Über Glykoproteideinschlüsse in den Trophoblastzellen der menschlichen Plazenta und die Frage ihres Zusammenhangs mit der Bildung von Gonadotropin

Summary In some of the basophilic trophoblastic cells of the basal plate and islands of the mature human placenta there are glycoprotein inclusions, many of which are within vacuoles. As established by the histochemical results these inclusions are composed of a neutral mucopolysaccharide and a basophilic protein with SH groups. They gradually increase in number from the 4th month of pregnancy and are not influenced by labor. From the first to the fourth month of gestation small vacuoles, initially infra-nuclear but later supranuclear, may be seen in the syncitial cells of the villi; these likewise contain glycoprotein granules. The same inclusions are to be found in some trophoblastic cells of the chorionic plate and of the chorion laeve, and in an occasional epithelial cell of the amnion.On the basis of the histochemical behavior of these inclusions, their similarity with the -granules of the hypophysis, and the obvious high activity of the trophoblastic cells which bear them, it is postulated that these inclusions contain gonadotropin. Their occurrence during pregnancy parallels the biochemical concentration and immunofluorescent histological demonstration of gonadotropin in the placenta. Thus, at the end of pregnancy the basal trophoblastic cells of the human placenta appear to form at least two different hormones: relaxin, probably represented morphologically by the acidophilic protein inclusions (described elsewhere), and gonadotropin most likely represented by the basophilic glycoprotein inclusions.     

Development of the discoidal hemochorial placenta in the black mastiff bat, Molossus ater: evidence for a role of maternal endothelial cells in the control of trophoblastic growth

In an effort to define further the factors that can influence trophoblastic growth, development of the discoidal chorioallantoic placenta was examined histologically in laboratory-bred black mastiff bats, Molossus ater. This placenta normally forms only at the cranial end of the right uterine horn. Its positioning was found to be related to the development of an unusual vascular tuft in the endometrium at this site following ovulation. When a decidual reaction occurred, the endothelial cells of the tuft vessels hypertrophied and their basal laminae became more prominent in sections stained for glycoproteins. Cytotrophoblast then proliferated preferentially around the tuft in the right horn and its vessels became surrounded by trophoblastic cuffs. A functional placenta was formed when trophoblastic tubules grew out from these cuffs, became interconnected, developed lumina, and began to carry maternal blood. Maternal endothelial cells generally persisted only in portions of the larger vascular tubules. These observations suggest that the endothelial cells of the tuft vessels may be secreting factors that influence early trophoblastic growth and are at least partially incorporated into the basal laminae of the endothelial cells. Evidence is reviewed which raises the possibility that endometrial endothelial cells might have a similar morphogenetic role in many other mammals with invasive trophoblast. Mitotic activity in the trophoblastic tubules of M. ater ceased when the tubules became patent and began to carry blood. Further growth was then accomplished by hypertrophy of the existing cytotrophoblast cells. Electron microscopic examination of near-term placentas confirmed that the interhemal barrier was hemomonochorial and lacked a continuous layer of syncytiotrophoblast.     

Observations on the fetus and placenta of a proboscis monkey (Nasalis Iarvatus)

The villous bidiscoid placenta in utero of a proboscis monkey with a fetus of 9.0 cm crown rump length was examined. The immature male fetus weighed 56 g and had a 5.4 cm head circumference. Of particular interest was the stomach, consisting of three parts, large fundus, a narrow tubular portion, and a small pyloric region. The fetal adrenal glands were remarkable in that they were larger than the kidneys. Thus, the adrenal/kidney weight ratio approximated 1.18. The marginally inserted umbilical cord measured 15 cm. Owing to the superficial implantation in the proboscis monkey, penetration of syncytiotrophoblast and a fibrin layer were not observed in the basal plate of the placenta; cytotrophoblastic cell columns attached to the basal plate, consisting of decidua basalis without invasion of trophoblastic giant cells. Intraarterial trophoblastic migration was found in the uterus.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Electron microscopic study of the chorioallantoic placenta of the rock hyrax (Heterohyrax brucei)

The interhaemal membrane consisted of only two cellular elements: a single layer of cellular trophoblast and the fetal capillary endothelium. The hyrax is therefore one of the few mammals known to possess the cellular haemomonochorial type of placenta. The trophospongium was also cellular while the basal trophoblastic cells were strongly phagocytic. The giant multinucleate cells at the feto-maternal junction were ultrastructurally different from the trophoblast cells and showed no signs of degeneration. Their appearance suggests that they are of maternal rather than fetal origin.     

