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.

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.

Migratory trophoblast cells express a newly identified member of the prolactin gene family.

Rodents possess an expanded prolactin (PRL) family of genes. These genes encode for a family of structurally related hormones/cytokines that are expressed most prominently in the anterior pituitary, uterus and placenta. In this study, we have identified a new member of the rat PRL family through a search of the National Center for Biotechnology Information expressed sequence database. The cDNA was sequenced and its corresponding mRNA characterized. On the basis of existing nomenclature, the rat cDNA was termed PRL-like protein-N (PLP-N). PLP-N has structural features indicative of its inclusion in the PRL family and is most closely related to PRL-like protein-F (PLP-F) and proliferin related protein (PLF-RP). A survey of PLP-N mRNA expression by Northern analysis indicated that PLP-N showed extensive expression in the metrial gland and minimal expression in the chorioallantoic placenta or other tIssues. Expression of PLP-N mRNA was restricted to migratory trophoblast cells. Junctional zone trophoblast cells isolated from day 13 of gestation placenta differentiated in vitro and exhibited a capacity for PLP-N expression. In summary, we have discovered a new member of the PRL family that is prominently expressed in migratory trophoblast cells residing in the metrial gland.

Placentation in the alpaca Lama pacos.

Reproduction in South American camelids is poorly studied. To extend our knowledge of the development and cellular physiology of the placenta in the alpaca Lama pacos, we have examined specimens from day 150 of pregnancy to term. Morphological investigations using light, transmission and scanning electron microscopy, the histochemical localization of iron, alkaline and acid phosphatase activity, and the immunodetection of placental lactogen hormone were performed. Throughout pregnancy there was a progressive increase in the depths of folds on the uterine mucosa surface together with a thickening of the endometrium. Glandular cells exhibited PAS and acid phosphatase (AcP) positive secretion granules. In the chorion, giant trophoblast polyploid cells gradually became more numerous and larger. Non-giant cells exhibited positive granules for PAS, alkaline phosphatase (AkP) reaction and immunostaining for bovine placental lactogen hormone (PLH). SDS -PAGE electrophoresis and Western blotting procedures also confirmed the presence of a bovine PLH-like glycoprotein in the fetal alpaca placenta. Over the glandular openings, the chorion formed typical areolae, where the trophoblast exhibited AcP and PAS positive reactions. At these sites, the fetal endothelial cells contained iron-storage granules in their cytoplasm. The trophoblast-epithelial interface exhibited a complex microvillous interdigitation, in which an AkP reaction was very prominent. The chorionic capillaries progressively indented adjacent trophoblast cells. These data suggest that although the epitheliochorial alpaca placenta is diffuse, various trophoblast cell types and specialized areas of the maternofetal interface give the placenta micro-regional functions where histiotrophic nutrition, hormone production and molecular exchange are prevalent.

Expression of vascular endothelial growth factor by granulated metrial gland cells in pregnant murine uteri.

Granulated metrial gland (GMG) cells are a characteristic uterine component belonging to a natural killer cell lineage. This study is aimed at revealing their kinetic and spatial relationship with vascular growth during pregnancy and the expression of vascular endothelial growth factor (VEGF). GMG cells and blood vessels were identified by periodic-acid-Schiff-reagent (PAS)-stained granules and positive staining for factor-VIII-related antigen, respectively. GMG cells were widely distributed in the decidua and metrial gland and showed a numerical increase with a peak at day 13 in parallel with the increase of vascular density. Preceding the maximal vascular development at day 13, microvessels with a narrow lumen representative of neovascularization prevailed at days 7-9, and the VEGF content in the decidua/metrial gland was significantly elevated at days 7-13 concurrently with mRNA expression. By immunolight microscopy combined with PAS staining, GMG cells with PAS-stained granules were positive for VEGF. Immunoelectron microscopy demonstrated that immunoreactions were diffuse in the cytoplasm but not localized in the granules. In contrast, fibroblast-like stromal cells were negative. These data indicate that GMG cells express VEGF and may play inducing roles in uterine neovascularization during pregnancy.

Characterization of the cells that migrate from metrial glands of the pregnant mouse uterus during explant culture.

