Human Chorionic Gonadotropin Secretion Research Articles

Manipulation of CD98 expression affects both trophoblast cell fusion and amino acid transport activity during syncytialization of human placental BeWo cells.

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein.

Homocysteine induces trophoblast cell death with apoptotic features.

Hyperhomocysteinemia has been suggested as a possible risk factor in women suffering from habitual abortions, placental abruption or infarcts, preeclampsia, and/or intrauterine growth retardation. However, little is known about the pathogenic mechanisms underlying the action of homocysteine. The present study investigated the in vitro ability of homocysteine to affect trophoblast gonadotropin secretion and to induce cell death. In primary human trophoblast cells, homocysteine treatment (20 micromol/L) resulted in cellular flattening and enlargement, extension of pseudopodia, and cellular vacuolization. Cellular detachment, apoptosis, and necrosis were favored. With in situ nick end labeling, we investigated DNA degradation, and we used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Cytochrome c release from mitochondria to the cytosol and DNA cleavage in agarose gel have been investigated. Homocysteine, but not cysteine, induced trophoblast apoptosis and significantly reduced human chorionic gonadotropin secretion. These findings suggest that trophoblast cell death might represent a pathogenic mechanism by which homocysteine may cause pregnancy complications related to placental diseases.

Hypoxia alters expression and function of syncytin and its receptor during trophoblast cell fusion of human placental BeWo cells: implications for impaired trophoblast syncytialisation in pre-eclampsia

The fundamental process of placental trophoblast cell fusion (syncytiotrophoblast formation or syncytialisation) which is a characteristic of this tissue is poorly understood. Pre-eclampsia is associated with placental hypoxia and suppressed syncytiotrophoblast formation. We therefore have studied the effect of low-oxygen tensions on the rate of cell fusion, relative abundance of mRNAs encoding syncytin and its receptor, amino acid transport system B 0, which are thought to be involved in trophoblast cell fusion (as well as the activity of amino acid transport through this system) in a cell model of syncytialisation (BeWo cells following forskolin treatment). Forskolin-induced cell fusion (determined by a quantitative flow cytometry assay) was reversibly suppressed in 2% oxygen compared to 20% oxygen. This was associated with suppressed secretion of human chorionic gonadotropin. Forskolin stimulated relatively less syncytin mRNA (determined by reverse transcription–polymerase chain reaction) in 2% than in 20% oxygen and there was no stimulation after 48 h in 2% oxygen. There was a spontaneous, time-dependent increase of amino acid transporter B 0 mRNA in vehicle, which was suppressed by 2% oxygen and by forskolin treatment in 20% oxygen. Forskolin-induced changes in amino acid transport system B 0 function were not seen in cells cultured for 48 h in 2% oxygen. These observations suggest that under conditions of low ambient oxygen, dysregulation of expression of syncytin and of its receptor may suppress the normal process of cell fusion necessary for syncytiotrophoblast formation and contributes to syncytiotrophoblast abnormalities characteristic of pre-eclampsia.

Epidermal growth factor enhances invasive activity of BeWo choriocarcinoma cells by inducing alpha2 integrin expression.

The trophoblast, an important component of the mammalian placenta, has several essential biological roles in the maintenance of pregnancy. First, trophoblast cells must attach to the uterine endometrium, and then they must invade to a depth at which the vascular network exists. Here, we investigated the effects of epidermal growth factor (EGF) on alpha2 integrin expression, adhesiveness to collagen, and invasive activity using human BeWo choriocarcinoma cells. EGF induced the expression of alpha2 integrin mRNA and protein, as shown by Northern blotting, Western blotting, and flow cytometry. Human chorionic gonadotropin (hCG) secretion was enhanced by the addition of EGF, which suggests that the BeWo cells functionally differentiated similarly to normal trophoblasts. EGF also dose-dependently stimulated the invasiveness of BeWo cells. Antibody against alpha2 integrin inhibited this effect, suggesting that it may be mediated by an increase of cell surface integrin. EGF had no effect on the adhesiveness of BeWo cells to collagen, whereas it stimulated the chemokinetic activity in a dose-dependent manner. The increase of chemokinetic activity was suppressed by antibody against alpha2 integrin. These results suggest that EGF may induce alpha2 integrin expression in trophoblast cells, thereby enhancing their invasiveness into the endometrium via an increase of their chemokinetic activity.

The effect of gonadotropin-releasing hormone-I (GnRH-I) and GnRH-II on human chorionic gonadotropin (hCG), progesterone (P4) and estradiol (E2) secretion by human placenta

Objective: GnRHs play a key role in the regulation of placental hCG synthesis and secretion. We investigated the secretion pattern of hCG, P4 and E2 in our organ culture system and compared the effects of GnRH-I and -II on hormonal release from placental explants during first and second trimester.

Neutral Endopeptidase/CD10 Expression during Phorbol Ester-induced Differentiation of Choriocarcinoma Cells through the Protein Kinase C- and Extracellular Signal-regulated Kinase-dependent Signalling Pathway

Neutral endopeptidase 24.11 (NEP) is identical to CD10, which is a differentiation antigen for early B-lymphoid progenitors in the B-cell differentiation pathway. This ectoenzyme is known to have a key role in the control of growth, differentiation, and signal transduction of many cellular systems by regulating bioactive peptides and cytokines. Recently, we demonstrated that NEP/CD10 is upregulated during forskolin-induced choriocarcinoma cell differentiation, suggesting that NEP/CD10 is a trophoblast differentiation marker. The purpose of this study was to clarify the enhancement of NEP/CD10 expression and its signal transduction pathway during phorbol ester (PMA)-induced differentiation of BeWo choriocarcinoma cells.PMA-induced differentiation of BeWo cells was confirmed by morphological change and human chorionic gonadotropin (hCG) secretion, which was completely blocked by a protein kinase C (PKC) inhibitor, Bisindolylmaleimide I (Bis I). On immunoblot analysis, PMA enhanced NEP/CD10 expression in a dose- and time-dependent manner, which was completely abolished by Bis I and a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, PD98059. PMA also induced phosphorylation of p44/p42 extracellular signal-regulated kinases (ERK) 1 and 2. These observations indicated that activation of PKC by PMA induced differentiation of BeWo cells, and that PMA activated MAPK/ERK, which resulted in the enhancement of NEP/CD10 expression. Furthermore, immunocytochemical analysis showed that NEP/CD10 expression was detected on the membranes of PMA-treated differentiated BeWo cells. In summary, we demonstrated that NEP/CD10 was enhanced during PMA-induced differentiation of BeWo choriocarcinoma cells through a PKC-dependent MEK/ERK signalling pathway. Our findings also suggest that NEP/CD10 may play a functional role in the process of trophoblast differentiation.

Enhancement of radiosensitivity of the MCF-7 breast cancer cell line with human chorionic gonadotropin.

Secretion of human chorionic gonadotropin (hCG) during pregnancy induces differentiation of the mammary gland, thereby making breast tissue less susceptible to carcinogenesis. HCG binds to specific hCG receptors on mammary epithelial cells inducing changes in gene expression that can inhibit cell proliferation and, therefore, interfere with tumorigenesis. Since breast cancer cells also contain a relatively high level of the hCG receptor, hCG has potential as a therapeutic agent. We postulated that hCG might also enhance the radiosensitivity of breast cancer cells and, therefore, be useful as an adjunctive therapy. In the present study, MCF-7 breast cancer cells grown in cell culture were treated with hCG (0.2-5 IU/ml) for 24 h prior to exposing the cells to 0 Gy, 3 Gy, 4 Gy, or 5 Gy of radiation. Following irradiation, the MCF-7 cells were incubated either in the presence or absence of hCG. Cell survival was monitored with an MTT assay 1 day, 4 days, and 7 days after irradiation. All of the concentrations of hCG tested enhanced radiosensitivity of MCF-7 cells. The maximum enhancement occurred with MCF-7 cells that had been exposed to 2 IU/ml of hCG for at least 24 h prior to irradiation with 4 Gy. The use of higher concentrations of hCG or a higher dose of radiation did not increase the enhancement effect. Treatment of MCF-7 cells with hCG for only 24 h was sufficient to achieve the maximum effect. However, maintaining the cells in hCG beyond 24 h increased the effectiveness of the lowest hCG concentration. Using a linear-quadratic equation to analyze the data, we determined that the use of hCG would result in an 8-10% reduction in MCF-7 cell survival at a dose of 2 Gy, a typical dose used in conventional cancer therapy.

Nausea and vomiting of pregnancy: endocrine basis and contribution to pregnancy outcome.

For a majority of women, nausea and vomiting of pregnancy (NVP) is an unpleasant but normal feature of pregnancy. NVP has a largely unknown etiology, but it is widely agreed that physiological, psychological, genetic, and cultural components are contributing factors. The occurrence of NVP may be of benefit in pregnancy, and NVP-associated pregnancies are less likely to result in miscarriage, preterm delivery, or intrauterine growth retardation. It has been suggested that NVP is promoted by secretion of human chorionic gonadotrophin (hCG), largely on the basis of the coincidence between the onset of NVP and hCG secretion and the peaks of both at around 12 to 14 weeks' gestation. Concentrations of hCG that are above or below the normal range are associated with poor pregnancy outcomes. It seems that NVP and hCG are related and that nutrient restriction during critical periods of embryonic development may ensure production of hCG at optimal levels, protect placental development, and optimize nutrient partitioning between maternal and fetal tissues.

Metallothionein protects against severe oxidative stress-induced apoptosis of human trophoblastic cells.

Oxidative stress induces cellular apoptosis. Many agents producing intracellular oxidative stress, including H(2)O(2) and steroid hormones, have also been found to induce metallothionein (MT) expression. Recently, MT has been recognized as potentially having antioxidant activity. This action may be essential for survival of terminally differentiated cells subject to oxidative stress, such as syncytiotrophoblasts, placental cells producing pregnancy hormones and forming the maternal-fetal barrier. We previously demonstrated an inverse relationship between basal MT expression and apoptotic incidence in the trophoblastic cell line, JEG-3. Using JEG-3 cells transfected with MT in sense or antisense orientation, we have examined here the effect of altered basal MT levels on trophoblastic function and apoptosis following treatment with H(2)O(2) or diethylstilbestrol (DES). Induction of MT mRNA was observed in control and transfected JEG-3 cells following exposure to severe oxidative stress. Changes in the localization of MT protein, however, were apparent after a low oxidative stress challenge. Exposure to H(2)O(2) resulted in a dose-dependent decrease in human chorionic gonadotropin secretion in all JEG-3 cultures regardless of basal MT expression, whereas no change was detected following DES treatment. With respect to apoptosis, a significant protective effect was observed proportional to the basal MT level. These results suggest that although MT does not ameliorate oxidative stress-induced perturbation of some trophoblastic functions, its expression is critical for protection of these cells from severe oxidative stress-induced apoptosis. MT thus appears to act as an anti-apoptotic antioxidant in trophoblastic cells.

Defect of Syncytiotrophoblast Formation and Human Chorionic Gonadotropin Expression in Down's Syndrome

The syncytiotrophoblast (ST) is a major component of the human placenta as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. We have studied the formation and function of the ST in normal and trisomy 21 (T21)-affected placenta using the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblast cells were isolated from first trimester, second trimester and term placentae. In vitro cytotrophoblast cells isolated from normal placenta fused to form the ST. This was associated with an increase in the transcript levels and the secretion of human chorionic gonadotropin (hCG). However, the secretion of hCG decreased through pregnancy. In T21-affected placentae, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of this hormone compared with cultured cells isolated from control age-matched placentae. These results were confirmed by a significant ( P< 0.05) decrease in transcript levels of α and β subunits of hCG in total homogenates of T21-affected placentae compared with controls. These results suggest a decrease in functional mass of ST in T21 placenta, and therefore a decrease in production of placental pregnancy-specific polypeptide hormones.

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants.

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.

The Expression and Localization of Neutral Endopeptidase 24.11/CD10 in Human Gestational Trophoblastic Diseases

Neutral endopeptidase 24.11 (NEP)/CD10 is a cell-surface peptidase that hydrolyzes various bioactive peptides. NEP is distributed in both normal and neoplastic cells and plays a functional role by modulating cellular responses to peptide substrates. Recently, NEP has been shown to be expressed in normal placental trophoblasts, suggesting its physiological role during pregnancy. In the present study, we investigated the expression of NEP in hyperplastic and anaplastic trophoblasts in gestational trophoblastic diseases (GTDs). Flow cytometric analysis demonstrated that NEP was expressed in all choriocarcinoma cell lines examined. The NEP enzyme activity in these cell lines correlated with cell-surface protein levels and was abolished by the NEP inhibitor phosphoramidon. On immunoblot analysis, NEP protein was detected in both hydatidiform mole and choriocarcinoma tissues as a double band of 95 and 100 kDa similar to that of the normal placental tissues. Immunohistochemical analysis revealed that NEP was present on syncytiotrophoblasts, while no or very faint NEP immunoreactivity was observed on cytotrophoblasts in the normal placenta. Similarly, NEP in hydatidiform mole and invasive mole was localized on the membrane of syncytiotrophoblasts, but not on hyperplastic cytotrophoblasts. In contrast, in choriocarcinoma, NEP was highly expressed not only on syncytiotrophoblastic cells but also on invading anaplastic cytotrophoblasts. In addition, NEP was also expressed on intermediate trophoblasts in placental site trophoblastic tumors. In summary, this is the first study demonstrating the expression of NEP/CD10 in GTDs. The differential localization of NEP among various trophoblastic tumors suggests that NEP may play a functional role in the regulation of trophoblast transformation and human chorionic gonadotropin secretion.

RECOGNIZING ABNORMAL MARKER RESULTS THAT DO NOT REFLECT DISEASE IN PATIENTS WITH GERM CELL TUMORS

No AccessJournal of UrologyCLINICAL UROLOGY: Original Articles1 Mar 2000RECOGNIZING ABNORMAL MARKER RESULTS THAT DO NOT REFLECT DISEASE IN PATIENTS WITH GERM CELL TUMORS MICHAEL J. MORRIS, and GEORGE J. BOSL MICHAEL J. MORRISMICHAEL J. MORRIS More articles by this author , and GEORGE J. BOSLGEORGE J. BOSL More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)67807-XAboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: The judicious use of serum α-fetoprotein (AFP), human chorionic gonadotropin (HCG) and lactate dehydrogenase is key to appropriate management of patients with germ cell tumors. Elevated AFP and/or HCG generally indicate active disease. We describe patients with germ cell tumors who had elevated serum AFP and/or HCG but no active disease, despite careful repeat evaluation. Materials and Methods: Histories of 6 cases of germ cell tumors that remained in remission despite abnormal serum AFP and/or HCG were reviewed. Results: Markers were only modestly elevated, remained constant or spontaneously normalized during repeat measurements, and there was no other clinical or radiographic evidence of disease. Patients were treated conservatively with physical examination, radiological tests and repeat marker assays, with no relapse to date. Conclusions: Stable, low increases in serum AFP and HCG may not represent active disease. Careful repeat evaluation will determine whether the markers increase. If no change is noted after appropriate studies have been reviewed by an experienced practitioner to exclude active disease from diagnosis, then consideration should be given to managing such cases with close surveillance to avoid unnecessary chemotherapy. References 1 : International Germ Cell Consensus Classification: a prognostic factor-based staging system for metastatic germ cell cancers. J Clin Oncol1997; 15: 594. Google Scholar 2 : Multivariate analysis of prognostic variables in patients with metastatic testicular cancer. Cancer Res1983; 43: 3403. Google Scholar 3 : Serum tumor marker half-life during chemotherapy allows early prediction of complete response and survival in nonseminomatous germ cell tumors. Cancer Res1990; 50: 5904. Google Scholar 4 : Does serum tumor marker half-life complement pretreatment risk stratification in metastatic nonseminomatous germ cell tumors?. Clin Cancer Res1996; 2: 1565. Google Scholar 5 : The role of tumour markers in the diagnosis and treatment of testicular germ cell cancers. Br J Urol1997; 79: 247. Google Scholar 6 : Microheterogeneity of human alphafetoprotein. Tumour Biol1988; 9: 3. Google Scholar 7 : Clinical relevance of alphafetoprotein microheterogeneity in alphafetoprotein-secreting tumors. Cancer Detect Prev1994; 18: 447. Google Scholar 8 : Alpha-fetoprotein-concanavalin. A binding as a marker to discriminate between germ cell tumours and liver diseases. Eur J Cancer1995; 31A: 2239. Google Scholar 9 : Falsely positive specific human chorionic gonadotropin assays in patients with testicular tumors: conversion to negative with testosterone administration. J Urol1979; 122: 126. Link, Google Scholar 10 : Pulsatile secretion of human chorionic gonadotropin in normal adults. N Engl J Med1987; 317: 1688. Google Scholar 11 : Commercial radioimmunoassay for beta subunit of human chorionic gonadotropin: falsely positive determinations due to elevated serum luteinizing hormone. Cancer1982; 49: 136. Google Scholar 12 : Gonadal function in men with testicular cancer. Semin Oncol1998; 25: 224. Google Scholar 13 : Gonadal dysfunction in patients treated for metastatic germ-cell tumors. J Clin Oncol1986; 4: 1500. Google Scholar 14 Bayer, Inc. Technicon Immuno 1 System Information. Publication No. DA4–1159J95, 1995 Google Scholar From the Department of Medicine, Division of Solid Tumor Oncology, Genitourinary Oncology Service, Memorial Sloan-Kettering Cancer Center and Department of Medicine, Weill Medical College of Cornell University, New York, New York© 2000 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 163Issue 3March 2000Page: 796-801 Advertisement Copyright & Permissions© 2000 by American Urological Association, Inc.Keywordsalpha-fetoproteinschorionic gonadotropin, beta subunit, humantesticular neoplasmstumor markers, biologicalneoplasms, germ cell and embryonalMetricsAuthor Information MICHAEL J. MORRIS More articles by this author GEORGE J. BOSL More articles by this author Expand All Advertisement PDF DownloadLoading ...

Biological action of keratinocyte growth factor in BeWo cells, a human choriocarcinoma cell line.

Previously, we reported that the expression of keratinocyte growth factor (KGF) is enhanced in secretory phase endometrial and decidual cells in early pregnancy as compared with the expression of KGF in proliferative phase endometrial cells, in humans. In order to clarify the role of KGF in embryo-endometrial interaction, we analyzed the in vitro effect of KGF on the human chorionic gonadotropin (hCG) secretion and on DNA synthesis in chorionic villi which are in close contact with the endometrium/decidua in the early stage of pregnancy. In this study, we used the BeWo cell line, a human choriocarcinoma cell line that possesses the biological features of secreting various placental hormones including hCG. Furthermore, we investigated the expression of KGF receptor (KGF-R) in these cells. KGF significantly stimulated hCG secretion in cultured BeWo cells but did not affect [3H]-thymidine incorporation. KGF-R mRNA was detected in BeWo cells by reverse transcriptase-polymerase chain reaction. These results suggest that the expression of KGF, which is induced in endometrial/decidual cells by progesterone, plays an important role in the embryo-endometrial/ decidual interaction by stimulating hCG secretion rather than affecting cell proliferation.

Modulation of human cytotrophoblastic leptin secretion by interleukin-1alpha and 17beta-oestradiol and its effect on HCG secretion.

