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Modulation of Copper/Zinc Superoxide Dismutase Expression and Activity with In Vitro Differentiation of Human Villous Cytotrophoblasts
Abstract
Due to the role of oxygen free radicals in trophoblast cell differentiation, we used the in vitro model of villous cytotrophoblast differentiation into the syncytiotrophoblast to investigate the modulation of the key antioxidant enzyme copper/zinc superoxide dismutase (SOD-1) in the human trophoblast during pregnancy. Cytotrophoblast cells were isolated from first-trimester and term placentae. SOD-1 mRNA levels were determined using real-time quantitative polymerase chain reaction, protein levels were determined by immunoblotting with a specific monoclonal antibody, and oxidoreductase activity was measured during syncytiotrophoblast formation in vitro. Interestingly, SOD-1 protein levels fell significantly (P< 0.001) during syncytiotrophoblast formation but no corresponding change in enzyme activity was observed. This apparent discrepancy may be related to different amounts of SOD-1 co-factor in the two cell types. Indeed the level of copper was significantly higher (P< 0.05) in syncytiotrophoblast as compared with cytotrophoblast. SOD-1 mRNA levels remained stable during cytotrophoblast differentiation. SOD-1 expression and activity were similar in cytotrophoblast cells isolated from first-trimester and term placentae, and in the differentiated syncytiotrophoblast in vitro. These results underline the need to determine SOD-1 protein expression and activity simultaneously in order to gain a better knowledge of its role in human trophoblast cell differentiation.
... Reference HCGB GCT-ACT-GCC-CCA-CCA-TGA-CC ATG-GAC-TCG-AAG-CGC-ACA-TC 94 J00117 Frendo et al. (2000) INHA CAA-GTA-TGA-GAC-AGT-GCC-C GCC-ATC-TAT-TTC-CCA-ACT-CTG 145 NM_002 ...
... In our study, we did not observe a sig- nificant increase in oxidative stress in cytotrophoblasts cultured under 21% O 2 for 4 days compared to undifferentiated cytotrophoblasts, and compared to cytotrophoblasts cultured under 8% O 2 . This result is comparable to the results published by Frendo et al. (2000). Nevertheless, there was a small decrease in SOD-1 mRNA expression when cells were cultured under 8% O 2 . ...
... This implies that higher copper concentrations in termite queens occur by trophic accumulation in castes. High copper concentrations are associated with increased Cu/Zn-SOD activity [43]. In the present study, we demonstrated that there were no significant differences in RsSOD1 and RsSOD3A expression among the termite castes although the queens had higher Cu/Zn-SOD activity than nonreproductive individuals. ...
... There are often discrepancies between enzyme activity and gene expression levels. A previous report indicated that copper is critical for Cu/ Zn-SOD activity and modulates enzymatic activity in the absence of changes in gene expression [43]. Therefore, the difference in copper concentrations between the queens and workers may be critical for their disparate levels of Cu/ Zn-SOD activity. ...
In most organisms, superoxide dismutases (SODs) are among the most effective antioxidant enzymes that regulate the reactive oxygen species (ROS) generated by oxidative energy metabolism. ROS are considered main proximate causes of aging. However, it remains unclear if SOD activities are associated with organismal longevity. The queens of eusocial insects, such as termites, ants, and honeybees, exhibit extraordinary longevity in comparison with the nonreproductive castes, such as workers. Therefore, the queens are promising candidates to study the underlying mechanisms of aging. Here, we found that queens have higher Cu/Zn-SOD activity than nonreproductive individuals of the termite Reticulitermes speratus . We identified three Cu/Zn-SOD sequences and one Mn-SOD sequence by RNA sequencing in R. speratus . Although the queens showed higher Cu/Zn-SOD activity than the nonreproductive individuals, there were no differences in their expression levels of the Cu/Zn-SOD genes RsSOD1 and RsSOD3A . Copper (Cu ²⁺ and Cu ⁺ ) is an essential cofactor for Cu/Zn-SOD enzyme activity, and the queens had higher concentrations of copper than the workers. These results suggest that the high Cu/Zn-SOD activity of termite queens is related to their high levels of the cofactor rather than gene expression. This study highlights that Cu/Zn-SOD activity contributes to extraordinary longevity in termites.
... For instance, trisomy 21 trophoblasts present SOD1 upregulation and unbalanced redox status, owing to the location of sod1 on chromosome 21. This redox imbalance is associated with defective cell differentiation and hormone secretion [7,35,57]. It has been reported that exposure of pregnant women to FA is associated with spontaneous abortions and low birth weight, in keeping with our observation of syncytial dysfunction leading to abnormal trophoblast differentiation and regeneration and defective pregnancy hormone secretion. ...
... This could be due to variations in the intracellular availability of GPx cofactors (selenium is the cofactor for GPx1-4 and 6). We have previously observed a similar discrepancy with SOD-1 protein expression and the intracellular SOD-1 cofactor (copper) concentration during trophoblast cell fusion [7,35,57]. Recent publications describe a protective effect of selenium against FA-induced oxidative stress [59,60]. However, more experiments are needed to quantify selenium during trophoblast differentiation and to confirm its protective effect against FA exposure of trophoblasts. ...
The chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST), which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT). Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused coty-ledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2). Moreover, we show that formaldehyde-exposed tropho-blasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac), an antioxidant.
... Both cytosolic Cu, Zn-SOD and mitochondrial Mn-SOD are expressed in human cytotrophoblasts [21,22]. We demonstrated a modulation of SOD-1 expression and activity with in vitro differentiation of human villous CT [23]. ...
... It is known that SOD-1 is located on human chromosome 21, and that it is overexpressed in different T21-affected cell types [24]. In T21 villous CT we demonstrated an overexpression of SOD-1 expression and activity, in accordance with a gene dosage effect [23,25], and a subsequent abnormal oxidative status of the villous CT as shown by a large increase in catalase activity [17]. ...
The syncytiotrophoblast layer plays a major role throughout pregnancy, since it is the site of numerous placental functions, including ion and nutrient exchange and the synthesis of steroid and peptide hormones required for fetal growth and development. Inadequate formation and regeneration of this tissue contributes to several pathologies of pregnancy such as intrauterine growth restriction and preeclampsia, which may lead to iatrogenic preterm delivery in order to prevent fetal death and maternal complications. Syncytiotrophoblast formation can be reproduced in vitro using different models. For the last ten years we have routinely purified villous cytotrophoblastic cells (CT) from normal first, second and third trimester placentas and from gestational age-matched Trisomy 21 placentas. We cultured villous CT on plastic dishes to follow the molecular and biochemical aspects of their morphological and functional differentiation. Taking advantage of this unique collection of samples, we here discuss the concept that trophoblast fusion and functional differentiation may be two differentially regulated processes, which are linked but quite distinct. We highlight the major role of mesenchymal-trophoblast cross talk in regulating trophoblast cell fusion. We suggest that the oxidative status of the trophoblast may regulate glycosylation of proteins, including hCG, and thereby modulate major trophoblast cell functions.
... Finally, we demonstrated by using specific antisense oligonucleotides that inhibition of syncytin1 expression lead to a decrease of trophoblast fusion and differentiation (Frendo et al., 2003b), hCG secretion in the culture medium of antisense treated cells being decreased by 5 fold. Furthermore the inhibition of trophoblast fusion by overexpression of superoxide dismutatse 1 (SOD-1) is associated with an absence of increase in HERV-W env mRNA (Frendo et al., 2001; Frendo et al., 2000a). All together, these results strongly support a direct role of syncytin1 in human trophoblastic cell fusion and differentiation. ...
... EGF (Alsat et al., 1993; Morrish et al., 1987) hCG (Cronier et al., 1994; Shi et al., 1993) cAMP (Cronier et al., 1997b; Keryer et al., 1998) GM-CSF (Garcia-Lloret et al., 1994) Macrophages and macrophage-conditionned media (Cervar et al., 1999; Khan et al., 2000) Dexamethasone (Cronier et al., 1998) Estradiol (Cronier et al., 1999b) TGF-β1 (Cronier et al., 1997a; Morrish et al., 1991) LIF (Nachtigall et al., 1996) Hypoxia, SOD-1 (Alsat et al., 1996; Frendo et al., 2001; Frendo et al., 2000a; Levy et al., 2000) Endothelin (Cronier et al., 1999a) 15ΔPGJ2 (Levy et al., 2000) TNFα (Leisser et al., 2006) TABLE 1 ...