Follicular placenta of the viviparous fish,Heterandria formosa: II. Ultrastructure and development of the follicular epithelium

Embryos of the viviparous poeciliid fish, Heterandria formosa, develop to term in the ovarian follicle where they undergo a 3,900% increase in embryonic dry weight. Maternal-embryonic nutrient transfer occurs across a follicular placenta that is formed by close apposition of the embryonic surface (i.e., the entire body surface during early gestation and the pericardial amnionserosa during mid-late gestation) to the follicular epithelium. To complement our recent study of the embryonic component of the follicular placenta, we now describe the development and fine structure of the maternal component of the follicular placenta. Transmission electron microscopy reveals that the ultrastructure of the egg envelope and the follicular epithelium that invests vitellogenic oocytes is typical of that described for teleosts. The egg envelope is a dense matrix, penetrated by microvilli of the oocyte. The follicular epithelium consists of a single layer of cuboidal cells that lack apical microvilli, basal surface specializations, and junctional complexes. Follicle cells investing the youngest embryonic stage examined (Tavolga's and Rugh's stage 5–7 for Xiphophorus maculatus) also lack apical microvilli and basal specializations, but possess junctional complexes. In contrast, follicle cells that invest embryos at stage 10 and later display ultrastructural features characteristic of transporting epithelial cells. Apical microvilli and surface invaginations are present. The basal surface is extensively folded. Apical and basal coated pits are present. The cytoplasm contains a rough endoplasmic reticulum, Golgi complexes, and dense staining vesicles that appear to be lysosomes. The presence of numerous apically located electron-lucent vesicles that appear to be derived from the apical surface further suggests that these follicle cells may absorb and process follicular fluid. The egg envelope, which remains intact throughout gestation and lacks perforations, becomes progressively thinner and less dense as gestation proceeds. We postulate that these ultrastructural features, which are not present in the follicles of the lecithotrophic poeciliid, Poecilia reticulata, are specializations for maternal-embryonic nutrient transfer and that the egg envelope, follicular epithelium, and underlying capillary network form the maternal component of the follicular placenta. © 1994 Wiley-Liss, Inc.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

B-cell lymphoma 6 promotes proliferation and survival of trophoblastic cells

Preeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity and its pathogenesis is not fully understood. B-cell lymphoma 6 (BCL6), a key regulator of B-lymphocyte development, is altered in preeclamptic placentas. We show here that BCL6 is present in all 3 studied trophoblast cell lines and it is predominantly expressed in trophoblastic HTR-8/SVneo cells derived from a 1^st trimester placenta, suggestive of its involvement in trophoblast expansion in the early stage of placental development. BCL6 is strongly stabilized upon stress stimulation. Inhibition of BCL6, by administrating either small interfering RNA or a specific small molecule inhibitor 79–6, reduces proliferation and induces apoptosis in trophoblastic cells. Intriguingly, depletion of BCL6 in HTR-8/SVneo cells results in a mitotic arrest associated with mitotic defects in centrosome integrity, indicative of its involvement in mitotic progression. Thus, like in haematopoietic cells and breast cancer cells, BCL6 promotes proliferation and facilitates survival of trophoblasts under stress situation. Further studies are required to decipher its molecular roles in differentiation, migration and the fusion process of trophoblasts. Whether increased BCL6 observed in preeclamptic placentas is one of the causes or the consequences of preeclampsia warrants further investigations in vivo and in vitro.     

alpha-, beta-, gamma-catenin, and p120(CTN) expression during the terminal differentiation and fusion of human mononucleate cytotrophoblasts in vitro and in vivo

The cadherins play key roles in the formation and organization of the mammalian placenta by mediating cellular interactions and the terminal differentiation of trophoblastic cells. Although cadherin function is regulated by the cytoplasmic proteins, known as the catenins, the identity and expression pattern(s) of the catenins present in the trophoblastic cells of the human placenta have not been characterized. In these studies, we have determined that alpha-, beta-, gamma-catenin, and p120(ctn) expression levels are high in villous cytotrophoblasts isolated from the human term placenta but decline as these cells undergo aggregation and fusion to form syncytium with time in culture. In contrast, the expression levels of these four catenin subtypes remained constant in non-fusing JEG-3 choriocarcinoma cells at all of the time points examined in these studies. alpha-, beta-, gamma-catenin, and p120(ctn) expression was further immunolocalized to the mononucleate cells present in these two trophoblastic cell cultures. Similarly, intense immunostaining for all four catenins was detected in the mononucleate villous cytotrophoblasts of the human first trimester placenta. Collectively, these observations demonstrate that the expression levels of alpha-, beta-, gamma-catenin, and p120(ctn) are tightly regulated during the formation of multinucleated syncytium in vitro and in vivo.     

Early stages in development of the Escherichia coli cell-division site

Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ^- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ^- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.     