Granulated metrial gland (GMG) cells are estrogen-receptor and Interleukin 2 (IL-2) receptor positive lymphocytes of the Natural Killer cell lineage found in the murine uterus during pregnancy. Functional studies of these cells, which are now more frequently called uterine NK (uNK) cells, have been limited due to technical difficulties. The cells are difficult to isolate and their proliferation and differentiation have not been achieved in culture. In 1988, Mukhtar and Stewart (Cell Tiss. Res., 253, 413-417) reported a method for explant culture of metrial glands isolated from pregnant rodents that yielded an almost pure population of uNK cells. This major technical advance has supported most of the subsequent functional and molecular studies of rodent uNK cells. However, the quality of the cells isolated by the explant culture procedure has not been established. A cytochemical approach was used to identify and quantify the cells migrating from metrial glands. At midpregnancy, almost all (> 90%) migrating nucleated cells were NK cells. Earlier in gestation, a significant proportion (25%) of cells having lymphoid morphology could not be assigned to the lineage. The viability of cells migrating from explants was assessed by DNA isolation and electrophoresis on days 6-16 of gestation. At all times evidence for apoptosis was found, even after culture intervals as brief as 4 h. Parallel analyses of histological sections of the metrial gland, using terminal deoxytransferase labelling to detect nuclear fragmentation, did not support significant levels of uNK cell death in situ prior to day 12 of gestation. Supplementation of the explant culture medium with estrogen, IL-2, various extracellular matrices, decidual cells or combinations of these did not lead to in vitro proliferation of uNK cells and usually did not extend the short term viability of these cells in serum supplemented or serum free media. Thus, the optimal culture conditions for uNK cells remain undefined.

The role of perforin-expression by granular metrial gland cells in pregnancy.

The pregnant uterus of humans and rodents contains a population of granulated lymphoid cells, which, in the mouse, are called granular metrial gland (GMG) cells and have been described to express high levels of perforin. Since there is evidence for cytolytic activity of these cells and since perforin is a crucial effector molecule for the lytic action of cytotoxic T cells and natural killer cells, we evaluated the function of perforin in the pregnant uterus by using perforin-deficient mice. Perforin-deficient female mice were found to reproduce as efficiently as normal control females when bred either with syngeneic or allogeneic males. However, perforin-deficient mice differed from normal mice in that the frequency of GMG cells was significantly higher within maternal blood spaces and within several compartments of the feto-maternal interface. Proliferating GMG cells, identified by [3H] thymidine incorporation, were observed during more advanced stages of pregnancy when compared to normal controls. In contrast to normal mice, perforin-deficient mice did not display GMG cells attached to degenerating trophoblasts; instead perforin-deficient GMG cells were often observed in association with small maternal lymphocytes. In addition, the lack of transmission of lymphocytic choriomeningitis virus from infected pregnant perforin-deficient mice to the fetuses argued against a role of perforin expression by GMG cells in prevention of virus transmission from the mother to the fetus. Our data indicate that functional perforin is not necessary for successful pregnancies. The morphological changes in the pregnant uterus of perforin-deficient mice might, however, point to a certain, as-yet undefined function of perforin in the uterus of pregnant normal mice, which is functionally compensated in perforin-deficient mice.

Preliminary characterization of lymphoid hybridoma cell lines derived from the pregnant mouse uterus.

Seven independent cell lines were derived from the fusion of migratory cells recovered from explant cultures of metrial glands to SP 2/0, a non-Ig secreting B cell myeloma. The migrating cells came from a pool of metrial glands from day 6-8 pregnant random bred CD1 mice and were assumed to be cells early in the differentiation pathway to granulated metrial gland (GMG) cells. The fused cells were cloned twice at the limiting dilution. Hybridization was confirmed by quantitation of cellular DNA using propidium iodide staining and by karyotyping. Electron microscopy revealed that each of the hybrid cell lines was composed of cells which were lymphoid in appearance, but lacked the granules found in mature GMG cells. The surface phenotype of all lines is CD45+, LGL-1-, asialo GM-1-, IgG-, IgM-, CD3- and CD25- (p55 of IL-2 receptor). Although the hybridomas lack those phenotypic markers which were used to show that GMG cells are related to the natural killer (NK) cell lineage (ie LGL-1, asialo GM-1), they do express the pan-leukocyte marker CD45 as well as the lytic protein, perforin, at levels intermediate to those of SP 2/0 cells and GMG cells. In addition, the hybridomas were observed to preferentially bind the NK target cell YAC and to be capable of lytic activity at temperatures below 30 degrees C. Because these hybridomas may represent fusion to an early progenitor cell of the NK/GMG cell lineage, their continued characterization is of merit.