Trophoblastic cell fusion is one essential step of the human trophoblast differentiation pathway and is a multifactorial and dynamic process finely regulated and still poorly known. Disturbances of syncytiotrophoblast formation are observed in numerous pathological clinical conditions such as preeclampsia, intrauterine growth retardation and trisomy 21. In this review, we summarize current knowledge of the different membrane proteins directly involved in trophoblastic cell fusion, which we identified by using the physiological model of primary culture of villous trophoblastic cells. Connexin 43 and gap junctional intercellular communication point to the role of molecular exchanges through connexin channels preceding membrane fusion. Zona occludens-1, which can interact with connexin 43, is also directly involved in trophoblast fusion. The recently identified fusogenic membrane retroviral envelop glycoproteins syncytin 1 (encoded by the HERV-W gene) and syncytin 2 (encoded by the FRD gene) and their receptors are major factors involved in human placental development . We describe the increasing number of factors promoting or inhibiting trophoblast fusion and differentiation and emphasize the role of human chorionic gonadotropin (hCG) and its receptor. Indeed, in trisomy 21 the dynamic process leading to membrane fusion is impaired due to an abnormal hCG signaling. This abnormal trophoblast fusion and differentiation in trisomy 21-affected placenta is reversible in vitro. Trisomy 21 trophoblastic cell culture may therefore be useful to identify the possible large number of prerequisite factors involved in trophoblast fusion, the limiting step of trophoblast differentiation.
... trophoblasts than by trophoblasts at later stages (Bidart et al., 2000; Frendo et al., 2000). 3. hCG may be subject to autocrine/paracrine regulation by a GnRH produced by the large population of cytotrophoblastic cells present in early pregnancy (Islami et al., 2001). ...
... Expression at this stage of pregnancy of the truncated hCG receptor isoform may impair the autocrine regulation of hCG biosynthesis (Licht et al., 1993). 2. The mechanisms regulating hCG gene transcription may differ in early and late placental development, as suggested by the higher levels of hCG production and secretion by early trophoblasts than by trophoblasts at later stages (Bidart et al., 2000; Frendo et al., 2000). 3. hCG may be subject to autocrine/paracrine regulation by a GnRH produced by the large population of cytotrophoblastic cells present in early pregnancy (Islami et al., 2001). ...
The placenta plays a key role in pregnancy, mediating exchanges between mother and fetus and maternal tolerance of fetopaternal antigens. In some species, it also produces hormones that ensure the maintenance of gestation and fetal well-being. This unique organ also has considerable potential for use as a model for various aspects of biology. Indeed, the use of transgenic mouse models has greatly improved our understanding of the genetic control of placental development in this species and has opened up new fields of investigation in developmental biology. Analogous cell types have been identified among human and murine trophoblasts: proliferative trophoblastic cells, invasive trophoblastic cells and cells differentiating into syncytium, but human and mouse placentas differ in both morphogenesis and endocrine function. Herein, the similarities and differences between the human and mouse models are reviewed, with a view to encouraging caution in the extrapolation of results from one model to the other.
... In our study, we did not observe a significant increase in oxidative stress in cytotrophoblasts cultured under 21% O 2 for 4 days compared to undifferentiated cytotrophoblasts, and compared to cytotrophoblasts cultured under 8% O 2 . This result is comparable to the results published by Frendo et al. (2000). Nevertheless, there was a small decrease in SOD-1 mRNA expression when cells were cultured under 8% O 2 . ...
Study question:
Is 8% O2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O2 traditionally used in cell culture?
Summary answer:
Human primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels.
What is known already:
Cell culture is traditionally done under 21% O2, which is equal to a pO2 of ~160 mm Hg. Based on the pO2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should under 8% O2, which is equivalent to 60 mm Hg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts.
Study design size, duration:
Cytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O2 and 8% O2 for 12 days. Several cellular parameters were assessed on days 2, 4, 8, and 12.
Participants/materials, setting, methods:
Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGβ, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H2O2 generation.
Main results and the role of chance:
There was no difference between 21% O2 and 8% O2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O2 compared to 8% O2. The expression level of hCGβ was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O2, while PlGF expression was inhibited compared to 21% O2. This latter result indicates that 8% O2 may be more hypoxic than normoxic for in-vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H2O2 generation) in our cell cultures.
Large-scale data:
Not applicable.
Limitations reasons for caution:
The in-vitro culture of cytotrophoblasts is artificial and does not reflect the in-vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%.
Wider implications of the findings:
It is possible to culture human primary term cytotrophoblasts for at least 12 days. The O2 percentage of the air does not negatively affect in-vitro cytotrophoblast differentiation. For in-vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%.
Study funding/competing interest(s):
This work was fully supported by 'Fetus for Life' charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper.
... Low levels of antioxidant proteins such as copper/zinc superoxide dismutase (SOD-1) and catalase in the syncytiotrophoblast are correlated with syncytial fusion [27]. In cases with trisomy 21 higher levels of such proteins are related to defective trophoblast fusion [28]. It has been speculated that high activity of antioxidant proteins in the trophoblast impairs syncytial fusion. ...
... However, little is known concerning a possible role for SOD-1 in human placental development. Recently, we demonstrated a modulation of SOD-1 expression and activity with in vitro differentiation of human villous cytotrophoblasts (33). Interestingly, we also have shown a failure of cytotrophoblast differentiation into syncytiotrophoblast in trisomy 21 (T21)-affected placentas (34,35). ...
The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast formation and a decrease in the production of pregnancy-specific hormones. Due to the role of oxygen free radicals in trophoblast cell differentiation, we investigated the role of the key antioxidant enzyme, copper/zinc superoxide dismutase, encoded by chromosome 21 in in vitro trophoblast differentiation. We first observed that overexpression of superoxide dismutase in normal cytotrophoblasts impaired syncytiotrophoblast formation. This was associated with a significant decrease in mRNA transcript levels and secretion of hCG and other hormonal markers of syncytiotrophoblast. We confirmed abnormal cell fusion by overexpression of green fluorescence protein-tagged superoxide dismutase in cytotrophoblasts. In addition, a significant decrease in syncytin transcript levels was observed in superoxide dismutase-transfected cells. We then examined superoxide dismutase expression and activity in isolated trophoblast cells from trisomy 21-affected placentas. Superoxide dismutase mRNA expression (P < 0.05), protein levels (P< 0.01), and activity (P < 0.05) were significantly higher in trophoblast cells isolated from trisomy 21-affected placentas than in those from normal placentas. These results suggest that superoxide dismutase overexpression may directly impair trophoblast cell differentiation and fusion, and superoxide dismutase overexpression in Down’s syndrome may be responsible at least in part for the failure of syncytiotrophoblast formation observed in trisomy 21-affected placentas.
... Although, data from many studies did not established a correlation between serum copper concentration during a routine and impaired pregnancies, but contrary to those reports maternal serum copper concentration can be used as prognostic value in predicting the out-come of pregnancy and growing fetus (Alvarez et al., 2007;Alebic-Juretic and Frkovic, 2005;Serdar et al., 2006;Merker et al., 2005;Knutson, 2007;Uriu-Adams and Keen, 2005;Zhang et al., 2004;Pathak and Kapil, 2004;McArdle et al., 2008;Gambling and McArdle, 2004;Schulpis et al., 2004;Meram et al., 2003;Baig et al., 2003;Frendo et al., 2000;Friel et al., 2005;Kantola et al., 2000). ...
