An immunogold, cryoultrastructural study of sites of synthesis and storage of chorionic gonadotropin and placental lactogen in human syncytiotrophoblast.

The sites of intracellular synthesis and storage of human placental lactogen (hPL) and human chorionic gonadotropin (hCG) are controversial. We have used one of the most sensitive methods, cryoultramicrotomy and immunogold labelling, to localise these hormones at the electron-microscopic level. In both 12-week and term placentas hCG and hPL are present throughout the rough endoplasmic reticulum cisternae, in the Golgi bodies, and in the infrequent small dense granules of the syncytiotrophoblast. Previous assays have shown that hCG is at a higher concentration in early pregnancy and hPL peaks in late pregnancy, and our results corroborate these findings. No significant localisation of either hormone was seen in the cytotrophoblast or villous stroma. The results suggest that both hCG and hPL are synthesised and packaged by the classical secretory pathway, although the level of hormone stored in granules at any one time is small.

500-Kilodalton calcium sensor regulating cytoplasmic Ca2+ in cytotrophoblast cells of human placenta.

Two monoclonal IgG antibodies E11 and G11, which react with parathyroid and kidney tubule cells, are in the present communication demonstrated to immunostain the surface of cytotrophoblast cells in human placenta. The G11 but not the E11 antibody has earlier been found to interfere with the sensing and gating of extracellular calcium in parathyroid cells. Microfluorometric measurement of the cytoplasmic calcium (Ca2+i) concentration was performed on suspended placental cells loaded with fura-2. The E11-positive placental cells displayed biphasic and parathyroid-like increases in Ca2+i in response to extracellular Ca2+. This increase was blocked by the G11 antibody and absent in the E11-negative placental cells. A sandwich enzyme-linked immunosorbent assay was constructed in which the G11 and E11 antibodies were shown to react with the same molecule. This calcium sensor was isolated and found to consist of a single, glycosylated polypeptide of approximately 500 kDa.

Intermediate trophoblast: further immunocytochemical characterization.

Intermediate trophoblast has been recently described as a distinctive subpopulation of trophoblast. Previous immunocytochemical studies demonstrated that these cells react with antibodies against human placental lactogen (hPL) but fail to react with antibodies against beta-human chorionic gonadotropin (beta-hCG). To further characterize the immunocytochemical features of intermediate trophoblast and to possibly identify a more sensitive marker, we studied 88 placentas and implantation sites ranging from 4 to 40 wk gestation with a panel of antibodies that included keratin, epithelial membrane antigen (EMA), placental alkaline phosphatase (PLAP), alpha-hCG, and prolactin. Keratin was found in all intermediate trophoblast cells throughout gestation, and EMA was present in intermediate trophoblast in the second and third trimesters but was less consistently expressed than keratin. Comparison with the distribution of hPL revealed that keratin and EMA were present in intermediate trophoblast cells that were hPL-positive as well as many that were hPL-negative. alpha-hCG showed reactivity in intermediate trophoblast in the first and second trimester. PLAP and prolactin showed little or no reactivity in intermediate trophoblast. Decidual cells, which may be difficult to distinguish from intermediate trophoblast at the implantation site, failed to react with any of the antibodies tested. Cytotrophoblast and syncytiotrophoblast were positive for keratin throughout gestation, but EMA was negative in cytotrophoblast and inconsistently expressed in syncytiotrophoblast. Thus, antibodies against keratin and EMA are more sensitive than those directed against hPL in identifying intermediate trophoblast and are therefore useful in distinguishing intermediate trophoblast from decidua.

Reliability of prenatal diagnosis of genetic diseases by analysis of amplified trophoblast DNA.

