Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta.

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta.

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and -beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.

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

Localization, secretion, and action of inhibin in human placenta.

Inhibin is a gonadal glycoprotein hormone that regulates the production of follicle-stimulating hormone (FSH) by the anterior pituitary gland and exhibits intragonadal actions as well. The present study shows that inhibin-like immunoreactivity (inhibin-LI) is present in cells of the cytotrophoblast layer of human placenta at term and in primary cultures of human trophoblasts. Human chorionic gonadotropin (hCG) stimulated secretion of inhibin-LI from these cultured placental cells. This effect was mimicked by 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP), forskolin, and cholera toxin, suggesting that the mechanism of hCG induction of placental inhibin-LI secretion is cAMP-dependent. Incubation with an antiserum that binds the alpha-subunit of human inhibin increased the secretion of hCG and gonadotropin-releasing hormone-like immunoreactivity (GnRH-LI) from trophoblast cells in culture, suggesting a local tonic inhibitory action of endogenous inhibin on hCG and GnRH-LI release. The action of inhibin on hCG secretion may partially require the presence of placental GnRH, as suggested by evidence that a synthetic GnRH antagonist partially reverses the hCG increase induced by inhibin immunoneutralization. Results suggest paracrine roles for both inhibin and GnRH in the regulation of placental hCG production.

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Presence of alpha 2-macroglobulin in normal but not in malignant human syncytiotrophoblasts.

alpha 2-Macroglobulin (alpha 2M) was demonstrated in normal syncytiotrophoblasts of both early and full-term human placentas using immunocytological staining. alpha 2M was also detected in hydatidiform moles, the benign tumors of proliferating syncytiotrophoblasts. In contrast, no alpha 2M was detected in invasive moles or choriocarcinomas. In culture conditions, both normal syncytiotrophoblasts and choriocarcinoma cells, identified by production of human chorionic gonadotropin, were negative when stained for alpha 2M or when studied using metabolic labeling and immunoprecipitation or radioimmunoassay. However, alpha 2M was taken up from added human serum by the cultured syncytiotrophoblasts, whereas choriocarcinoma cells remained negative also under these conditions. The possible role of alpha 2M in the regulation of proteolysis in cell invasion is considered.

Calcium-dependant binding proteins associated with human placental syncytiotrophoblast microvillous cytoskeleton.

Isolated human placental syncytiotrophoblast microvillous plasma membrane vesicles were extracted with Triton X-100 to yield a detergent-insoluble residue. The residue contained approx. 50% of the total membrane protein and was qualitatively different from untreated trophoblast on SDS-polyacrylamide gel electrophoresis, Western blots and dot-immunobinding assay. Three major proteins, with molecular weights of 68, 36 and 34 kDa, dissociated from this non-ionic detergent-insoluble submembranous cytoskeletal fraction in the presence of calcium chelators. They were immunologically related to human lymphocyte cytoskeletal calcium-binding proteins, and the 36 kDa component reacted with antisera to the phospholipase A2 inhibitor, lipocortin II. Anti-lipocortin I sera did not recognise the 34 kDa protein, but did react with a series of trophoblast cytoskeletal proteins in the 34-37 kDa region. Incubation of epidermal growth factor with isolated trophoblast membrane vesicles stimulated the phosphorylation of a 36 kDa protein on tyrosine residues. Immunoprecipitation studies further showed there was no phosphorylation of the 34 kDa protein, but the 68 kDa protein was a major phosphorylated component of isolated syncytiotrophoblast membranes. p68 was principally phosphorylated on serine with slight tyrosine phosphorylation which showed an apparent increase after epidermal growth factor treatment. These results indicate a family of calcium-dependant binding proteins, some of which are phosphorylated, associated with the submembranous cytoskeleton of syncytiotrophoblast microvilli.

The apical and basal plasma membranes of the human placental syncytiotrophoblast contain different erythrocyte membrane protein isoforms. Evidence for placental forms of band 3 and spectrin.