Copper is an integrated parts of metal-protein required far varieties of oxide-reductive metabolic pathways in human. Copper deficiency is considered as risk factors in some pregnancies. Premature rupture of membrane is a pregnancy complication with major adverse effects and is believed maternal Copper deficiency can also be considered as interventional factors. This study was done to evaluate if there is a correlation between maternal serum Copper concentration and premature rupture of membrane in pregnancy. In this case-control study 60 pregnant women with Premature Rupture of Membrane (PROM) were selected as case group including term and pre term the control group consist of 60 pregnant women with normal delivery of term and pre term states. Both group were matched for maternal and pregnancy age. In case and control group the pregnancy at term and pre-term were grouped independently as well. In general the maternal mean serum Copper concentration were 192.4±78.2 and 201.08±82.06 in case and control groups, respectively but this differences statistically was not significant. Data in this study revealed that the absolute value of maternal serum Copper concentration of term or pre term in case groups was slightly lower than related controls. Drop in maternal Copper concentration in some disturbed pregnancies such as premature rupture of membrane is previously demonstrated and based on our data the absolute Copper serum concentration of women with premature rupture of membrane was also slightly lower compared to healthy pregnancy but it was not statistically significant.
... The primer sequences for all transcripts examined are listed in Table 1. Primer sequences for cyclophilin 1A were from Frendo et al. [6] and purchased from Integrated DNA Technologies (IDT, Coralville, IA). All other primer sets were designed using Gene Fisher software [7] and purchased from IDT. ...
Pregnancy induces rapid, progressive and substantial changes to the cardiovascular system. The low recurrence risk of preeclampsia, despite familial predisposition, suggests an adaptation associated with pregnancy that attenuates risk for subsequent preeclampsia. We aimed to evaluate the persistent effect of pregnancy on maternal cardiovascular physiology.
Forty-five healthy nulliparous women underwent baseline cardiovascular assessment preconception and repeated an average of 30 months later. After baseline evaluation, 17 women (Preg) conceived singleton pregnancies and all delivered at term. The remaining 28 women comprised the non-pregnant control group (NP). We measured mean arterial blood pressure (MAP), cardiac output (CO), plasma volume (PV), pulse wave velocity (PWV), uterine blood flow (UBF), and flow-mediated vasodilation (FMD) at each visit.
There was a significant decrease in mean arterial pressure from the prepregnancy visit to postpartum in women with an interval pregnancy (prepregnancy: 85.3±1.8, postpartum: 80.5±1.8 mm Hg), with no change in NP subjects (visit 1: 80.3±1.4, visit 2: 82.8±1.4 mm Hg), (p = .002). Pulse wave velocity was significantly decreased in women with an interval pregnancy (prepregnancy: 2.73±0.05, postpartum: 2.49±0.05 m/s), as compared to those without an interval pregnancy (visit 1: 2.56±0.04, visit 2: 2.50±0.04 m/s), (p = .005). We did not observe a residual effect of pregnancy on cardiac output, plasma volume, uterine blood flow or flow-mediated vasodilation.
Our observations of decreased mean arterial pressure and reduced arterial stiffness following pregnancy suggest a significant favorable effect of pregnancy on maternal cardiovascular remodeling. These findings may represent a mechanism by which preeclampsia risk is reduced in subsequent pregnancies.
Copyright © 2015 Elsevier Inc. All rights reserved.
... Low levels of antioxidant proteins such as copper/zinc superoxide dismutase (SOD-1) and catalase in the syncytiotrophoblast are correlated with syncytial fusion [27]. In cases with trisomy 21 higher levels of such proteins are related to defective trophoblast fusion [28]. It has been speculated that high activity of antioxidant proteins in the trophoblast impairs syncytial fusion. ...
... The total RNA concentration was determined at 260 nm and its integrity was checked in a 1% agarose gel. The relative mRNA levels of the different genes were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), essentially as previously described (Frendo et al., 2000a), using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem, USA) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The nucleotide sequences of the primers used are listed in Table 1. ...
Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.
... In conclusion, we have demonstrated that: (1) there is an abnormal formation of the syncytiotrophoblast in Down's syndrome; (2) there is, therefore, a decrease in production hCG by the placenta in Down's syndrome. In addition, we recently demonstrated that the synthesis and secretion of other pregnancy specific hormones such as human placental lactogen (hPL), placental growth hormone (PGH) and leptin, synthesized by the syncytiotrophoblast, are decreased in T21 placentae (Frendo et al., 2000b). This finding will enhance understanding of the maternal hormonal changes of placental origin that are used as markers of fetal Down's syndrome and will assist in finding new markers of placental origin. ...
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 alpha and beta 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.
... However, little is known concerning a possible role for SOD-1 in human placental development. Recently, we demonstrated a modulation of SOD-1 expression and activity with in vitro differentiation of human villous cytotrophoblasts (33). Interestingly, we also have shown a failure of cytotrophoblast differentiation into syncytiotrophoblast in trisomy 21 (T21)-affected placentas (34,35). ...
The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast formation and a decrease in the production of pregnancy-specific hormones. Due to the role of oxygen free radicals in trophoblast cell differentiation, we investigated the role of the key antioxidant enzyme, copper/zinc superoxide dismutase, encoded by chromosome 21 in in vitro trophoblast differentiation. We first observed that overexpression of superoxide dismutase in normal cytotrophoblasts impaired syncytiotrophoblast formation. This was associated with a significant decrease in mRNA transcript levels and secretion of hCG and other hormonal markers of syncytiotrophoblast. We confirmed abnormal cell fusion by overexpression of green fluorescence protein-tagged superoxide dismutase in cytotrophoblasts. In addition, a significant decrease in syncytin transcript levels was observed in superoxide dismutase-transfected cells. We then examined superoxide dismutase expression and activity in isolated trophoblast cells from trisomy 21-affected placentas. Superoxide dismutase mRNA expression (P < 0.05), protein levels (P < 0.01), and activity (P < 0.05) were significantly higher in trophoblast cells isolated from trisomy 21-affected placentas than in those from normal placentas. These results suggest that superoxide dismutase overexpression may directly impair trophoblast cell differentiation and fusion, and superoxide dismutase overexpression in Down's syndrome may be responsible at least in part for the failure of syncytiotrophoblast formation observed in trisomy 21-affected placentas.
... The total RNA concentration was determined at 260 nm, and RNA integrity was confirmed by 1 per cent agarose gel electrophoresis. Relative mRNA levels of the different genes of interest were measured by quantitative RT-PCR assay, essentially as described in (Frendo et al., 2000b), using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystem). The nucleotide sequences of the primers are given in Table 1. ...
Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.
... The total RNA concentration was determined at 260 nm and its integrity was checked in a 1% agarose gel. The relative mRNA levels of the different genes were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), essentially as previously described (Frendo et al., 2000a), using an ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystem, USA) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The nucleotide sequences of the primers used are listed in Table 1. ...
The syncytiotrophoblast is the principal component of the human placenta involved in feto-maternal exchanges and hormone secretion. The syncytiotrophoblast arises from the fusion of villous cytotrophoblasts. We recently showed that functional gap junctional intercellular communication (GJIC) is an important prerequisite for syncytiotrophoblast formation and that connexin 43 (Cx43) is present in cytotrophoblasts and in the syncytiotrophoblast. To determine whether Cx43 is directly involved in trophoblast fusion, we used an antisense strategy in primary cultures of human villous cytotrophoblasts that spontaneously differentiate into the syncytiotrophoblast by cell fusion. We assessed the morphological and functional differentiation of trophoblasts by desmoplakin immunostaining, by quantifying hCG (human chorionic gonadotropin) production and by measuring the expression of specific trophoblast genes (hCG and HERV-W). Furthermore, we used the gap-FRAP (fluorescence recovery after photobleaching) method to investigate functional GJIC. Cytotrophoblasts treated with Cx43 antisense aggregated and fused poorly. Furthermore, less HERV-W env mRNA, hCGbeta mRNA and hCG secretion were detected in Cx43 antisense-treated cytotrophoblasts than in cells treated with scrambled antisense. Treatment with Cx43 antisense dramatically reduced the percentage of coupled trophoblast cells. Taken together, these results suggest that Cx43 is directly involved in human trophoblast cell-cell communication, fusion and differentiation.