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.

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.

The distribution of perforin in normal tissues.

We describe the production of monoclonal antibodies to murine and human forms of the lymphocyte pore-forming protein (perforin, PFP, or cytolysin), a major granule-localized cytolytic mediator of CTL and NK cells. Antibodies were raised against both murine perforin purified from a CTL line, and human perforin expressed in bacteria as a fusion protein with the Escherichia coli TrpE protein. Antibodies raised against either immunogen inhibited the hemolytic activity of murine perforin, and thus may enable us to identify the pore-forming or self-associative domain of perforin. One mAb, MP1, was used to study the distribution of perforin in murine tissues under physiological conditions. We found that perforin was expressed in the granular metrial gland (GMG) cells of the pregnant murine uterus, but not in other tissues examined. These results further support the view that perforin is induced only in activated cytolytic lymphocytes, and raise the question whether perforin-containing GMG cells represent an effector of a maternal immune response to the fetus.

Secretory function of cells in the rat metrial gland.

The immunochemical determination of antigens, products of the secretory activity of metrial gland cells, in the serum of pregnant rats is described. In order to prepare a specific immune antiserum, rabbits were immunised with homogenates of the rat metrial gland removed at day 15 of pseudopregnancy. The resultant immune antisera were adsorbed by glutaraldehyde-polymerised sera from nonpregnant rats until the Ouchterlony test ceased to give a positive reaction with sera of nonpregnant rats. These adsorbed specific sera continued to react with sera of pregnant and pseudopregnant animals, indicating that the latter both contain one or more antigens identical to those produced by the cells of the metrial gland, and against which the specific antisera had been raised. When 10% and 15% fresh serum from nonpregnant females was added to molten agar or was initially dropped into wells in the agar, the positive reaction between the specific antisera and the sera of pregnant and pseudopregnant rats was not eliminated. An antigen identical to the serum antigen was also found in amniotic fluid, in sera, and in extracts of metrial glands from pregnant and pseudopregnant laboratory animals. The data obtained indicate that cells in the metrial gland have a secretory function. It is proposed that the serum antigen of pregnant and pseudopregnant rats is secreted by the granular cells in the metrial gland.

[Glycogen-containing cells in the maternal and embryonic portions of the placenta in the rat and the common vole Microtus subarvalis]. / Glikogensoderzhashchie kletki v materinskoi i zarodysevoi chasti platsenty krysy i seroi polevki Microtus subarvalis.

Differentiation sequences and further transfiguration of glycogen-rich cells during placenta development were investigated for the rat and field vole Microtus subarvalis (11-20 day gestation). The presence of glycogen is a characteristic feature of decidual cells located in the region of lateral sinusoids, as well as of metrial gland cells, secondary giant trophoblast cells and trophoblast cells in the connective zone of placenta. Glycogen-containing metrial gland cells and trophoblast cells of connective zone of placenta are found to underlie the layer of tertiary giant trophoblast cells that cover the wall of the central arteria. Thus, both maternal and embryo-derived glycogen-containing cells always accompany the tertiary giant trophoblast cells that penetrate deeply into the maternal part of placenta but do not contain glycogen. In the field vole placenta the cells of peripheral trophoblast subpopulation of the connective zone of placenta attaching to the decidua basalis are stained by PAS-reaction more intensely than deeply situated ones. These data, as well as other phenomena revealed here, show that maternal and trophoblastic cells attaching to each other in placenta contain, as a rule glycogen. Glycogen cells in rat placenta and trophoblast cells of peripheral subpopulation of connective zone of placenta are similar in many respects. In this connection, a possible protective role of glycogen-containing cells, that probably favour the co-existence of maternal and embryo-derived cells in placenta, is discussed.

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.

Detection and immunohistochemical localization of alpha 1-pregnancy-associated protein (alpha 1-PAP) in rats.