Using immunochemical techniques, we identified forms of erythrocyte membrane proteins in apical and basal plasma membranes of human placental trophoblast. A wheat germ agglutinin-binding intrinsic protein was present in the microvillous (maternal facing) but not the basal (fetal facing) membrane of the syncytiotrophoblast epithelium. Conversely, erythrocyte-related proteins of the basal membrane included two intrinsic membrane proteins, a 95,000 Mr band 3 isoform and a form of spectrin. These four proteins were all absent from the microvillous membrane. The basal membrane spectrin isoform was also present in basal membrane skeletons. A 70,000 Mr polypeptide which reacted with antibodies to band 3 was present in both microvillous and basal plasma membranes. Therefore, certain isoforms of red cell membrane proteins are polarized between the two surfaces of the human placental syncytiotrophoblast. We propose that the localization of spectrin to the basal membrane is related to the less bundled organization of microfilaments at this membrane compared with that of the microvillous membrane. The band 3 isoforms are candidates for participation in maternofetal anion transport.

Rabbit placental relaxin: ultrastructural localization in secretory granules of the syncytiotrophoblast using rabbit placental relaxin antiserum.

Although relaxin has been isolated from the placenta of the human, rabbit, horse, and cat, this study represents the first ultrastructural localization of the hormone in placental tissue. Placentas were removed from rabbits on days 15, 23, and 30 of pregnancy, and the tissues were prepared for light and electron microscopies. The cytoplasm of the syncytiotrophoblast from all stages of pregnancy studied showed positive staining for the hormone at the light level using guinea pig antirabbit relaxin serum and the avidin-biotin technique. Ultrastructurally, the syncytiotrophoblast was found to contain membrane-bounded granules (150-400 nm in diameter) which formed at the Golgi and were seen in close association with the cell membrane. Exocytosis involving the incorporation of the granule membrane into the cell membrane was observed. These granules labeled positively for relaxin after treatment with guinea pig antirabbit relaxin serum and goat antiguinea pig immunoglobulin G-colloidal gold. Control sections in which the relaxin antiserum was absorbed with purified rabbit relaxin or substituted with normal guinea pig serum contained no gold-labeled granules. Cross-reactivity of the rabbit relaxin antiserum with porcine relaxin was demonstrated by labeling of the relaxin-containing granules in the pregnant pig corpus luteum with the rabbit relaxin antiserum and by inhibiting the labeling of rabbit placental and pig corpora luteal granules by absorbing the rabbit relaxin antiserum with porcine relaxin. We have previously described the labeling of rabbit placental relaxin with porcine relaxin antiserum. This study suggests that relaxin is synthesized and secreted from the syncytiotrophoblast of the rabbit placenta, with the subcellular site of storage being membrane-bounded granules.

Immunohistochemical localization of placental lactogen in binucleate cells of bovine placentomes.

Bovine placental lactogen (bPL) has been isolated from bovine trophoblast and characterized as a 32 K mol wt protein which exists in three different forms which differ in their isoelectric point values and their amino acid compositions. Two of the three forms have been shown to have both bovine GH (bGH)- and bovine PRL (bPRL)-like activities equal on a molar basis to bGH and BPRL in radioreceptor assays. It has been postulated that, in sheep, PL is delivered to the maternal circulation by the migration of fetal binucleate cells from the trophoblast across the fetal-maternal boundary into the uterine epithelium. To determine whether an analogous situation exists in the cow, antibodies to bPL were used to localize bPL in bovine placentomes and to measure its concentration in fetal and maternal sera. For cytology, bPL was localized on sections of placentomes from midgestation and term bovine placentas using an indirect immunoperoxidase technique. Stained binucleate cells were demonstrated throughout the trophoblast, often in close association with the microvillous boundary which separates the trophoblast from the maternal epithelium. In cross-sections of fetal villi, binucleate cells with cytoplasmic processes extending into and through the uterine epithelium were immunostained as well as cells within the plane of the uterine epithelium in close approximation or apposition to the maternal basement membrane. RIA demonstrated bPL to be present in maternal sera in concentrations of 1-2 ng/ml and in fetal sera at 5-12 ng/ml. These data are consistent with the hypothesis that binucleate cell migration accomplishes the delivery of bPL to the maternal circulation.

