has been classified by using morphological criteria into two types: necrosis and apoptosis. To study the mechanism involved in the peripartum regression of the rat metrial gland, we examined the mode of cell death in the gland during the last week of gestation. We identified apoptotic cells in the regressing metrial gland by using DNA fragmentation, in situ DNA 3'-end labeling, and electron microscopy. Expression of progesterone receptor (PR) and estrogen receptor (ER) was also demonstrated by immunohistochemistry in the gland. The mean weight of metrial gland nodes decreased after day 18 of pregnancy. The apoptotic granulated metrial gland (GMG) cells that were detected by using the in situ DNA 3'-end labeling method were observed on day 16 of pregnancy, and they increased in number after day 20 of pregnancy. Intense fragmentation of DNA was also found from day 20 to day 22 of pregnancy. Electron microscopy demonstrated apoptotic GMG cells in the regressing metrial glands, confirming the results of the labeling studies. Immunohistochemical study revealed that expression of PR and ER, which were localized mainly in fibroblast-like stromal cells but not in GMG cells, was almost unchanged during late pregnancy. Apoptotic cell death is the major mode of rat metrial gland cell death in the peripartum loss of metrial gland cells.

Antibody-dependent cellular cytotoxic activity of cells in the rat metrial gland in pregnancy.

Antibody-dependent cellular cytotoxic (ADCC) activity of cells in rat metrial glands at days 13 and 20 of pregnancy has been examined. A 51chromium-release cytotoxicity assay was carried out using Yac-1 cells and P815 cells as targets in the presence of the corresponding, heat inactivated rat antibody or non-immune rat serum. No killing was observed when effector metrial gland cells and target cells were cultured in the presence of non-immune rat serum. In the presence of rat antibody to Yac-1 cells, or rat antibody to P815 cells, cells from the metrial glands of rats killed at day 13 of pregnancy demonstrated ADCC activity, but cells from the metrial glands of rats killed at day 20 of pregnancy showed no ADCC activity. Spleen and peritoneal exudate cells displayed ADCC activity. Immunohistological and immunocytological studies showed OX-6, OX-34, OX-35 and OX-42 positive cells were present at days 13 and 20 of pregnancy, in sections of metrial glands and in the cell suspensions prepared for the cytotoxicity assays. It is suggested that macrophages are the cells most likely to be responsible for the ADCC activity in the rat metrial gland.

Natural killer cytotoxicity and antibody-dependent cytotoxicity of cells of rat metrial glands.

Cell suspensions of rat metrial gland tissue, containing healthy granulated metrial gland (GMG) cells, were assessed for their ability to lyse natural killer (NK) cell target Yac-1 myeloma cells in a [51Cr]-release cytotoxicity assay. Cytotoxicity indices were found to be low even after in vivo stimulation of NK cytotoxic activity by polyinosilic-cytidilic acid and the addition of interferon to test cultures. It is concluded that cell suspensions of rat metrial gland, and rat GMG cells in particular, do not exhibit NK cytotoxicity. However, when a heat inactivated rabbit anti-Yac-1 serum was added to assay cultures, cytotoxicity indices above background levels were obtained, indicating that rat metrial glands contain cells capable of antibody-dependent cytotoxicity.

Investigations on cell metabolism of rat term decidua and metrial gland using a combination of cryo- and plastic sections

Investigations on cell metabolism of rat term decidua and metrial gland using a combination of cryo- and plastic sections

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 granualted metrial gland (GMG) cells. They contained large amounts of glycoprotein and displayed the leucocyte common antigen. Other cells which migrated from the explans 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.

Fine structure and functions of the rat metrial gland: I. Effect of prostaglandin F2.ALPHA. on the granulated metrial gland cells.

The morphological changes of granulated metrial gland (GMG) cells were investigated in 10-day, 14-day, 19-day pregnant, and post partum rats, using light and electron microscopy. The effect of Prostaglandin F2α (PGF2α) on the pregnant uterus was also examined. The GMG cells began to appear at day 10 and reached their maximum number at day 14. On the 19th day of pregnancy myelination and vacuolation of the granules were noted in these cells, which subsequently seemed to degenerate. After parturition, most of the degenerated GMG cells were shown to be phagocytized by macrophages. PGF2α given at day 14, caused similar, but more abrupt, changes of the GMG cells. In addition, it was demonstrated that some of the remaining GMG cells were replaced by myoblasts which contained myofilaments in the cytoplasm.An autoradiography (ARG) technique was employed to detect the PGF2αreceptors. Developed silver grains were found in the GMG cells and in smooth muscle cells, but there were no receptors found in the interstitial tissue.Immunohistochemical staining with rabbit anti-rat relaxin serum showed a very strong reaction in luteal cells, as has been reported in the literature. The GMG cells had a similar reaction, although weak compared to the luteal cells.These results may indicate that the GMG cells originate from the smooth muscle cells and are associated with contraction and relaxation of the pregnant uterus.