A protein exhibiting immunochemical cross-reactivity with the murine alpha 1-pregnancy-associated protein (alpha 1-PAP) has been detected in the sera of female rats. The protein has an alpha 1-electrophoretic mobility, an estimated molecular weight of 150,000 and is readily detectable in the sera of nonpregnant female rats of each strain examined. During pregnancy, the serum concentration increases up to five-fold, reaching maximal levels at the same gestational stage as alpha 1-PAP in the mouse. Immunohistochemical studies revealed a staining pattern for the rat protein similar to that seen for alpha 1-PAP in the mouse, with positive cells being observed in lumbar lymph nodes, the lamina propria of gut mucosa, Peyer's patches, intracellularly in some hepatocytes and, during pregnancy, also in the placenta and metrial gland. On the basis of these immunochemical, physicochemical and immunohistochemical findings, it is proposed that the protein detected in rat sera represents the analogue of the murine alpha 1-PAP, and that the rat strains examined more closely resemble high endogenous alpha 1-PAP-producer mouse strains. It is proposed that alpha 1-PAP, and a number of other rodent pregnancy proteins recently described, may be used in functional studies of human alpha 2-PAG.

Effects of exogenous progesterone following ovariectomy on the metrial glands of pregnant mice.

The numbers of granulated metrial gland cells, the percentage of them incorporating tritiated thymidine and the numbers of granulated metrial gland cell precursors in a defined area of the metrial gland, and the cross sectional area of the metrial gland, have been determined in mice ovariectomised on Day 8 of pregnancy. The effects, on these parameters and on fetal survival, of progesterone treatment, given at two levels (0.5 mg or 1 mg twice daily) and started immediately after ovariectomy or after 6, 10 or 24 hours delay, have been studied. Granulated metrial gland cell number, DNA synthetic activity and metrial gland size decreased rapidly after ovariectomy but there was a significant increase in the number of granulated metrial gland cell precursors in the first 24 hours. Progesterone treatment, at the higher dose level started immediately after ovariectomy, resulted in significantly more granulated metrial gland cells in the area analysed two days after ovariectomy although a normal proportion of them were in DNA synthesis. Immediate progesterone treatment, at the higher dose level, was able to prolong the DNA synthetic activity of granulated metrial gland cells until Day 19 and this probably accounted for the significantly greater numbers of granulated metrial gland cells in these mice than in control mice, at this time. Delaying progesterone treatment at the higher level by 24 hours significantly reduced the size of the metrial glands at Day 10 but there was no effect on the cellular composition of the area analysed. Delaying the lower dose of progesterone treatment by 24 hours also resulted in small metrial glands and was associated with significantly fewer granulated metrial gland cells in the area analysed but significantly more of them were synthesising DNA. The observations are discussed and an attempt is made to relate the effects on individual fetal and placental survival to the effects on the corresponding metrial glands.

Estrogen and progesterone receptors in the endometrium, myometrium, and metrial gland of the rat during the decidualization process.

Uterine cytosolic and nuclear receptors for estradiol (E2) and progesterone (P), were simultaneously determined in pseudopregnant rats subjected to a traumatic decidualizing stimulus (knife-scratch). The entire decidualizing endometrium, myometrium, and metrial gland were separated, homogenized, and studied on alternate days beginning with day 1 post trauma (pt) and extending to day 13 pt when even the metrial gland is regressing. The rise and fall in E2 and P receptors during the earlier stages of deciduoma differentiation confirmed the work of others. Splitting of the deciduoma on day 5 pt into antimesometrial and mesometrial segments showed a higher concentration of cytosolic P receptor in the well differentiated antimesometrial segment, whereas there was a higher concentration of E2 cytosolic receptor in the still differentiating mesometrial segment. The differences, measured as femtomoles per microgram DNA or per mg protein were statistically significant. The receptor changes in the myometrium were not as pronounced as in the endometrium especially with regard to the low myometrial levels of nuclear E2 and P. The metrial gland which comes into prominence while the deciduoma is regressing, showed sustained high concentrations of cytosolic P receptor. It is suggested that although initiated by the same decidualizing stimulus, the hormonal and receptor interplay in the metrial gland may be somewhat different from that of the deciduoma. The metrial gland merits further study because of an implied immunological role.

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.