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S Peel

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Distribution of 125iodine-labelled mouse immunoglobulin G injected intravenously in pregnant mice.

Mouse immunoglobulin G (IgG) was iodinated with 125iodine (I) and injected intravenously into pregnant mice in order to examine whether mouse granulated metrial gland (GMG) cells are able to take up IgG in vivo. The mice were injected intravenously on days 8, 12 or 16 of pregnancy and killed either 5 min, 2 h or 24 h after injection. Implantation sites and spleen, thymus, liver and para-aortic lymph nodes were fixed and autoradiographs of sectioned (1 micron) material prepared to examine the distribution of labelled IgG. In general, at all stages of pregnancy and time intervals examined after injection of the 125I IgG, radioactivity was detected at higher levels in blood vessels than in tissue spaces of the same regions. No evidence for the uptake of radioactive IgG by normal GMG cells in the decidua basalis, metrial gland or in the maternal blood spaces of the labyrinthine placenta was found. The only GMG cells which had accumulations of silver grains showed signs of pyknosis. The uptake of IgG by stromal cells in close proximity to GMG cells and the distribution of radioactivity in the extravascular tissues showed that the intravenously injected 125I IgG was available to the GMG cells. Accumulations of silver grains were a prominent feature of the regions immediately adjacent to most GMG cells in the placental labyrinth and some were clearly associated with degenerate layer 1 trophoblast cells. The radioactivity detected in degenerate GMG cells and degenerate layer 1 trophoblast cells may be the result of nonspecific uptake as a consequence of the cells' death.

Animals

The uterine response in pregnant inbred and non-inbred rats.

Comparisons were made between fetal, placental and metrial gland weights and the cellular composition of the placentae and metrial glands of pregnant inbred (Agus), outbred (Agus x Wistar) and random-bred (Wistar) rats. Fetal and placental weight differences between inbred and outbred rats provided evidence of hybrid vigour. Metrial gland weight differences between inbred and outbred rats showed that the Agus mothers responded to hybrid (Agus x Wistar) fetuses by producing heavier metrial glands than if they were carrying inbred fetuses, up to and included Day 18 of pregnancy. Histological examination revealed some differences between the 3 groups of rats at corresponding stages of pregnancy. Granulated metrial gland (GMG) cells formed a larger proportion of cells in the metrial gland in Agus rats carrying hybrid fetuses than in Agus rats carrying inbred fetuses up to Day 14 of pregnancy, but the situation was reversed after Day 14. Degenerative changes were more apparent in the metrial glands from Agus rats carrying hybrid fetuses than in rats from the other 2 groups from Day 15 of pregnancy onwards. Clusters of lymphocytes were a prominent feature of the placental labyrinths of Agus rats (mated with Agus or Wistar males) around Day 15 of pregnancy. Examination of single cell suspensions of metrial gland showed variations in the proportion of immunoglobulin G (Fc gamma) receptor bearing cells during pregnancy and between rats in the 3 groups of corresponding stages of pregnancy. The significance of the differences occurring in the metrial gland during pregnancy and between the groups at corresponding stages of pregnancy may be related to the functional involvement of GMG cells in cytotoxic activity directed against placental trophoblast.

Animals

Changes in the metrial gland of the rat uterus following ovariectomy on day 10 of pregnancy.

Rats were subjected to ovariectomy or a control operation on day 10 of pregnancy and light and electron microscope studies carried out to examine the cells of the decidua basalis and the mesometrial triangle. Degeneration of the decidua basalis was rapid but proliferation in the mesometrial triangle and differentiation of the typical granulated cells continued for some time, and resulted in the formation of a metrial gland. The earliest effect noted in the metrial gland was the appearance of inclusions in the fibroblast-like stromal cells one day after ovariectomy. During the next four days many stromal cells became packed with a variety of inclusions and it is suggested that some of these may represent phagocytosed cellular debris. Some debris appeared to be in intercellular spaces and was probably derived from granulated cells. Some granulated cells appeared to develop apparently empty vacuoles and this may be a preliminary stage in degeneration. Although granulated cells decreased to become very few in number five days after ovariectomy, the precise mechanism of their loss and the relationship of this to the hormonal environment could not be established.

Animals

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.

Animals

The effects on the rat uterus and placenta of ovariectomy at day 10 of pregnancy.