Immunological characterisation of surface receptors on rat metrial gland cells

Cells from metrial glands of pregnant rats were examined for surface receptors. No E, EAμ or EAC receptors were demonstrated. Fcγ receptors were detected by EAγ rosette formation, using sheep red blood cells sensitised with the IgG fraction of rabbit or rat antisera. Significantly more of the metrial gland cells, and of the rat peritoneal exudate and spleen cells examined as controls, formed rosettes with red cells sensitised with rabbit IgG than with those sensitised with rat IgG. The proportion of metrial gland cells forming EAγ rosettes decreased significantly between day 12 and day 15 of pregnancy but increased by day 19. Metrial gland cells from deciduomata formed EAγ rosettes, and the proportions varied during pseudopregnancy. At day 13 of pregnancy a greater proportion of metrial gland cells displayed Fcγ receptors in multiparous rats than in primigravid rats. The binding affinity of the Fcγ receptors was characterised by inhibition studies with homologous and heterologous IgGs. Maximal inhibition occurred when the inhibitory IgG was homologous to the IgG used to sensitise the red cells. EAγ rosette formation by cells from the metrial gland was inhibited by both monomeric and heat-aggregated IgGs.

Localisation of immunoglobulin (IgG) within the rat metrial gland

An immunohistological method was used to assess the IgG content of the rat metrial gland at different stages of pregnancy. The result apparently varied according to the type of fixative used. Saturated alcoholic mercuric chloride was found to produce the most consistent demonstration, with the IgG located in cells which also contained diastase-fast, PAS-positive granules. Using single cell suspensions prepared from metrial glands, significantly more cells were shown to contain cytoplasmic IgG at day 13, compared to day 14 of pregnancy. However, there were no other significant differences at other stages of pregnancy examined. Surface IgG was detected on a small proportion of the cells and the findings are discussed in relation to the hypothesis of a lymphocytic origin for the granulated metrial gland cells.

The characterisation of Fc receptors on cells from the metrial gland of the pregnant rat uterus

Using an EA rosetting technique on cells from the rat metrial gland at days 12–15 of pregnancy, approximately 30% of the cells were found to possess immunoglobulin (Fc) receptors. At day 14 a comparison was made between cells from the metrial gland and cells from the peritoneal exudate; binding affinity of each for homologous and heterologous immunoglobulins was assessed by the resulting inhibition of rosette formation. Cells from the metrial gland were inhibited to a greater degree than cells from the peritoneal exudate by low concentrations of rabbit, human and rat immunoglobulin. The Fc receptors observed on cells from the metrial gland show some resemblance to those described by other workers on yolk sac cells and certain foetal placental cells, but their function is at present unknown.

Ultrastructural changes in the rat metrial gland in the latter half of pregnancy

The ultrastructural changes occurring in the metrial gland in the latter half of pregnancy in the rat have been studied. Typical palely stained granulated cells are present in the metrial gland up to day 20 but many granulated cells show variations in appearance which may be associated with degeneration. In some the cytoplasm is more darkly stained and such cells often have apparently empty areas of cytoplasm adjacent to the granules. From day 14 onwards many areas of the gland show cellular debris, apparently resulting from lysis of the granulated cells. However, occasionally normal granulated cells are present in blood vessels and have been observed apparently penetrating the vascular endothelium. A variety of changes was also noted in the stromal cell population. Inclusions became apparent in many of these cells; some of these consisted of lipid while others resembled granules from the typical granulated cells. Other cells with numerous inclusions appeared to be macrophages. Lysis of granulated cells in situ is compatible with suggestions that the metrial gland produces a holocrine secretion, though some normal granulated cells enter blood vessels. The stromal cells may have a phagocytic role in late pregnancy but evidence for this was inconclusive.

Bioassay of rat metrial gland extracts for relaxin using the mouse interpubic ligament technique.

Extracts of rat metrial gland tissue were assayed for relaxin using the mouse interpubic ligament technique. Relaxin preparations from pregnant rat and sow ovaries as well as Warner-Lambert Reference Relaxin served as controls. Dose-response analysis revealed that the metrial gland apparently is not a significant source of bioactive relaxin in the pregnant rat. Sow ovary extracts prepared in this laboratory had a dose responsecurve similar to that of Warner-Lambert Reference Relaxin. The dose-response of rat ovary preparations was significantly different from that of the sow ovary. The results are discussed in relation to the primary source of relaxin in the pregnant rat. (Endocrinology 94: 567, 1974)