Detection of vitamin D binding protein on the surface of cytotrophoblasts isolated from human placentae.

Vitamin D binding protein (DBP), a Mr 56,000-58,000 alpha 2-glycoprotein, is the major serum protein involved in the transport of vitamin D sterols. Recently it has been suggested that DBP may also be involved in immunoglobulin G binding to cells. Because the trophoblast is involved in the transport of molecules such as vitamin D and immunoglobulin G to the fetus, we asked whether DBP could be detected on the surface of human placental trophoblast cells. Cytotrophoblasts purified from human term placentae were fixed and made permeant with Triton X-100 and examined by indirect immunofluorescence after incubation with a monoclonal antibody to DBP. Greater than 90% of these cells stained positively, whereas no staining was observed with nonimmune antiserum. The presence of DBP on/in the surface of cytotrophoblasts could also be demonstrated by fluorescent cytometry. When cell surface-associated proteins of cytotrophoblasts were radioiodinated, a Mr 57,000 radiolabeled protein could be immunoisolated from the cell lysate with a purified monospecific polyclonal antibody to DBP. Immunoisolation of this radiolabeled protein was prevented by the addition of excess unlabeled human DBP to the cell lysate before incubation with antibody. This Mr 57,000 radiolabeled protein could also be isolated by affinity chromatography selecting for proteins that bind to globular actin. When cytotrophoblasts were incubated with [35S]methionine for 3 or 18 h, active synthesis of DBP could not be demonstrated by immunoisolation techniques. These studies demonstrate the presence of DBP on the surface of well washed, human cytotrophoblasts. This DBP may be maternally derived, since active synthesis of DBP could not be demonstrated.

Effects of maternal iodine deficiency on the L-thyroxine and 3,5,3'-triiodo-L-thyronine contents of rat embryonic tissues before and after onset of fetal thyroid function.

Female rats were placed on a low iodine diet (LID) or LID supplemented with KI. They were mated 3-6 months later. Maternal and embryonic tissues were obtained both before the onset of fetal thyroid function, at 11 and 17 days of pregnancy, and at 21 days of gestation. T4 and T3 concentrations were measured by RIA. T4 concentrations were very low in the plasma, liver, and lung of LID dams and in all embryonic samples obtained from such mothers, namely 11-day-old embryotrophoblasts, 17-day-old placentas and embryos, 21-day-old placentas, embryos, plasma, liver, lung, and carcass (whole embryos minus the trachea, thyroid, blood, liver, and brain). T3 was low in 17-day-old placentas and embryos and in all fetal tissues obtained at 21 days of gestation from LID dams. These results show that when iodine deficiency is severe enough to result in very low maternal plasma T4 levels, embryonic tissues are deficient in T4 and T3 both before and after the onset of fetal thyroid function. This finding might be relevant to the etiopathology of human iodine deficiency disorders.

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.

Evidence for the presence of oncoplacental SP1-like protein in normal nonpregnant serum.

To seek the pregnancy-specific beta 1-glycoprotein (SP1) in nonpregnant serum, normal human serum was applied to immunoadsorbent containing monoclonal anti-SP1 antibodies. SP1 eluted with 8 M urea was further analyzed by sodium dodecyl sulfate-gel electrophoresis and immunoblotting. A SP1-positive band with the same electrophoretic mobility as purified placental SP1 was found. The results suggest that serum from normal nonpregnant subjects contains material closely related to the placental protein SP1. The mean serum concentrations of SP1 were similar in men and women, ranging from 1.1-3.4 ng/ml.

Immunohistochemical demonstration of relaxin in the genital tract of pregnant and nonpregnant women.

The biotin-avidin immunoperoxidase staining method and antisera against highly purified porcine relaxin were used to localize relaxin in the genital tract of pregnant and nonpregnant women. Formalin-fixed tissue specimens from normal placenta, decidua, myometrium, vagina, corpus luteum, and Fallopian tubes were studied. In pregnant women, relaxin was found in the placental syncytiotrophoblast, decidua, and corpus luteum. In nonpregnant women, relaxin was identified in the corpus luteum and endometrium in the secretory, but not in the proliferative, phase. Myometrium, cervix, vagina, and Fallopian tubes were negative for relaxin. This is the first report describing relaxin in the nonpregnant corpus luteum, and we also confirm results of an early disputed study claiming that endometrium in the secretory phase contains relaxin. The origin and biological role of human endometrial relaxin remain to be studied.

