Granzymes D, E, F, and G are regulated through pregnancy and by IL-2 and IL-15 in granulated metrial gland cells.

Granulated metrial gland (GMG) cells are NK cells that proliferate and differentiate within the murine uterus during pregnancy. They have been predicted to play important roles in nurturing the embryo, normal placentation, and uterine tissue remodeling. GMG cell differentiation is manifested by the accumulation of the cytolytic mediators, perforin, granzyme A, and granzyme B, within cytoplasmic granules. The signaling mechanisms required for GMG cell differentiation are largely unknown, although recent in vitro assays have implicated IL-15 in these events. In this report, we demonstrate that granzymes D, E, F, and G (granzymes D-G) are also expressed in GMG cells but at a later stage in pregnancy when compared with granzyme A expression. Whereas granzyme A is expressed in early to mid-gestation, the expression of granzymes D-G peak in mid- to late gestation. In addition, we show that the expression patterns of IL-2Rbeta and the IL-2Rgamma mRNAs overlap with that of granzyme D-G mRNAs in the pregnant uterus. Finally, we demonstrate that granzymes D-G are up-regulated by IL-2 and IL-15 in primary cultures containing GMG cells. Taken together, these results suggest that IL-2 and/or IL-15 may regulate GMG cell differentiation in vivo, and that granzymes D-G may have different functions than granzyme A during pregnancy.

Granulated metrial gland cells contain nitric oxide synthases during pregnancy in the rat.

Nitric oxide synthase (NOS) enzyme activity is present in rat decidua and metrial gland, and the activity decreases near the end of pregnancy. In this study, inducible and endothelial NOS isoforms were immunolocalized to rat granulated metrial gland (GMG) cells using anti-NOS antibodies proven to be isoform specific. These NOS-positive GMG cells are of the natural killer cell lineage as they stained positively for NKR-P1 cell surface receptor, and for perforin. The number of NOS-positive GMG cells corresponded with the level of decidual and metrial gland NOS enzyme activity. NOS activity declined when GMG cells containing NOS decreased in number. Uterine arteriolar vascular smooth muscle also stained positively for inducible NOS and the staining did not change with advancing gestation. Only a minority of uterine myocytes stained positively for inducible NOS and these were subjacent to the placental attachment site. Neuronal NOS immunostaining was not present in the decidua and the metrial gland.

Enzyme histochemistry of the regressing rat decidua and metrial gland.

In order to understand more about participation of the basal placental zones in processes of regression and degradation as well as separation on the cellular level, the cell metabolism of the rat decidua and metrial gland was investigated enzyme histochemically in cryosections for activities of oxyradical-forming enzymes and hydrolyzing enzymes. Additionally, plastic sections were studied to facilitate the recognition of cell types. Decidual stromal cells and fibroblasts formed the vast majority amongst many cell types in the decidua and metrial gland. High activities of enzymes involved in purine degradation and oxyradical generation were demonstrated in decidual stromal cells and fibroblasts. Microsomal alanyl aminopeptidase and various acid hydrolases were shown to be extremely active in decidual stromal cells. The abundance of these enzyme activities in the decidua and metrial gland in contrast to other placental areas suggests, that these enzymes may have specialized functions in connection with regression and degradation processes finally contributing to placental separation.

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.

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.

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.

Histochemical study of metrial gland cells through the last week of pregnancy in the albino rat.

The metrial gland cells of the rat were studied from day 16 to 21 of pregnancy through enzyme histochemistry. The following enzyme activities were tested: 2 carboxylic esterases, 4 phosphatases, 1 aminopeptidase and 10 dehydrogenases including 4 which are involved in steroid metabolism. Although acid phosphatase activity was strong in mesenchymal cells, it remained undetected in large, sometimes binucleate cells lining central and subplacental vessels of the mesometrial triangle. 5-Bromo-indoxyl acetate esterase was likewise absent from the parietal vascular cells of the central artery. The study of 6 enzyme activities, i.e., GPDH, 3-OU-BDH, G6PDH, 3-B-HSDH, primary and secondary alcohol dehydrogenases, allowed to conclude that metrial gland cells are actively involved in steroidogenesis and lipid catabolism. Maternal origin of this gland can be sustained with reference to the pattern of acid phosphatase, indoxyl-esterase and some oxidoreductases II activities distribution which appears to be different from what is found in the trophospongium area.