A study was made of the changes in morphology and in nuclear incorporation of tritiated thymidine in the rat uterus and placenta after ovariectomy at day 10 of gestation. There was some individual variation between different animals in the results of the ovariectomy, but in general the effects on both maternal and fetal tissues were more severe than those reported to follow ovariectomy at later stages of pregnancy. Even in the animals where there was extensive placental survival 2 or 3 days after the ovariectomy, normal differentiation of the placental labyrinth did not occur and the fetuses failed to survive. Ovariectomy had a pronounced effect on the proliferative activity of uterine epithelial cells. The high labelling index in the control animals up to day 12 was markedly reduced in the ovariectomized animals; after day 13, however, the labelling index of the controls was reduced to a level lower than that of the ovariectomized animals. Nuclear labelling occurred in the glandular epithelium from two days after ovariectomy, but was never present in the controls. There was a marked reduction in the percentage of labelled nuclei in the uterine muscle and in the metrial gland after ovariectomy. In the metrial gland this was associated with a reduction in the number of typical granulated cells and with the appearance of numerous small round cells. It is suggested that the latter represented metrial gland cell precursors which had undergone impairment of their normal differentiation process.

Animals

Metrial gland cells in deciduomata of pseudopregnancy.

Light and electron microscope studies were carried out on the metrial gland cells of deciduomata of pseudopregnancy in the rat. At the light microscope level these cells were characterised by a comparatively pale staining cytoplasm with numerous glycoprotein granules. Some also had extensive cytoplasmic deposits of glycogen. In electron micrographs the metrial gland cell cytoplasm as a whole was much less electron-dense than that of fibroblasts and decidual cells, but it contained densely stained pleomorphic granules. There were stacks of rough endoplasmic reticulum, oval or round profiles of fibroblasts and decidual cells, but it contained densely stained pleomorphic granules. There were stacks of rough endoplasmic reticulum, oval or round profiles of mitochondria, and an extensive Golgi network. The metrial gland cells were roughly spherical in appearance, with irregular surface projections. In all these features, and in the sequence of their appearance, first in the decidua basalis, and later in the mesometrial triangle, the granulated metrial gland cells of deciduomata resembled the metrial gland cells of pregnancy. In addition to granulated metrial gland cells, decidual cells and fibroblasts, a number of other cell types were identified. These cells could be placed in a series, with each member of the series minimally different in morphological appearances from its neighbours. It is proposed that the series represents the differentiation of granulated metrial gland cells, in situ, from lymphocyte-like cells. The differentiation from a lymphocyte precursor appears to follow a sequence in deciduomata formation similar to that previously described in pregnancy.

Animals

Uptake of marker proteins by glycoprotein-containing cells of the pregnant rat uterus and placenta.

A study was made of cells in the pregnant rat uterus and placenta known to contain glycoprotein inclusions to investigate their ability to endocytose marker proteins (fluorescein conjugated serum and horseradish peroxidase) injected into the maternal circulation. The visceral endoderm showed marked uptake of both proteins, though in later pregnancy this was restricted to the area of yolk sac adjacent to the chorio-allantoic placenta. The intracellular distribution of the endocytosed marker proteins resembled that of the glycoprotein inclusions. In the earlier stages some of the giant cell inclusions contained glycoprotein, some showed staining with the Dunn-Thompson technique for haemoglobin, and some showed peroxidase activity. There was endocytosis of both marker proteins by giant cells, and apparently this occurred independently of ingestion of red blood cells. Uptake by the giant cells persisted to a later stage in the area round the margin of the chorio-allantoic placenta than in the other giant cells. Endocytosis occurred in the labyrinthine trophoblast, and the glycoprotein inclusions found in this situation may represent material being transmitted or digested. The glycoprotein-containing granulated metrial gland cells showed no evidence of endocytotic activity, but there was uptake of both marker proteins by the associated stromal cells.

Animals

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

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.

Animals

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

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 mu in diameter, mono- or binucleate and the glycogen rich cytoplasm typically contains many large glycoprotein granules which may be up to 5 mu 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.

Animals

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Proliferation and differentiation of trophoblast in the establishment of the rat chorio-allantoic placenta.

The morphological differentiation of the elements contributing to the chorioallantoic placenta of the rat was studied between days 6 and 16 of gestation, and DNA synthetic activity was investigated in animals killed 1 hour after injection of tritiated thymidine. The chorionic lamina is formed from the epithelial cells lining the ectoplacental cavity, though it is possible that a contribution is made by ectoplacental cone cells at the mesometrial pole of the cavity. From day 8 onwards a high proportion of cells was labelled in both cone and lamina, with the labeliing index reaching a peak of about 80% at 10-11 days. There was no corresponding peak in mitotic activity, which remained at a high level in all regions up to day 12. The amount of thymidine incorporation, as judged by grain count per nucleus, varied. At 9 days a significantly higher grain count was obtained in a central core of cells in the ectoplacental cone, and at day 10 the grain density of labelling in the cone was very much heavier than in the lamina. This difference in labelling intensity was maintained until day 12, and there was no evidence of any influx of heavily labelled cells from the cone into the developing labyrinth.

Allantois

The fine structure of the rat metrial gland in relation to the origin of the granulated cells.

The fine structure of the metrial gland of the rat was examined at days 12, 14, 16, 18 and 20 of pregnancy and at all these stages two well differentiated cell types were seen. A relatively pale-staining cell which contained numerous dense granules was readily distinguished from a darkly stained fibroblast-like cell. Numerous cells could be identified, however, which did not fit either of these categories. On the basis of their morphological appearances these cells were placed in a series, with the members of the series showing a transition from a small cell with a high nuclear/cytoplasmic size ratio to a readily identifiable metrial gland cell containing a few typical granules. The small cell, apparently the precursor of the granulated metrial gland cell, was characterized by a markedly heterochromatic nucleus and few cytoplasmic organelles, although there were occasional mitochondria and Golgi bodies. On morphological grounds this cell appeared to be from the lymphocyte series and was probably a small lymphocyte.

Animals

Proliferative characteristics of epithelial cells in the pregnant rat uterus.

In the pregnant rat, killed at about mid gestation and 1 h after injection of tritiated thymidine, 40% of the cells in the epithelium lining the uterine lumen at the implantation site were labelled. Between implantation sites fewer than 20% of the surface epithelial cells were labelled. A series of rats was given tritiated thymidine on day 12 of pregnancy and killed at intervals in the next 30 h. A percentage labelled mitoses analysis of the epithelium between implantation sites (interconceptual) and within the implantation site (conceptual) showed that cells in either region spent 7 h in DNA synthesis and 1-5 h in the G2 + 1/2 MITOSIS PHASES. The epithelial cells in the conceptual region spent 1-5 h in the G1 + 1/2 MITOSIS PHASES WHEREAS CELLS IN INTERCONCEPTual regions spent at least 11-5 h in these phases. The average cycle times of cells in conceptual regions was 10 h: in interconceptual regions minimum cycle time was 20 h and the average appeared to be considerably longer. The grain count of the epithelial cells in the conceptual region was rapidly reduced during the 30 h after injection of tritiated thymidine suggesting successive rounds of cell division. In contrast the grain count distribution of cells in interconceptual regions changed only slowly during this time. The percentage of labelled epithelial cells was determined in the animals killed up to 30 h after injection of tritiated thymidine. In both conceptual and interconceptual regions these percentages increased initially as labelled cells produced labelled progeny. In the conceptual region the increase was not maintained after 7 h as cells initially in G1 divided to give unlabelled progeny. In the interconceptual region the increase in the percentage of labelled cells continued for 14 h; thereafter the percentage did not significantly alter. The interpretation of these results is discussed in relation to the differences in the kinetic characteristics of the epithelial cells in the two regions and in relation to the morphology of the epithelium lining the uterus during pregnancy.

Animals

The effects of late ovariectomy on the proliferation and differentiation of the uterus of the pregnant rat.

The effects of ovariectomy at day 12 of pregnancy on the proliferative activity of the uterine epithelium, metrial gland and uterine muscle in the rat were studied by autoradiography in animals killed one hour after a single injection of tritiated thymidine. The results were amplified by histological and histochemical studies. One day after operation epithelial proliferation was impaired in the ovarectomized animals. From 16 days onwards, however, proliferative activity was at a lower level in controls than in the ovariectomized animals. Three days after ovariectomy uptake of tritiated thymidine by metrial gland cells was less than in the controls. The metrial gland became smaller after ovariectomy and showed histological and histochemical changes from the normal pattern. The increased thickness of the uterine muscle in the ovariectomized animals from the 16 day stage onwards was not associated with any significant change in the labelling index of the muscle cells. The results are discussed in relation to the changes in endocrine environment which may occur after ovariectomy.

Animals

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.

Animals