Indirect immunofluorescent localization of prolactin to the cytoplasm of decidua and trophoblast cells in human placental membranes at term.

An indirect immunofluorescent technique was used to determine the localization of cytoplasmic human PRL (hPRL) in fresh and incubated human placental membranes at term. In both fresh and 8-h incubated samples of amnion, amniochorion decidua, or chorion decidua obtained from three placentas, we found specific reproducible localization of hPRL to the cytoplasm of decidua and trophoblast cells. The decidua cells appeared to be the most intensely fluorescent. No specific hPRL immunofluorescence was noted in the amniotic epithelium of fresh or incubated samples of amnion and amniochorion decidua. These data suggest that the trophoblast decidua cell layer is the site of PRL localization and possibly synthesis in placental membranes at term and may be the origin of amniotic fluid PRL in humans.

Immunocytochemical localization of relaxin in human decidua and placenta.

Specimens from 20 human term placentas were stained with 4 different antisera produced against porcine relaxin (Rlx) using the avidin-biotin immunoperoxidase procedure. Cells of the parietal decidua adherent to the fetal membranes, cells of the chorionic cytotrophoblast, as well as cells of the placental basal plate consistently stained with all 4 anti-Rlx sera. Occasionally, Rlx was detected in epithelial cells lining the amniotic membrane. The syncytiotrophoblast stained for Rlx in 2 specimens only. This response was seen only in syncytiotrophoblast that lined villi in close proximity to the basal plate. Syncytiotrophoblast of the chorionic villi either did not stain at all or gave very weak positive immunostaining with the anti-Rlx sera in all specimens. No difference was noted in staining patterns among placentas delivered by elective cesarean section or vaginal delivery.

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.

Trophoblastic cells of the hydatidiform mole contain a beta 1-subtype adrenergic receptor.

Both beta 1- and beta 2-adrenergic receptors have been previously described in normal human placental homogenates; the cells upon whose surface membranes these receptors reside have not been identified. In order to show that a beta 1-adrenergic receptor is present on trophoblastic cells, the cells which mediate maternal-fetal transport and produce placental hormones, beta-adrenergic receptors were demonstrated in membrane fractions of human hydatidiform mole. Microscopic sections of the mole samples used demonstrated edematous villi lined by trophoblastic cells with minimal nontrophoblastic (stromal or vascular) contamination compared with placenta. (--)-[3H]Dihydroalprenolol [(--)-[3H]DHA] binding to molar membranes was reversible and saturable to a single class of sites (Kd = 0.97 +/- 0.12 nM; n = 7; maximum binding capacity, 72.9 +/- 6.4 fmol/mg protein). (--)-[3H]DHA binding was associated with catecholamine-stimulated adenylate cyclase activity. Agonist competition for the molar beta-adrenergic receptor showed the order of potency to be (--)isoproterenol much greater than norepinephrine = epinephrine, characteristic of a beta 1-adrenergic receptor subtype. Competition for (--)-[3H]DHA binding to trophoblastic membranes by the beta-adrenergic receptor subtype-specific agents metoprolol (beta 1 selective) and zinterol (beta 2 selective) was also characteristic of a homogeneous subtype of beta 1-adrenergic receptors. Because beta 1-adrenergic receptors alone were seen on trophoblast cells, the beta 2-adrenergic receptor in placenta must reside on nontrophoblastic elements (stromal or vascular endothelium). No differences in beta-adrenergic receptor binding were seen related with ploidy (2 or 3 N), the presence or absence of a fetus, or the progression of the mole to choriocarcinoma. Two choriocarcinoma cell lines, BeWo and JEG-3, however, showed no specific (--)-[3H]DHA binding. Human trophoblast contains beta 1-adrenergic receptors coupled to catecholamine-sensitive adenylate cyclase, supporting a role for catecholamines in the regulation of placental metabolism.

Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres.

A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability.