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.

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.

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.

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.

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.

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.

[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 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.

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.

Laminin in human trophoblast--decidua interaction.

Laminin was localized by immunohistology to stromal cells of the human non-pregnant endometrium as well as to similar cells of the decidua. Only small amounts of laminin were detected during the proliferative phase, but significant quantities had accumulated by the mid-secretory phase which persisted in decidua if pregnancy occurred. This cyclical variation suggested hormonal control of laminin production. The presence of laminin could also be demonstrated in decidual cells in culture, most of which was intracellular or pericellular with very little liberated into the culture supernatant. Human first trimester trophoblast cells were observed to attach preferentially to laminin-coated surfaces in vitro. The role of laminin in trophoblast-decidua interaction in vivo is discussed.

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Two fetal antigens (FA-1 and FA-2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: localisation in the fetus and adult female genital tract.

Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.

Comparative study of placental protein 19, human chorionic gonadotrophin and pregnancy-specific beta 1-glycoprotein as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease.

PP19, a new placental tissue protein, has alpha 1-beta 1 electrophoretic mobility, a molecular weight of 36,500 and 3.9% carbohydrate. To study immunocytochemical PP19 localization in extravillous trophoblast, we obtained formalin-fixed specimens from extravillous tubal pregnancy at gestational weeks (GW) 7-9 (12 blocks); four early intrauterine pregnancies at GW 7-13 (12 blocks); four late pregnancies at GW 28-38 complicated with intramural uterine myoma, placenta increta and abruptio placenta (8 blocks); four invasive complete moles (9 blocks); and seven primary and metastatic gestational choriocarcinomas (12 blocks). Immunohistochemical staining was done for PP19, pregnancy-specific beta 1-glycoprotein (SP1) and human chorionic gonadotrophin (hCG) using the indirect-labeled antibody method [purified PP19 (Lot no. 225/242) and antibody against PP19 (Lot no. 632ZA) prepared by H. Bohn, antibodies against hCG (Behringwerke, Marburg, FRG) and SP1 (Dakopatts, Copenhagen, Denmark)]. In both early and late intrauterine pregnancies, the extravillous syncytiotrophoblastic cell (XST) showed positive staining for hCG and SP1 in the cytoplasm, as well as for PP19, which stained more intensively in the nucleus than in the cytoplasm. The three proteins were not seen in the evtravillous cytotrophoblastic cell (XCT) in the trophoblastic cell column and shell. The interstitial cytotrophoblast-like cell (ICT), which infiltrated into the decidua and myometrium, and their blood vessels, was immunoreactively positive for PP19 but negative for hCG and SP1 with the exception of SP1-positive ICT in the myometrium in late pregnancy. XST and ICT in the endosalpinx of tubal pregnancy stained for all three proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytogenetic, endocrinological and immunological studies of tissue cultures from ectopic pregnancies]. / Cytogenetische, endokrinologische sowie immunologische Studien an kultiviertem Gewebe ektoper Schwangerschaften.

The world-wide increase in extrauterine pregnancies induced us to grow trophoblastic elements obtained from ectopic locations in tissue cultures in order to determine whether the tubal implantation of the blastocyst could be caused by chromosomal aberrations. In addition, we investigated for what period of time trophoblastic tissue from ectopic sites is capable of producing HCG in culture. This was done in view of an eventual use of this method as a model for the testing of the effect of antitrophoblastic agents in vitro. Finally, the concentration of several tumor markers in the culture medium was measured. The karyotype could be determined in 16 out of 20 tissue specimen subjected to tissue culture; in one case a chromosomal aberration was recognized, two times there was evidence of increased fragility of the chromosomes. The ratio of female to male karyotypes was 10 to 6. HCG was found to be produced for 6 to 9 days in the tissue cultures. The concentration of the tumor markers CEA, CA 19-9, CA 12-5 and CA 15-3 in the culture medium was low as compared to cultures containing additional tubal mucosa. We are concluding that chromosomal aberrations do not seem to play a causal role in the genesis of EUP. The assay of HCG production by trophoblastic cells in tissue cultures could eventually be used for the testing of agents which might ultimately be employed for the non-surgical treatment of certain cases of EUP.

Control of the steroidogenic machinery of the human trophoblast by cyclic AMP.

Human cytotrophoblasts express adenylate cyclase activity and possess membrane-bound regulatory proteins that bind guanine nucleotides (G proteins). Stimulation of the cyclase by forskolin or addition of 8-bromo-cAMP augments progesterone secretion by cultured cytotrophoblasts at least in part, by promoting accumulation of components of the cholesterol side-chain cleavage system. This is the consequence of increased synthesis of the proteins participating in steroidogenesis as a result of the 8-bromo-cAMP-provoked increase in their respective mRNAs. We propose that progesterone synthesis by cytotrophoblasts is up-regulated by cyclic AMP, which acts to increase expression of genes encoding the steroidogenic machinery. Paracrine or autocrine factors may initiate this cascade by stimulating the cytotrophoblast adenylate cyclase.

Localization of alpha-interferon in the human feto-placental unit.

The distribution of alpha-interferon in human placental tissue was investigated by immunocytochemical study of paraffin wax-embedded tissue sections using a sheep alpha-interferon antiserum. Fifty-eight placentas of gestational ages from 8 to 40 weeks were examined. alpha-Interferon was present in the syncytiotrophoblast of the chorionic villi of all placentas and was also in macrophages in 28 cases. The appearances suggest production of interferon in human placental trophoblast and, in view of its diverse biological effects, support the concept of a role for alpha-interferon in the complex series of events required for successful gestation.