Indoleamine 2,3-dioxygenase is regulated by IFN-gamma in the mouse placenta during Listeria monocytogenes infection

The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is expressed in macrophages that have been differentiated in the presence of CSF-1 and is important in the containment of intracellular pathogens. IDO also appears to play a role in suppression of T cell responses in a variety of contexts. In the placenta, its enzymatic activity is believed to establish a chemical barrier that protects the fetal allograft from T cell-mediated immune aggression. We have studied the regulation of IDO in the utero-placental unit of mice following infection with the Gram-positive, intracellular bacterium Listeria monocytogenes that has a predilection for replication in the decidua basalis. IDO mRNA and protein expression is enhanced in the utero-placental unit following infection with L. monocytogenes. However, in contrast to the human where IDO is expressed by the CSF-1R-positive syncytial trophoblast, IDO is not expressed in murine trophoblastic tissue but instead is found in stromal cells of the decidua basalis and metrial gland and following infection, in endothelial cells. Using mice carrying null mutations in cytokine/growth factor genes, we explored the regulation of IDO in the placenta. Consistent with the absence of CSF-1R expression in the IDO-expressing cells of mice, neither the basal levels of IDO nor its induction following infection is affected by the absence of CSF-1. However, although the basal level of IDO is normal, the enhanced expression during Listeriosis is completely abrogated in the absence of IFN-gamma, a cytokine required for the resolution of this infection. These data suggest that IDO plays a role in resolving bacterial infection in the placenta while at the same time maintaining a barrier to T cells whose presence might result in fetal rejection.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.     

Glycogen-containing cells in the maternal and embryonic portions of the placenta in the rat and the common vole 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.     

Analysis of ultrashort feedback regulation in human placenta: synthesis and secretion of GnRH by human trophoblastic cells.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) presumably controls placental growth and functions by autocrine/paracrine mechanisms, and is therefore an important part of the neuroendocrine network in human placenta. AIM: Our earlier work had indicated that GnRH was expressed in human placenta; in extension to these findings, we wanted to analyse synthesis and release of GnRH by trophoblastic cells. GnRH-associated peptide, co-linearly synthesised with GnRH, was used as indicator of actual peptide synthesis. METHOD: First, we immunised rabbits with lipopeptides containing partial sequences of GnRH-associated peptide (GAP) and developed antibodies for immunohistochemical staining. Second, we set up a competitive enzyme immunoassay to measure GnRH: Non-biotinylated GnRH, GnRH analogues or trophoblastic cell culture supernatants were used to inhibit binding of biotinylated des-pGlu1-GnRH to a monoclonal anti-GnRH antibody. RESULTS: a) Placental sections stained positive for GAP in the layers of trophoblastic cells. b) GnRH could be detect by a competitive EIA in supernatants of placental cultures in concentrations between 200 and 5 nM. CONCLUSIONS: GnRH is synthesised and released by trophoblastic cells.     

A Proteomic Analysis of Placental Trophoblastic Cells in Preeclampsia–Eclampsia

To explore the proteomic changes of placental trophoblastic cells in preeclampsia–eclampsia (PE), placental trophoblastic cells from normally pregnant women and women with hypertension during gestational period were prepared by laser capture microdissection (LCM), and proteins isolated from these cells were subjected to labeling and proteolysis with isotope-coded affinity tag reagent. A qualitative and quantitative analysis of the proteome expression of placental trophoblastic cells was made using two-dimensional liquid chromatography tandem mass spectrometry (2D LC–MS/MS). A total of 831 proteins in placental trophoblastic cells were identified by combined use of LCM technique and 2D LC–MS/MS. The result was superior to that of conventional two-dimensional electrophoresis method. There were marked differences in 169 proteins of placental trophoblastic cells between normally pregnant women and women with PE. Of 70 (41.4 %) proteins with more than twofold differences, 31 proteins were down-regulated, and 39 were up-regulated in placental trophoblastic cells of the woman with PE. Laminin expression in placenta trophoblastic cells of women with PE was significantly down-regulated as confirmed by Western blot analysis. These findings provide insights into the proteomic changes in placental trophoblastic cells in response to PE and may identify novel protein targets associated with the pathogenesis of PE.     

Increased CD11b+ Gr‐1+ cell population in the placenta after infection with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.     

Immunoelectron microscopy localization of immunoglobulin G in human placenta

Immunoelectron microscopy of IgG molecules in human mature placenta has shown that IgG bound to microvillar surfaces and the inner wall of endocytotic vesicles of syncytiotrophoblasts. The endocytotic vesicles, containing both bound and unbound IgG molecules, tended to fuse with each other or with other cellular organelles, particularly with lysosomes. The phagolysosomes were more abundant in the basal regions of the cells. Apparently some IgG molecules were not digested by lysosomal enzymes. Vesicles with residual IgG were found to fuse with the basal and basolateral cell membrane and to discharge their contents into the extracellular space by exocytosis. It is suggested that IgG molecules were transported through the trophoblastic basement membrane and the interstitial space by diffusion to the endothelial basement membrane. The IgG molecules then migrated into the fetal vascular lumen via endothelial gaps and interendothelial spaces.     

Localization of hepatocyte growth factor activator inhibitor type 1 in Langhans' cells of human placenta

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-I in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-I in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.