To investigate the role of leptin during pregnancy, we assessed leptin production by pure cultured human cytotrophoblastic cells (CTB), its regulation by cytokines and 17beta-oestradiol and its effects on human chorionic gonadotrophin (HCG) secretion. Purified CTB from first trimester placenta were incubated in duplicates in the presence or absence of cytokines or 17beta-oestradiol. Medium was harvested on day 2 and the culture stopped on day 4. Results were corrected for protein content of each individual well and expressed as percent of controls per day (mean +/- SEM). Basal CTB leptin production was 25.2 +/- 2.6 (ng/mg prot). In comparison with controls, leptin production was stimulated to 320 +/- 16% (P < 0.0001) and 195 +/- 3.2% (P < 0.0004) by 3 and 10 ng/ml of interleukin-1alpha respectively. 17beta-oestradiol 10(-6) to 10(-9) mol/l increased basal leptin production 5-9-fold, while 10(-5) mol/l had no such effect. Basal CTB HCG secretion was 5722 +/- 1055 (mIU/mg prot). There was a dose-dependent leptin-induced increase in HCG secretion (P = 0.0039) reaching a 5-fold increase with a leptin concentration of 1 microg/ml (P < 0.006). Gonadotrophin-releasing hormone (GnRH) 8.5 x 10(-8) mol/l significantly increased HCG secretion to 140 +/- 21% of controls (P = 0.031). Cetrorelix (0.1 microg/ml) inhibited leptin-induced HCG secretion (P = 0.0028).

Effects of interleukin-6 (IL-6) on cytotrophoblastic cells.

Tumour invasion and trophoblastic invasion share the same biochemical mediators: the matrix metalloproteinases (MMP) and their inhibitors. In contrast to tumour invasion of a host tissue, trophoblastic invasion during implantation and placentation is stringently controlled both in tissue localization and developmental stage. The factors responsible for these important regulatory processes are unknown, but in-vitro studies point to endometrial cytokines and growth factors as possible candidates. Here we examined the possibility that interleukin-6 (IL-6), a trophoblastic and endometrial cytokine, represents such a regulatory factor. Purified first trimester cytotrophoblastic cells (CTB) were cultured for 4 days in presence or absence of increasing concentrations of IL-6. MMP-2 and MMP-9 bioactivity (zymography) and immunoactivity were measured in the culture supernatants together with total human chorionic gonadotrophin (HCG), fetal fibronectin (FFN) and leptin. IL-6 did not change the cytotrophoblastic secretion of FFN or total HCG. In contrast, this cytokine induced a dose-dependent stimulation of the leptin secretion and increased the activity, but not the immunoreactivity, of MMP-9 and MMP-2. These results indicate that IL-6 could be considered as an endometrio-trophoblastic regulator of cytotrophoblastic gelatinases.

Expression and functional analysis of endothelial nitric oxide synthase (eNOS) in human placenta.

We have investigated the expression and localization of endothelium-derived nitric oxide synthase (eNOS) and the effect of eNOS on placental human chorionic gonadotrophin (HCG) release. eNOS mRNA was found to be expressed in all tissues, with its expression significantly (P<0.05) increased across gestation. Compared to normal term gestation, placentae from term pregnancies with fetal retardation, or maternal diabetes, but not with maternal hypertension, displayed significantly more (P<0.05) eNOS mRNA. By immunocytochemistry, we found staining for eNOS in both the cyto- and syncytiotrophoblasts of first trimester and a loss of cytotrophoblast eNOS staining in term placentae, while syncytiotrophoblasts at term were strongly eNOS positive. Additional staining was found in endothelium surrounding the vascular tree. HCG was found to colocalize with eNOS in trophoblasts, but not in endothelia. When placental explants were perifused, exposure to the NOS substrate, the NO donor, I-arginine and trinitroglycerol evoked a prompt, albeit transient, increase of HCG release. The NOS inhibitor delayed, but did not block arginine-induced HCG release. Thus, eNOS is expressed in the human placenta at increasing levels during gestation with further increases during some pathological conditions. A role for NO in the acute endocrine modulation of the placenta is suggested by the colocalization of eNOS with HCG in human trophoblasts and the prompt secretion of HCG in response to agents which increase NO concentrations.

Regulation of human chorionic gonadotropin secretion and messenger ribonucleic acid levels by follistatin in the NUCC-3 choriocarcinoma cell line

NUCC-3 choriocarcinoma cell line was used as anin vitro placental cell model to investigate the effects of follistatin on GnRH-stimulated hCG secretion and its subunit mRNA level and stability. Follistatin alone did not affect basal hCG secretion and its subunit mRNA level. GnRH increased hCG secretion and hCG β-mRNA level in a dose-dependent manner. Follistatin significantly suppressed GnRH-stimulated hCG secretion and hCG α- and β-mRNA level. It inhibited hCG secretion in response to PMA, and did not affect the stability of hCG α- and β-mRNA. The authors suggest that follistatin inhibits GnRH-stimulated hCG secretion as well as hCG α- and β-RNA level by decreasing the rate of transcription through the second messenger transduction system —protein kinase-C.

Identification of pregnancy-associated genes during implantation in rat uterus

The human placenta and its associated membranes are vital to the maintenance, nutrition, and protection of the developing fetus. During placental development some cytotrophoblasts give rise to the chorionic membrane whereas others fuse to form a differentiated syncytium of cells that are responsible for placental protein and steroid hormone production. The mechanisms involved in the differentiation of the trophoblasts are unknown; however, an intermediate stage with a characteristic phenotype has been documented in vivo. We have observed that chemically dissimilar xenobiotic agents induced BeWo choriocarcinoma cells to change their usual cytotrophoblastic phenotype and acquire morphologic and functional characteristics typical of intermediate trophoblast. Incubation of BeWo cell cultures in the presence of 1 μmol/L methotrexate for 48 hours stimulated human chorionic gonadotropin secretion (2.3-fold) and aromatase activity (4.9-fold). Morphologic findings associated with these hormonal changes, including increased nuclear size and cytoplasmic expansion, were also observed. With the use of a computer-interfaced image analyzer, planimetric morphometry of the nuclear area of the cells revealed a 1.8-fold increase after incubation with methotrexate. The effect of forskolin, a direct activator of adenylyl cyclase, was also evaluated by means of this model of cytotrophoblast differentiation. The addition of 10 μmol/L forskolin to BeWo cultures also resulted in dramatic changes in trophoblast cell phenotype. Increases in human chorionic gonadotropin synthesis (7.3-fold), aromatase activity (13.5-fold), and nuclear area (3.0-fold) were induced over those of untreated cells. In addition, increases in [3H]thymidine incorporation (1.7-fold) were afforded by both treatments. These results suggest that biochemical and cytologic changes associated with human trophoblast differentiation can be induced in vitro via activation of the adenylyl cyclase pathway by forskolin and through unknown and apparently independent signals by methotrexate.

Sex hormone-binding globulin stimulates chorionic gonadotrophin secretion from human cytotrophoblasts in culture.

The effects of sex hormone-binding globulin (SHBG) on the secretion of human chorionic gonadotrophin (HCG) and cAMP by cultured human cytotrophoblasts were investigated. Cytotrophoblasts obtained from normal term placentae were cultured in serum-free medium with or without the addition of human SHBG. The presence of SHBG in the medium increased the release of HCG and the accumulation of cAMP. Ligand-free SHBG was able to raise both HCG and cAMP concentrations and the maximal response was observed with 1 nM of the steroid-binding globulin. Addition of either oestradiol or 5alpha-dihydro-testosterone (DHT) to cultures previously incubated with SHBG in a final molar ratio of 1:10 resulted in a further increase of HCG and cAMP concentrations. This effect was blocked when cultured placental cells were exposed to SHBG that was previously saturated with DHT or when incubated in the presence of steroids only. The results of the present study provide evidence for the in-vitro regulation of HCG secretion by SHBG and further support the concept that this steroid-binding protein may act as a mediator of steroid action at the cellular level. Finally, the increase in cAMP suggests that SHBG receptor located in the surface of syncytiotrophoblast membranes is coupled to adenylate cyclase as part of the G-protein receptor family. Our results may provide new insights into the biological implications of extracellular steroid-binding proteins as well as new perspectives on the endocrinology of pregnancy.