Dot blot analysis on enzymatically amplified trophoblast DNA with allele specific oligonucleotide probes is currently used for the prenatal diagnosis of single gene disorders characterised at the molecular level, such as the beta thalassaemias, phenylketonuria, sickle cell anaemia, and alpha 1-anti-trypsin deficiency. A potential problem with the use of this procedure is the co-amplification of maternal sequences, which may obscure the diagnosis in the fetus. To address this question, we carried out prenatal diagnosis of beta thalassaemia in 300 couples at risk by dot blot analysis on enzymatically amplified DNA with 32P or horseradish peroxidase labelled allele specific oligonucleotide probes. We verified the diagnosis obtained by this procedure with oligonucleotide hybridisation on electrophoretically separated non-amplified trophoblast DNA fragments. We detected no co-amplified maternal sequences, even with a faint signal, in the dot blot of trophoblast DNA from those fetuses diagnosed as normal or homozygotes, nor in those diagnosed as heterozygotes, who were born to parents carrying different mutations and had inherited the paternal mutation. These results indicate that, when careful dissection of trophoblast tissue from maternal decidua is carried out, amplification of chorionic villi DNA is not associated with amplification of maternal DNA sequences. We may thus conclude that dot blot analysis of trophoblast DNA is a very reliable procedure for prenatal diagnosis.

HIV-1 in trophoblastic and villous Hofbauer cells, and haematological precursors in eight-week fetuses.

AIDS in children is usually caused by vertical transmission of human immunodeficiency virus type 1 (HIV-1). Aborted eight-week fetal and placental tissue from HIV-1 positive and negative (enzyme-linked immunosorbent assay and Western blot) women was analysed by immunocytochemistry and in-situ hybridisation. Maternal decidual leucocytes, villous trophoblastic derivatives, villous mesenchymal cells, and embryonic blood cell precursors in tissues from seropositive patients all stained for HIV-1 antigen and hybridised for nucleic acids. These observations suggest that a cytological pathway for vertical transmission of HIV-1 is established by eight weeks gestational age.

Human relaxin in the amnion, chorion, decidua parietalis, basal plate, and placental trophoblast by immunocytochemistry and northern analysis.

Immunocytochemistry and Northern analysis were used to show that relaxin is a product of intrauterine tissues of pregnancy. In addition, tissues from a patient without ovaries had similar results on both immunocytochemistry and Northern analysis as tissues from intact patients. The parietal decidua was clearly the major source of relaxin within the uterus and the relaxin mRNA (1.2 kilobases) from this tissue was detected with a 48-mer oligonucleotide probe designed to hybridize with both H1 and H2 relaxin gene transcripts. The mRNA isolated from the placental trophoblast was slightly smaller (1.1 kilobases), and the placental basal plate which has both maternal and fetal cells contained relaxin mRNAs of both sizes. Two monoclonal antibodies (Mabs) raised to synthetic human relaxin (H2) gave different patterns of localization in the fetal membranes, decidua and placenta. One Mab (RLX8) stained the chorionic cytotrophoblast in the fetal membranes and all of the cells in the placental basal plate. The other Mab (RLX6) stained the chorionic cytotrophoblast in some instances and selectively stained the decidua-like cells of the placental syncytiotrophoblast, whereas Mab RLX8 failed to detect this relaxin. Tissues obtained after spontaneous labor and delivery contained significantly less relaxin mRNA than tissues obtained at elective cesarean section without labor, but their hormone contents, as judged by immunocytochemistry, were not different. We conclude that the relaxin gene (H2) is expressed in intrauterine tissues, but that expression and hormone synthesis are not ubiquitous. Whether the relaxin gene H1 is expressed has not been determined.

Immunohistochemical localization of chorionic gonadotrophin on baboon placenta, dispersed trophoblast cells and those derived from blastocysts grown in vitro.

An immunohistochemical technique using a high specificity antiserum against baboon CG was used to demonstrate the presence of a CG-like material on: (1) fixed baboon placental sections collected between 31 and 39 days of gestation, (2) trophoblast monolayers derived from hatched embryos grown in vitro for 15 days and (3) trophoblast cells derived from cells dispersed from placentae collected between Days 31 and 39 of pregnancy. A specific radioimmunoassay was used to detect concentrations of baboon CG in daily spent medium. Immunohistochemical studies showed that material cross-reacting with CG was present on all the three sources of trophoblast. The embryos secreted CG from attachment onwards and immunoactive CG was measurable in daily spent medium collected from placenta-derived trophoblast cultures. It is concluded that baboon CG is localized in the syncytiotrophoblast of fixed placental sections and cellular trophoblast derived from cultured embryos and placental cells.

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst.

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.

Evaluation of tumor and trophoblastic marker concentration in breast cyst fluid.

In our study we have examined 314 samples of cyst fluid taken from women suffering from fibrocystic breast disease (gross cystic disease). We have subdivided the cyst fluid with respect to epithelial coating and we have related trophoblastic protein content of the cyst fluid with age, seriousness of illness, and cytology of epithelial lining. We have performed RIA analysis of the trophoblastic proteins betahCG, beta1-SP-1, and alphahCG and in a smaller (n=84) group of specimens we have also tested for CEA, TPA, and ferritin. Trophoblastic proteins were positive in cystic fluids but the biological meaning of this is not known and the values are not related to clinical manifestations, except in a group of patients with apocrine metaplasia in which we tried to find a relationship between fertile age and increased betahCG. This finding presumably has a prognostic meaning that can be further understood by epidemiologic studies (of dietary intake and evaluation of lipid metabolites) and by information about inflammatory state of cystic fluid.

Keratin 7 is a marker for a subset of trophoblastic cells in human germ cell tumors.

Human testicular germ cell tumors were studied immunohistochemically with the monoclonal antibody to the 54-kd keratin polypeptide (keratin 7) to determine whether this antibody could be used selectively to identify trophoblastic cells. The antibody reacted with the intermediate filaments in the cytoplasm of some cells in choriocarcinoma cell lines, and in trophoblastic cells in mixed germ cell tumors and a seminoma. It did not react with classic seminoma cells, embryonal carcinoma, yolk sac carcinoma, or somatic tissues of mixed germ cell tumors. On the basis of these data we conclude that monoclonal antibody to keratin 7 is a marker for a subset of trophoblastic cells in human germ cell tumors.

Molecular alterations induced by hypertension in pregnancy on syncythiotrophoblast plasma membranes from human placenta.

To evaluate the possible functional relationships between hypertensive status and syncythiotrophoblast plasma membrane behaviour we have carried out a freeze-fracturing and biochemical investigation to assess: 1) ultrastructurally, relations between number and diameter of Intramembrane Particles (IMP) and hypertensive conditions; 2) biochemically, actin content of microvilli in this pathological status. Our data in vitro show a decrease of IMP in hypertension before and after Ca++ addition and a decrease of actin in microvilli of hypertensive placenta. These observations seem to be in agreement with the hypothesis of a possible structural immaturity in hypertensive placenta and may represent morphological signs of placental insufficiency.

Characterization of baboon pregnancy-specific beta 1-glycoprotein.

Immunostaining of baboon placental tissues with anti-human pregnancy-specific beta 1-glycoprotein (SPI) antibodies demonstrated that an SP1-like molecule was present in the syncytiotrophoblasts. Staining was observed on the membrane and in the cytoplasm, but the nucleus was devoid of any staining. Western blot analysis further demonstrated the presence of five protein species in baboon placental extract, whereas four protein bands were detected in human placental extract. Culture medium of baboon placental villi also contained five SP1-like molecules with sizes slightly different from those present in the placental extract. Amniotic fluid and culture medium of decidua basalis and chorioamniotic tissue contained lesser quantities and fewer species of SP1-like molecules. However, an 87 kDa band was present in all samples examined. Northern blot analysis of baboon placenta with a human placental SP1 cDNA probe demonstrated the presence of a 1.65 Kb band, whereas two hybridizing bands (1.65 Kb and 2.25 Kb) were present in human placenta. Southern blot analysis of baboon genomic DNA further demonstrated the presence of multiple bands hybridizing with a human placental SP1 cDNA probe. These results showed the presence in baboons of multiple genes encoding mRNAs and proteins highly similar to human placental SP1.

Hormonal and basement membrane markers for immunoidentification of cultured human trophoblast cells.

Improvement of human chorionic villi cells in vitro culture has been obtained by supplementing the medium with 30% human fetal cord serum, instead of fetal calf serum. Expression of human chorionic gonadotropin, estrogen and progesterone receptors, laminin, laminin receptors, and type IV collagen has been studied on first subculture passages (4-7 weeks) by immunoperoxidase method. A minority of the cultured cells were positive for estrogen receptors, the majority were positive for progesterone receptors, while all cells were negative for human chorionic gonadotropin. Cultured cells showed variable positive immunostaining for basement membrane markers like laminin, and type IV collagen, and for laminin receptors. Detection of both progesterone receptors and laminin, or type IV collagen, excluded fibroblast contamination and could then be useful for quick identification of cultured trophoblast cells.

Subinvolution of the uteroplacental arteries in the human placental bed.

Subinvolution of the uteroplacental arteries of the placental bed is a recognized cause of post partum haemorrhage causing significant morbidity. Whilst the physiological changes in these arteries during pregnancy and the part played by endovascular trophoblast migration are well documented, the sequence of events during involution and the pathophysiology of subinvolution are unknown. Using immunohistochemical techniques we have studied uteroplacental arteries in the placental bed in 25 cases of post partum haemorrhage and compared the subinvoluted vessels with normally involuted vessels. Non-involuted vessels were present in 22 test cases; these abnormal vessels were filled with thrombus and no endothelial lining was detected. Extravillous perivascular trophoblast was usually present in the walls of these abnormal vessels and in some cases was seen in an endovascular position. Subinvolution of placental site vessels may represent an abnormal interaction between maternal uterine cells and fetal trophoblast.

[Analysis of trophoblast subpopulations on touch smears of chorionic villi during normal pregnancy].

Trophoblasts of normal human pregnancy are classified into syncytiotrophoblasts (STs), cytotrophoblasts (CTs) and intermediate trophoblasts (ITs) which exist in cell columns and so forth. We analyzed the trophoblast subpopulations which appear on touch smears of chorionic villi morphologically and immunohistochemically, using the uterine contents of 37 cases of induced abortion. Troma 1, a rat monoclonal antibody, was utilized as a trophoblast marker and a mouse monoclonal antibody to HLA-A,B,C was employed to discriminate ITs from CTs. The results were as follows: 1. Two sorts of cells were positive for Troma 1 and therefore were considered to be trophoblastic: Multinuclear cells of various sizes and relatively large mononuclear cells. 2. Multinuclear trophoblasts were thought to be STs because of their characteristic cytomorphology and negative reaction for HLA-A,B,C. 3. The lower the gestational age was, the more ITs were observed. 4. The cellular and nuclear size of ITs varied and their chromatin was coarsely granular with aggregates. In addition one or more marked nucleoli were noted. Consequently it would be important to take care not to misdiagnose ITs as trophoblasts of a malignant nature.

[The DNA content in the cell nuclei of trophoblastic tumors]. / Soderzhanie DNK v iadrakh kletok opukholei trofoblasta.

The DNA content in cytotrophoblast (CTB) and syncytiotrophoblast (STB) cell nuclei was assayed in tissue sections of 7 hydatidiform moles (HM) and 27 choriocarcinomas (CH). The procedure involved Feulgen's reaction and scanning cytophotometry. The analysis of summarized histograms showed the DNA distribution in CTB cell nuclei, on the one hand, and that in STB, on the other, to differ significantly in both the tumors. The HM studied cases were referred to as two subtypes on the basis of such parameters as modal class value, its ploidy and degree of nuclear poly- and heteroploidy of CTB and STB. These characteristics were used to identify three patterns of CH. A pronounced modal class (2c--4c) was typical of type 1. A wider range of modal class (2c--10c or 4c--8c) was observed in type 2. Type 3 of tumor was characterized by a pronounced polyploidy with the absence of the modal class. The analysis of individual CTB and histograms showed no significant differences between HM and CH with respect to the DNA content. An increase in the share of highly polyploid cells was associated with a shorter survival of patients.

Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen

Immunoregulatory activity in supernatants from cultures of normal human trophoblast cells of the first trimester.

Supernatants from isolated trophoblast cell cultures (trophoblastic fluid) derived from first-trimester human placentas were assessed for immunoregulatory activity. Trophoblastic fluid at different days of culture consistently inhibited the blast transformation of allogenic lymphocytes. This suppressor effect had no apparent correlation with biosynthesis of human chorionic gonadotropin by trophoblast cells, since this hormone was secreted into the culture fluid only for the initial 3 days. However, the culture fluids of such purified trophoblast cells contained an immunosuppressive factor, pregnancy-associated plasma protein A, which was measurable throughout the culture period of 8 days. The presence of pregnancy-associated plasma protein A in significant amounts in trophoblastic fluid collected at daily intervals indicated a continuous secretion ability of pregnancy-associated plasma protein A by trophoblast cells in culture parallel to the suppressive immunoregulatory effect of the fluid. Such immunosuppressive effect was absent in the culture fluids of control BeWo malignant trophoblast cells; the BeWo cell culture fluids had markedly reduced levels of pregnancy-associated plasma protein A. The culture supernatant of normal trophoblast cells of placentas from first-trimester pregnancy activated in vitro the generation of a population of suppressor lymphocytes. This effect is generally considered responsible for immunologic tolerance. Therefore demonstration of immunosuppressive effects and the presence of relatively high levels of pregnancy-associated plasma protein A in trophoblastic fluid indicate that such proteins secreted by the trophoblast cells may be important in the local immunoregulatory processes of the fetal allograft.

Localization of fetal major histocompatibility complex antigens and maternal leukocytes in murine placenta. Implications for maternal-fetal immunological relationship.

An immunohistochemical study of the days 14-19 murine placenta and uterus using a panel of antibodies specific for maternal and paternal class I major histocompatibility complex (MHC) antigens, specific leukocyte subsets, and other lineage-specific markers was performed to elucidate the nature of the maternal tolerance of the fetal implant in the uterus. Fetally derived trophoblast lining maternal blood spaces lacked MHC antigens, but other interstitial trophoblasts expressed fetally derived polymorphic class I MHC determinants. The latter class I MHC-bearing trophoblasts were most prominent in the maternal decidua basalis where they were mixed with maternal cells. Our results identify two factors that may be relevant for understanding why these alloantigen-bearing fetal cells are not rejected by the mother: a paucity of la-bearing antigen presenting cells, macrophages, and mature T and B lymphocytes and the presence of large numbers of Thy-1+ asialo-GM-1+ CD4- CD8- bone marrow-derived (CLA/Ly5+) cells of maternal origin in the decidua basalis. These latter cells correspond to previously described granulated metrial gland cells that may be involved in local immunoregulation.

Localization of mRNA for the oncotrophoblastic protein oncomodulin during implantation and early placentation in the rat.

The mRNA for the oncodevelopmental calcium-binding protein oncomodulin (MW 11,700) has been detected in tissues of the rat conceptus by in situ hybridization using biotinylated RNA probes. Oncomodulin mRNA was detected in the basal zone and labyrinth of rat placenta, following a similar distribution to that shown for oncomodulin by immunohistochemistry. Oncomodulin mRNA was also detected in rat ectoplacental cone at ten days and in amnion and PYS, but not VYS from 11 days onward. Previously oncomodulin was not detected embryonically from day 14 to birth, but in the present study of oncomodulin mRNA and protein, both were detected in implantation stages from blastula through egg cylinder. Staining was also present on decidual tissue. The suggestion is made that the oncomodulin gene is initially active in all cell types, but later its activity is confined to extraembryonic tissues.