... The relative mRNA levels of the different genes of interest were measured by quantitative RT-PCR, essentially as described in [21], using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems) and the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied Biosystems). The nucleotide sequences of the primers have been previously published [22,23]: hCG (+) 5#-TCCCACT CCACTAAGGTCCAA-3# and hCG ( ) 5#-CCCCATTA CTGTGACCCTGTT-3#; hPL (+) 5#-GCATGACTCCCA GACCTCCTT-3# and hPL ( ) 5#-TGCGGAGCAGCTCT AGATTGG-3#; PGH (+) 5#-AGAACCCCCAGACCTCC CT-3# and PGH ( ) 5#-TGCGGAGCAGCTCTAGGTT AG-3# CYT7 (+) 5#-GGACATCGAGATCGCCACCT-3# and CYT7 ( ) 5#-ACCGCCACTGCTACTGCCA-3#; PPAR (+) 5#-AGTGGGGATGTCTCATAATGCC-3# and PPARg ( ) 5#-AGGTCAGCGGACTCTGGATTC-3#; RXR (+) 5#-CCTTTCTCGGTCATCAGCTC-3# and RXR ( ) 5#-CTCGCAGCTGTACACTCCAT-3#; PPIA (+) 5#-GTCAACCCCACCGTGTTCTT-3# and PPIA ( ) 5#-CTGCTGTCTTTGGGACCTTGT-3#. ...
Human placenta extracts are widely used in clinical and fundamental research, particularly to study the hormonal and exchange functions of the placenta. However, very little is known about the distribution of the main hormone mRNAs in the placenta as a whole. Total placenta extracts are heterogeneous in their cellular components, as they contain material of both fetal and maternal origin, and in their structure. Results vary greatly depending upon the location of the biopsy and the number of biopsies performed. We used real-time quantitative RT-PCR to determine whether transcripts corresponding to the main hormones secreted by the human placenta (e.g. hCG, HPL and PGH) are equally distributed within and between term placentae. We also measured cytokeratin 7 transcripts, which are specifically expressed in the trophoblast, and transcripts corresponding to nuclear receptors PPARgamma and RXRalpha. A comparison of the results obtained with 12 different samples from each of four normal term placentae revealed that the amounts of transcripts differ considerably within and between each placenta. This emphasizes the need to study large numbers of samples when looking for significant differences in gene expression.
... The primer sequences for all transcripts examined are listed in Table 1. Primer sequences for cyclophilin 1A were from Frendo et al. [6] and purchased from Integrated DNA Technologies (IDT, Coralville, IA). All other primer sets were designed using Gene Fisher software [7] and purchased from IDT. ...
Trophoblast cell lines are important research tools used as a surrogate for primary trophoblast cells in the study of placental function. Because the cellular origins of transformed trophoblasts are likely to be diverse, it would be of value to understand the unique and shared phenotypes of the cells on a global scale. We have compared two widely used cell lines, BeWo and JEG3, by microarray analysis in order to identify differentially expressed genes. Results indicated that approximately 2700 genes were differentially expressed between the cell lines, with principal differences observed in the biological processes of response to stress, cell adhesion, signal transduction, and protein and nucleobase metabolisms. These data suggest that BeWo and JEG3 cell lines, and perhaps other trophoblast cell lines, are sufficiently dissimilar from each other such that they will be differentially suited for specific experimental paradigms.
... Syncytiotrophoblast formation in vivo and in vitro arises from villous cytotrophoblast fusion and 40 differentiation. Several factors modulate villous trophoblast differentiation, including EGF (epidermal 41 growth factor) and EGF receptor expression (Morrish et al., 1987; Alsat et al., 1993), hypoxia (Alsat et 42 al., 1996), cAMP-dependent protein kinase (PKA) (Keryer et al., 1998), granulocyte-macrophage 43 stimulating factor (Garcia-Lloret et al., 1994), transforming growth factor (TGF) (Morrish et al., 1991) and oxidative stress due to overexpression of copper zinc superoxide dismutase (Frendo et al., 45 2000a, 2001). The molecular mechanisms underlying trophoblast membrane fusion are poorly 46 understood. ...
Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation.
... Primer sequences, annealing temperatures and amplicon lengths are given inTable 1. All primers but the pairs for SOD1 [31] and Rplp0 [32] were designed using the Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Alpha DNA. Primer pairs used to amplify the genes of interest through real-time RT-PCR (Table 1) were designed to avoid cross-amplification with other similar genes, such as MT2A and the other members of the MT family. ...
The limits of copper homeostatic regulation in humans are not known, making it difficult to define the milder effects of early copper excess. Furthermore, a robust assay to facilitate the detection of early stages of copper excess is needed. To address these issues, we assessed changes in relative mRNA abundance of methallothionein 2A (MT2A), prion (PrP), amyloid precursor-like protein 2 (APLP2), Cu/Zn superoxide dismutase (SOD1) and its copper chaperone (CCS) in peripheral mononuclear cells (PMNCs) from healthy adults representing the 5% highest and lowest extremes in the distribution curve of serum ceruloplasmin (Cp) concentrations of 800 individuals. The intracellular Cu content was also determined. PMNCs were isolated from individuals before and after exposure to a single daily dose of 10 mg Cu (as CuSO(4)) for 2 months. Results showed that although there were fluctuations in serum Cp values of the samples assessed before copper exposure, no significant differences were observed in cell copper content or in the relative abundance of MT2A, PrP and APLP2 transcripts in PMNCs. Also, these values were not modified after copper supplementation. However, CCS and SOD1 mRNA levels were reduced in PMNCs after copper supplementation in the individuals with the high Cp values, suggesting that they should be further explored as biomarkers of moderate copper overload in humans.
Le développement du placenta humain lors d’une grossesse associée à une trisomie 21 est caractérisé par différentes anomalies, conduisant notamment à un défaut de formation et de fonctionnalité du syncytiotrophoblaste. Le syncytiotrophoblaste, qui provient de la fusion et de la différenciation des cytotrophoblastes, joue un rôle essentiel dans le maintien de la grossesse en assurant le transport des nutriments indispensables au développement fœtal et en sécrétant dans le sang maternel les hormones de la grossesse comme l’hCG. Les cytotrophoblastes issus de placentas trisomiques fusionnent peu ou avec un certain retard et le syncytiotrophoblaste résultant est à l’origine d’une synthèse et d’une sécrétion d’hCG diminuée, anormalement glycosylée et biologiquement moins active par rapport aux cellules issues de placenta normal. Cependant, lors d’une grossesse associée à une trisomie 21, l’hCG est retrouvée augmentée dans le sang maternel. Nous sommes donc confronté à un paradoxe: d’un côté une synthèse d’hCG diminuée et de l’autre, un taux sérique maternel augmenté. Nous avons montré que ce paradoxe ne s’explique pas par une anomalie de clairance de l’hCG anormalement glycosylée, ni par une augmentation de sa demi-vie plasmatique après expulsion du placenta. Il semblerait donc qu’il existe une élimination anormale de l’hCG par le placenta dans les grossesses affectées par une trisomie 21. Pour mieux comprendre le défaut de formation du syncytiotrophoblaste observé dans les cellules trophoblastiques trisomiques et le paradoxe observé entre la faible production d’hCG et son taux sérique maternel anormalement élevé, nous avons étudié la fonctionnalité et l’intéraction de l’hCG anormale avec son récepteur (R-LH/CG). Nous avons montré pour la première fois que l’expression du R-LH/CG est régulée au cours de la différenciation trophoblastique. De plus, nous avons caractérisé d’un point de vue biochimique et moléculaire le R-LH/CG dans les cytotrophoblastes trisomiques, montrant qu’il existe une très nette diminution de l’expression de la forme mature du R-LH/CG dans les cellules trophoblastiques trisomiques. L’utilisation de RNAi inhibant spécifiquement l’expression du R-LH/CG dans les cytotrophoblastes normaux, nous permet de mimer le phénotype observé dans les cellules trophoblastiques trisomiques (le défaut de formation syncytiale). Mais plus intéressant, nous avons montré que le défaut de différenciation observé dans les cellules trisomiques est réversible par l’action d’une hCG recombinante fonctionnelle, entraînant la fusion et la différenciation des cytotrophoblastes issus de grossesses associées à une trisomie 21. L’ensemble de nos travaux montre que la diminution de l’expression du R-LH/CG associée à une hCG anormalement glycosylée et biologiquement moins active semble être impliquée dans le défaut de formation syncytiale et dans le recaptage de l’hormone par le placenta, permettant alors d’expliquer le paradoxe de l’hCG dans la trisomie 21.
Introduction:
Vascular endothelial growth factor A (VEGF-A) is one of the main pro-angiogenic factors regulating placental vasculogenesis and angiogenesis. Along with placental growth factor (PlGF), and soluble VEGF receptor 1 (sVEGFR1), it is produced and secreted by the syncytiotrophoblasts covering the surface of the placental villi bathing in the maternal blood circulating within the placental cavity.
Unfortunately, during in vitro cultures of primary human term cytotrophoblasts, we and others have been unable to detect VEGF-A secreted in the supernatants of these cells, while PlGF and sVEGFR1 were easily detected and quantified. One hypothesis for the ELISA not being able to detect VEGF-A is that VEGF-A is immediately and completely bound to its soluble receptor after secretion, and cannot be recognized by the antibodies used in the commercial ELISA kits.
We decided to verify this hypothesis by measuring VEGF-A expression during in vitro cultures of primary term cytotrophoblasts.
Methods: Primary term cytotrophoblasts were isolated and purified from term placentas obtained after cesarean delivery (n≥5). The cells were cultured for 4 days under 21% O2, but also under hypoxia (2.5% O2) to stimulate VEGF-A expression. VEGF-A transcripts were quantified by real-time PCR using a standard curve. VEGF-A protein was detected by western blotting in total protein extracts and in supernatants previously concentrated.
Results: When the cells were cultured under 21% O2, the copy number of VEGF-A transcripts decreased from 3.55x105 copies/µg total RNA (±0.54x105) on day 0 to 1.16x105 copies/µg total RNA (±0.37x105) on day 4. To the contrary, when the cells were cultured under hypoxic conditions (2.5% O2), there was a two-fold increase on day 1 (7.8x105 ±1.4x105), but then the copy number started to decrease to reach the same copy number measured on day 0. PlGF transcripts were also measured. As expected, PlGF copy number increased under 21% O2, while PlGF mRNA expression was inhibited by hypoxia.
Surprisingly, VEGF-A protein was detected by western blotting in all the total protein extracts tested. There were no differences in expression between extracts prepared from cells cultured under 21% O2 and cells cultured under 2.5% O2. Finally, no VEGF-A could be detected in concentrated supernatants.
Conclusion: Primary human term cytotrophoblasts do not secrete VEGF-A either under 21% O2 or 2.5% O2, while they are still able to synthesize and produce it at a basal level. Our results indicate that, at the term of pregnancy, cytotrophoblasts are not the source of VEGF-A, while they still produce and secrete PlGF and sVEGFR1 proteins.
The occurrence of breast cancer during pregnancy is a dramatic event reaching roughly 1/3000 to 1/10000 pregnancies, this type of cancer being the most frequent in pregnant women. Regarding therapeutic options, some anticancer agents may be used, especially taxanes (paclitaxel and docetaxel). If most of retrospective data appear to be reassuring, little is known regarding their transplacental transfer. Moreover, to our knowledge, potential effects of taxanes on human placenta, especially on placental transport function are unknown. Our aims were to 1) provide a transcriptional expression cartography of various placental drug transporters throughout pregnancy, using primary trophoblast culture model, 2) assess the comparative transplacental transfer of taxanes and their accumulation in cotyledons, using the perfused placental model, 3) assess potential effects of paclitaxel on human placenta, especially on drug transporter expression, not only using above-described models, but also cotyledons from pregnant-cancer patients treated with paclitaxel during pregnancy. Here, we finally provided an original transcriptional cartography of various drugs transporters in human normal placenta all along pregnancy. Moreover, we found a low and comparable transplacental transfer of paclitaxel and docetaxel that led to a moderate accumulation in cotyledons. Finally, we evidenced a significant effect of paclitaxel on human placenta, especially by modulating drug transporter expression.
The placenta produces a number of hormones that are not otherwise synthesized in the organism, suggesting that a distinct set of hormones are required to bring about the physiological changes of pregnancy rather than simply producing more of certain hormones. Therefore, placental hormones may also enter the maternal and fetal circulation, where they are often present at concentrations far in excess than what is found in the nonpregnant adult, to play a pivotal role in the maternal–fetal hormonal dialog. The placenta, decidua, amnion, and chorion actively participate in human reproductive physiology in allowing implantation, the regulation of fetal-placental blood flow, the growth of the conceptus throughout gestation, and in directing the appropriate signals for the timing of parturition. Some of the placental hormones are gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH) and somatostatin (SST). GnRH—a decapeptidic hormone—is produced by the hypothalamus that controls reproduction in vertebrates through the hypothalamic–pituitary–gonadal axis. GHRH and SST—hypothalamic releasing factors—are involved in the regulation of the complex process of growth hormone (GH) synthesis and secretion in the anterior pituitary. While GHRH functions as a specific stimulator of GH secretion, on the contrary, SST acts as the inhibitory counterpart.
Recent studies performed with null mice suggested a role of either RXRα or PPARγ in murine placental development. We report here that both PPARγ and RXRα are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPARγ (but not for PPARα or PPARδ) increase both human CGβ transcript levels and the secretion of human CG and its free β-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPARγ ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPARγ/RXRα heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CGβ gene (β5) were found to bind RXRα and PPARγ from human cytotrophoblast nuclear extracts, suggesting that PPARγ/RXRα heterodimers directly regulate human CGβ transcription. Altogether, these data show that PPARγ/RXRα heterodimers play an important role in human placental development.
Copper is essential micronutrient and has an important role in the human body. The serum copper increases during pregnancy and is doubled at full term. Lower levels of serum copper in pregnancy are connected with some pathological conditions.
The aim of this study was to estimate the levels of serum copper in normal and pathological pregnancies, comparing them with values of serum copper in non-pregnant women, to determine if serum copper is lower in some pathological pregnancies and if this is of some importance.
A total of 2170 plasma samples for copper analyses were made in the following groups: healthy non-pregnant women; healthy pregnant women from the 5th-40th gestational week, during the first delivery stage and during the first three postpartum weeks, in pregnant women with habitual abortion, imminent abortion, abortion in progress, missed abortion (9th-24th weeks), missed labour and premature rupture of membranes (29th-40th weeks). Levels of serum copper were determined by colorimetric technique of bathocuproin with disulphate as a chromogen.
Serum copper values in non-pregnant women range from 11.6-25.8 micromol/L. In healthy pregnant women, there is a constant trend of the increase of serum copper. The mean serum copper values revealed three significant peaks at the 22nd, 27th and 35th gestational week. Serum copper values in the patients with some pathological pregnancies in relation to the serum copper values of the healthy pregnant women were significantly lower.
Serum copper values can be used as an indicator of some pathological pregnancies.
An unavoidable consequence of aerobic respiration is the generation of reactive oxygen species (ROS). These may negatively impact development. Nevertheless, a certain amount of oxidative stress is required to allow for the normal progression of embryonic and fetal growth. Alterations in placental oxidative stress results in altered placental function and ultimately altered fetal growth and/or developmental programming leading to long-term consequences into adulthood. This article reviews the role of redox in fetal development and will focus on how developmental programming is influenced by the fetal and placental redox state as well as discuss potential therapeutic interventions.
During human placental development trophoblast follows two differentiation pathways: the extravillous (EVCT) and the villous cytotrophoblasts (VCT) that display different phenotypes and functions. It is well established that human chorionic gonadotropin hormone (hCG) is mainly secreted by the endocrine VCT (syncytiotrophoblast) into the maternal compartment and stimulates the formation of the syncytiotrophoblast (ST) in an autocrine manner. We recently reported that the invasive EVCT also produces hCG that promotes trophoblast invasion in vitro. Herein, we compared hCG gene expression in primary culture of villous and extravillous trophoblasts obtained from the same first trimester human chorionic villi and differentiated in vitro into ST and invasive EVCT, respectively. Total hCG, free alpha and free beta subunits were quantified in cell supernatants by immunometric assays and normalized to DNA content. alpha and beta transcript levels were quantified by Q-PCR and normalized to cytokeratin 7. We show that free alpha-, free beta-subunits and total hCG are differently expressed and secreted by the two trophoblast subtypes during their differentiation in vitro. We found an alpha/beta ratio 100 times lower in invasive EVCT in comparison to the ST suggesting that beta subunit may not be step limiting for hCG production in EVCT. Finally we investigated the regulation of hCG gene expression by PPARgamma, a nuclear receptor that controls trophoblast differentiation and invasion. Interestingly, activation of PPARgamma by the agonist rosiglitazone gave opposite results in the endocrine VCT and invasive EVCT: alpha and beta subunit transcript levels and protein secretions were up regulated in VCT, whereas they were down regulated in EVCT. Our results demonstrated that hCG gene expression is differentially regulated in the two trophoblast lineages during their in vitro differentiation and modulated in an opposite way by PPARgamma.
It has been known for more than 150 years that syncytial fusion is a normal feature in biological systems. In humans there are two larger syncytial tissues: skeletal muscles fibers and placental syncytiotrophoblast. Other fusion events take place as well from fertilization of the oocyte to infection of human cells by enveloped viruses (however, the latter does not necessarily lead to syncytium formation).
Although knowledge of the fusion process is incomplete, it is clear that membranes do not fuse easily; specific proteins and other factors are required and are selectively activated. In this chapter, we describe the classic proteins, such as the syncytins, assumed to be involved in the fusion process. We also describe other factors that may play roles in the fusion process or in the preparation of the cells to fuse, such as charged phospholipids, divalent cations, and intracellular proteases. Finally, we speculate on why trophoblast cells fuse in vitro and deal with in vitro models of trophoblast fusion and how their fusion rates can be quantified.
Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.
We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation
of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein
(Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation.
To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous
cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that
HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic
gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast
cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition
of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture
medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support
a direct role for Env-W in human trophoblast cell fusion and differentiation.
Oxidative stress has been implicated in a wide variety of diseases and degenerative states including cancer, rheumatoid arthritis, cardiovascular disease and ageing. There is now considerable evidence to suggest that pregnancy leads to the generation of an increased oxidative burden, but whether this overwhelms the anti-oxidant capacity within the placenta and/or the peripheral circulation remains a point of conjecture. There is little doubt that oxidative stress is a significant contributor in the pathogenesis of preeclampsia. The use of exogenous anti-oxidants such as vitamins C and E in the prevention of preeclampsia is the subject of several large clinical trials currently being conducted in many countries around the world. The results of these studies are eagerly awaited, but what of the endogenous anti-oxidant systems that have evolved to combat the oxidative burden associated with living in an aerobic environment? This review will focus on several important anti-oxidant enzyme systems, their role in pregnancy and the evidence to suggest that endogenous anti-oxidants are important in the development of complications of pregnancy such as preeclampsia.
The fatty liver Shionogi (FLS) mouse, a unique model for nonalcoholic fatty liver disease (NAFLD), is an inbred strain that develops spontaneous hepatic steatosis without obesity or diabetes mellitus. Peroxisome proliferator-activated receptor (PPAR) alpha controls fatty acid metabolism. In the present study, we investigated the effect of fenofibrate, a PPARalpha agonist, on hepatic steatosis in FLS mice.
Thirteen-week-old FLS mice were fed a diet with 0.1% fenofibrate (w/w) for 12 days. The degree of hepatic steatosis was estimated by histological examination and hepatic triglyceride levels. Expression levels of genes involved in fatty acid turnover, including Acox1, Cpt1a, Fabp1, Acadl, and Acadm, were determined by Northern blot analyses. We measured levels of lipid peroxidation, glutathione, and anti-oxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, in the liver.
Treatment of FLS mice with fenofibrate improved hepatic steatosis by activating expression of genes involved in fatty acid turnover and decreased hepatic lipid peroxidation. Fenofibrate increased the activity of catalase by upregulating its mRNA levels.
Fenofibrate, which is currently used in therapy of hyperlipidemia, might also be useful for treating patients with NAFLD even in cases where NAFLD is not associated with obesity or diabetes mellitus.
Placental development is markedly abnormal in women bearing a fetus with trisomy 21, with defective syncytiotrophoblast (ST) formation and function. The ST occurs from cytotrophoblast (CT) fusion and plays an essential role by secreting human chorionic gonadotropin (hCG), which is essential to placental development. In trisomy of chromosome 21 (T21) pregnancies, CTs do not fuse and differentiate properly into STs, leading to the secretion of an abnormal and weakly bioactive hCG. In this study we report for the first time, a marked decrease in the number of mature hCG receptor (LH/CG-R) molecules expressed at the surface of T21-affected CTs. The LH/CG-R seems to be functional based on sequencing that revealed no mutations or deletions and binding of recombinant hCG as well as endogenous hCG. We hypothesize that weakly bioactive hCG and lower LH/CG-R expression may be involved in the defect of ST formation. Interestingly, the defective ST formation is mimicked in normal CT cultures by using LH/CG-R small interfering RNA, which result in a lower hCG secretion. Furthermore, treatment of T21-affected CTs with recombinant hCG overcomes in vitro the T21 phenotype, allowing CTs to fuse and form a large ST. These results illustrate for the first time in trisomy 21 pathology, how abnormal endogenous hCG signaling impairs human placental development.
During pregnancy, the trophoblast develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. As the exchange of molecules through gap junctions is considered to play a role in the control of cell and tissue differentiation, the cell to cell diffusion of a fluorescent dye was investigated in human trophoblastic cells differentiating in culture. The fluorescence recovery after photobleaching technique was used to estimate the transfer of 6-carboxyfluorescein from contiguous cellular elements into photobleached cells. Fluorescence recovery follows a slow exponential time course when the cell to cell exchange process is rate limited by the presence of gap junctional channels between contiguous cells, contrasting with a much faster step-like course in the case of fusion of the plasma membranes. In the presence of 10% fetal calf serum, Percoll-purified cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 24-48 h after plating. During this in vitro differentiation, fluorescence recoveries after photobleaching with a time course typical for gap junctions were observed between aggregated cytotrophoblastic cells, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous syncytiotrophoblasts. The maximum percentage of gap junctional coupling occurs on the fourth day. This fluorescence recovery is attributed to the diffusion of dye through gap junctions, because it can be interrupted by exposure to a known junctional uncoupler (3 mM heptanol). The effects of hCG on this gap junctional communication during trophoblast differentiation were investigated. In the presence of 500 mIU/ml hCG in the culture medium, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days of culture vs. 4 days in control medium. Moreover, the diffusion rate constant k (the inverse value of the time constant measured on recovery curves) was also significantly increased in the presence of chorionic hormone. It is concluded that during trophoblast differentiation, the development of a cell to cell communication through gap junctions precedes the formation of a morphological syncytium by cell fusion. This gap junctional communication is promoted by hCG. Furthermore, our study confirms the differentiating role and the autocrine action of hCG in the physiology of the trophoblast.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.
Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.
The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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
Cu2Zn2-superoxide dismutase (CuZn-SOD) was purified from chicken liver. The liver enzyme had a subunit Mr of 16900 and contained equimolar amounts of copper and zinc [0.26% (w/w) for each]. Aortic CuZn-SOD had the same Mr as estimated by gel filtration and cross-reacted with antibodies to the liver enzyme. Both enzymes were inhibited by 1.0 mM-NaCN. Within 24-72 h after hatching, total SOD activity in aorta rose 3-fold over the day-1 level and stayed elevated for 10 days. With low dietary copper, the total SOD activity rose as before, but then decayed progressively to non-detectable levels in 10 days. Both the cyanide-sensitive (CuZn-SOD) and insensitive (mangano-SOD) activities fell, but not at the same rate. When the 10-day-old deficient chicks were injected with 0.5 mumol of CuSO4 intraperitoneally, SOD activity in aorta was restored to control levels in about 8 h. Despite non-measurable SOD activity in aorta, extracts from the 15-day-old-deficient-chick tissue contained as much, or slightly more, immunoreactive CuZn-SOD protein as age-matched control tissue. The data show clearly that dietary copper regulates SOD activity in the aortas of young developing animals. They further suggest that a copper deficiency suppresses CuZn-SOD activity without inhibiting synthesis or accumulation of the CuZn protein in this tissue.
Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.
During pregnancy, the trophoblast develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. As the exchange of molecules through gap junctions is considered to play a role in the control of cell and tissue differentiation, the cell to cell diffusion of a fluorescent dye was investigated in human trophoblastic cells differentiating in culture. The fluorescence recovery after photobleaching technique was used to estimate the transfer of 6-carboxyfluorescein from contiguous cellular elements into photobleached cells. Fluorescence recovery follows a slow exponential time course when the cell to cell exchange process is rate limited by the presence of gap junctional channels between contiguous cells, contrasting with a much faster step-like course in the case of fusion of the plasma membranes. In the presence of 10% fetal calf serum, Percoll-purified cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 24-48 h after plating. During this in vitro differentiation, fluorescence recoveries after photobleaching with a time course typical for gap junctions were observed between aggregated cytotrophoblastic cells, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous syncytiotrophoblasts. The maximum percentage of gap junctional coupling occurs on the fourth day. This fluorescence recovery is attributed to the diffusion of dye through gap junctions, because it can be interrupted by exposure to a known junctional uncoupler (3 mM heptanol). The effects of hCG on this gap junctional communication during trophoblast differentiation were investigated. In the presence of 500 mIU/ml hCG in the culture medium, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days of culture vs. 4 days in control medium. Moreover, the diffusion rate constant k (the inverse value of the time constant measured on recovery curves) was also significantly increased in the presence of chorionic hormone. It is concluded that during trophoblast differentiation, the development of a cell to cell communication through gap junctions precedes the formation of a morphological syncytium by cell fusion. This gap junctional communication is promoted by hCG. Furthermore, our study confirms the differentiating role and the autocrine action of hCG in the physiology of the trophoblast.
The -1389 to +588 region of the human genomic glutathione peroxidase gene (hgpx1) was amplified using the polymerase chain reaction. This DNA fragment was cloned and sequenced, and various deletion constructs derived from the hgpx1 5'-flanking region were fused to the chloramphenicol acetyltransferase gene. These reporter genes were analyzed in transient transfection assays using primary cultured human ventricular cardiomyocytes obtained from patients with tetralogy of Fallot. Two distinct regions upstream from the transcription start site, which was determined using S1 nuclease analysis, were identified to be responsive to the oxygen tension in culture (pO2 values of 150 or 40 mm Hg). Methylation interference footprinting assays revealed proteins closely apposed to two sequences located at -1232 to -1213 and -282 to -275. We have designated these oxygen responsive elements ORE1 and ORE2, respectively. Gel mobility shift assays using double-stranded oligonucleotides corresponding to each site have demonstrated the formation of specific complexes using both cultured human cardiomyocyte and HeLa nuclear extracts. ORE1 and ORE2 bind disparate proteins with equal precision as bound complexes could be competed away with identical sequences but not with either the other ORE or an unrelated sequence. Insertion of these oxygen responsive elements into a reporter gene governed by a SV40 promoter similarly regulated chloramphenicol acetyltransferase activity according to the oxygen tension in culture.
Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.
Human trophoblast cells offer a unique in vitro model for the study of aspects of the dynamic processes occurring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the addition of cyclic AMP analogs, 8-bromo-cyclic-AMP or Sp-8-bromo-cyclic AMPS, promotes syncytiotrophoblast formation, as shown by the disappearance of immunostained E-cadherin and desmoplakin, and increased numbers of nuclei per syncytium. An antagonist of cyclic AMP, Rp-8-bromo-cyclic AMPS, and an inhibitor of the cyclic AMP-dependent protein kinase catalytic subunit, H-89, impair cell fusion. This led us to study the pattern of expression and subcellular localization of cyclic-AMP-dependent protein kinase subunits during syncytium formation. Cytotrophoblasts expressed the RIalpha and RIIalpha regulatory subunits and the Calpha and Cbeta catalytic subunits. RIalpha was down-regulated during syncytium formation. No change in RIIalpha protein levels was observed, but there was a drastic subcellular redistribution. RIIalpha located in the Golgi-centrosomal area of cytotrophoblasts was scattered throughout the cytoplasm of the syncytiotrophoblast. Interestingly, an accumulation of RIIalpha was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. This suggests a key role of cyclic AMP-dependent protein kinase type IIalpha during cell fusion and microvilli formation, both of which are essential for the secretory and transfer functions of the syncytiotrophoblast.
Nearly all maternofetal and fetomaternal exchange takes place in the placental villi. There is only a limited contribution by the extraplacental membranes. In addition, most metabolic and endocrine activities of the placenta have been localized in the villi (for review see Gröschel-Stewart, 1981; Miller & Thiede, 1984; Knobil & Neill, 1993).
During trophoblast differentiation, a gap junctional intercellular communication (GJIC) precedes the trophoblastic cell fusion and the formation of the syncytiotrophoblast. The end of GJIC could thus be considered as an objective criterion of trophoblast differentiation. Maternal cortisol level increases during gestation and glucocorticoid receptors have been identified in trophoblastic cells. Therefore, the effects of dexamethasone on intercellular dye diffusion have been investigated by means of the Fluorescence Recovery After Photobleaching (gap-FRAP) technique in human trophoblast in culture. In parallel, trophoblast differentiation, hCG and hCS production were assessed. Dexamethasone stimulated the syncytium formation and significantly increased the percentage of coupled cells (2.5 fold at 0.5 μM). Simultaneously, hCG release was increased. After 2 days, hCS expression, was also increased by 35% compared to control. The glucocorticoid action was specific, since the stimulatory effects were blocked by RU486 (1 μM). Dexamethasone could act by the stimulation of hCG production or by the stimulation of GJIC and trophoblast differentiation. An excess of hCG antibody with dexamethasone did not significantly affect the stimulatory effect of the glucocorticoid, suggesting a self-sufficient effect of the steroid. In conclusion, dexamethasone stimulated intercellular communication, subsequent differentiation and concomitant endocrine functions of the trophoblast.
Human trophoblast cells offer a unique in vitro model to study aspects of the dynamic processes occuring during cell fusion and syncytium formation. In the human placenta, mononuclear cytotrophoblasts aggregate and fuse to form a multinucleated syncytiotrophoblast. In vitro, the presence of 8-bromo-cAMP promotes syncytiotrophoblast formation, while an inhibitor of the cAMP dependent protein kinase (PKA) catalytic subunit, H-89, impairs cell fusion. This led us to review the pattern of expression and the subcellular localization of PKA subunits during syncytium formation as well as the role of specific A-kinase anchoring proteins in cytotrophoblast differentiation. Cytotrophoblasts expressed the RIα and RIIα regulatory subunits and Cα and Cβ catalytic subunits. RIα was down-regulated during syncytium formation. No change in RIIα mRNA or protein expression was observed, but there was a drastic subcellular redistribution. RIIα, located in the centrosomal Golgi area of cytotrophoblasts, was scattered throughout the cytoplasm in syncytiotrophoblast. Interestingly, an accumulation of RIIα was observed underneath the apical membrane of syncytiotrophoblast in vitro and in situ. As shown by overlay assay, RIIα appeared to bind to two major binding proteins, one of them being ezrin, an actin-binding protein. This suggests a key role of PKA type IIα in the secretory and transfer functions of the syncytiotrophoblast, but also in membrane reorganization associated with cell fusion and microvilli formation.
Plasma copper and zinc levels were studied in 15 non-pregnant,176 normal pregnant, 19 pregnancy induced hypertension (PIH) and 7 eclamptic women in different trimesters of pregnancy and also in 89 pairs of cord and maternal blood samples. Plasma zinc levels showed a progressive decrease with copper levels showing an increase with increasing gestational age. Plasma copper and zinc levels were low in both PIH and eclampsia. While both maternal and cord plasma levels of copper showed a decrease, maternal plasma zinc levels showed an increase with increasing birth weight, with higher percentage of low birth weight (LBW) mothers having zinc levels below 50 μg/dl. The mechanism of high plasma zinc level in cord blood of LBW infants is not clearly understood.
The oxygen radical may induce peroxidation of unsaturated fatty acids in vivo. We studied changes in superoxide dismutase (SOD) activity and lipid peroxidation using human placental tissue. Lipid peroxidation was marked in early stages (2 to 4 months) of gestation but decreased with growth and was very small by the end of pregnancy. The SOD activity of placental tissue in early gestation was approximately 250 to 500 U per gram of wet weight. The activity increased with growth of the placenta and reached a level of 400 to 1,500 U per gram of wet weight by the end of gestation. These results for SOD activity suggest that the oxygen requirement in the placenta at early stages of gestation is low compared with that at the end of gestation.
The isolated human trophoblast was used as a system to analyze the effects of different physiological ligands on cellular uptake of copper. The results show that the uptake of copper by these cells follows a similar pattern for the ligands tested (histidine, albumin and ceruloplasmin) as that for copper chloride. The process follows a typical hyperbolic curve at 37 degrees C. The initial phase of uptake follows a linear pattern during 30 min at 37 degrees C and at least 60 min at 4 degrees C from which the uptake rate is calculated. However, a significant decrease in the uptake rate is observed for albumin. The effect of histidine on stimulating copper transport is observed in the presence of serum, a phenomenon which is considered to be due to the release of copper that is bound to albumin. These results support the role of ceruloplasmin as a copper transport protein which releases copper at the cell surface, and a subsequent transport of the released copper in a manner similar to that of copper chloride or copper-histidine complexes.
The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in vivo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependent on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation.
The effect of PTH on the epidermal growth factor (EGF) receptor was analyzed during the in vitro differentiation of human cytotrophoblasts. The cytotrophoblasts were isolated by a trypsin-DNase method from first trimester and term placentas and purified on a Percoll gradient. In culture, these cells aggregated and fused together to form a syncytium. This in vitro differentiation was associated with a 2-fold increase in 125I-EGF binding after 48 h of culture. The addition of 0.1 microM PTH (PTH-treated cells) to the culture medium induced a significant 2- to 3-fold increase (P less than 0.005) in EGF binding. The effect was dose related with a maximum obtained at a 1 nM concentration. Scatchard analyses revealed that PTH-treated cells possess a 2-fold higher number of high affinity sites as compared to control cells from early placenta (0.71 +/- 0.06 pmol/mg protein and 0.34 +/- 0.04 pmol/mg protein, respectively) and from term placenta (1.24 +/- 0.10 pmol/mg protein and 0.61 +/- 0.07 pmol/mg protein, respectively). The apparent Kd values for high affinity sites (0.15 nM) and for low affinity sites (4 nM) were not altered either by the gestational age of the cells or by PTH treatment. With respect to the EGF-dependent phosphorylation in membranes of trophoblast cells in culture, it was found that the phosphorylation of two major proteins of 175 kilodaltons and 35 kilodaltons, is greatly increased in PTH-treated cell membranes in the presence of EGF. This PTH-induced effect on EGF receptors was associated with an augmented functional response of trophoblastic cells to EGF. PTH increased the EGF-stimulated secretion of hCG. These results demonstrate that PTH increases the number of biologically active EGF receptors during the in vitro differentiation of human trophoblast cells. This PTH-induced effect suggests a role for this hormone in the regulation of the growth and the endocrine functions of these cells.
Substantial advances in our understanding of placental function have resulted from recent establishment of in vitro approaches, such as cell culture, and application of molecular methods to study placental steroidogenesis. Insight into the processes of placental cell differentiation and hormonal function has been gained from culture of relatively pure preparations of cytotrophoblast. Various factors, e.g. cAMP and peptide growth factors, have been shown to have striking effects on progesterone and estrogen formation by placental tissue under in vitro conditions. Using advanced molecular approaches, the genes governing specific enzymes critical to placental steroidogenesis have been identified. Regulation of the mRNAs encoding specific enzyme peptides and thus expression of the genes by factors, such as cAMP, have been elucidated by Northern analysis and other techniques. It is critical that these contemporary approaches continue to be implemented aggressively to further elucidate placental function. However, it is clear from a survey of the literature, particularly of the past decade, that the vast majority of investigation in the area has been conducted in vitro. It is essential to determine whether the factors that have been observed to regulate placental endocrine function in vitro are operable in vivo. It is only with in vivo study that the dynamics of steroidogenesis and the complex functional relationships between placenta, fetus, and mother will be uncovered and understood. It is increasingly evident that the regulation of placental steroidogenesis involves autocrine and/or paracrine mechanisms, similar to those integral to hormone biosynthesis within other reproductive organs, e.g. ovary and testis. For example, as discussed above, estrogen regulates LDL uptake and P-450scc, and thus apparently is involved in generating substrate for progesterone production within the placenta. Conversely, progesterone has effects on 17 beta-hydroxysteroid oxidoreductase and thus the metabolism of estradiol, while androgens exert marked inhibitory effects on placental progesterone formation, at least in vitro. Not surprisingly, the regulation of placental progesterone and estrogen formation also is multifactorial. Thus, aromatase activity is stimulated synergistically by cAMP and phorbol esters, an effect that is suppressed by peptide growth factors. Therefore, the autocrine/paracrine and multifactorial regulation of hormone biosynthesis that has been relatively well documented in other tissues should be recognized as important in the primate placenta. Finally, the basic mechanisms underlying regulation of steroidogenesis within the fetoplacental unit during primate pregnancy appear similar, in important ways, to those of widely used laboratory animals, such as the rat and rabbit.(ABSTRACT TRUNCATED AT 400 WORDS)
We utilized the Amicon micropartition system (MPS-1) to prepare ultrafiltrates of serum for evaluating non-protein-bound copper (NPBCu). The method is rapid (a usual turnaround time less than 2 h), practical (only 1 mL of serum is required), and reproducible (average CV = 5.7% within assay and 9.3% between assays). Using this technique, we determined that (a) NPBCu in serum appeared to be fairly stable, showing little variation among subjects in different physiological conditions (normal female, pregnancy, or fetal samples); and (b) of all copper fractions studied, only NPBCu appeared to correlate between maternal concentrations and cord counterparts at delivery. Thus the NPBCu fraction may be appreciably involved in the overall mechanism of copper nutritional supply from mother to fetus.
The concentration of lipoperoxides in maternal blood increases as gestation progresses. The concentration in pregnant women at 40 weeks gestation is 1.6 times higher than in nonpregnant women. The concentration in the cord blood, however, is 70% lower than that in maternal blood. To study the role of placental tissue in the difference in the lipoperoxide concentration between the cord blood and maternal blood, we investigated the lipoperoxide concentration, antioxidant activities and in vitro lipoperoxide formation in placental tissue during pregnancy. The lipoperoxide concentration was 50% lower in placental tissue of 40 weeks gestation than in tissue of 5-11 weeks gestation. Catalase and superoxide dismutase activities in placental tissues increased as gestation progressed, while glutathione peroxidase activity and alpha-tocopherol concentration did not change significantly during the gestational period. The in vitro formation of lipoperoxides in placental tissue decreased as gestation progressed. These results show that placental tissue suppresses lipoperoxide formation in the late gestational age, lowers the concentration of lipoperoxides in the blood and protects the fetus against oxygen toxicity.
Several libraries of monoclonal antibodies have been produced against epitopes that reside on hCG, alpha hCG, and beta hCG. Having characterized them physically, we explored their use in the construction of highly specific and sensitive immunoradiometric assays. There were several important immunochemical considerations with respect to developing assays that accurately detect low levels of free subunits in serum in the presence of high concentrations of the native hormone. These include physical properties and specificities of the monoclonal antibodies, choice of capture antibody on the solid phase support, assay design, and purity of hormone standards. Using such assays, we found early pregnancy (in vitro fertilization) to be characterized by the sequential appearance of hCG, followed by beta hCG and then alpha hCG. Molar ratios of beta hCG to alpha hCG and beta hCG to hCG were highest in early gestation. However, there was a reversal of the beta hCG to alpha hCG ratio at 12-13 weeks gestation, and an excess of free alpha hCG was observed thereafter. Except for values obtained in very early pregnancy, the beta hCG to hCG ratio remained remarkably constant at approximately 0.5% throughout gestation. In contrast, choriocarcinoma was distinguished by absolute serum beta hCG concentrations 3-100 times greater than the maximum values observed during pregnancy and, more importantly, by exceedingly high beta hCG to hCG ratios. For comparison, we studied hCG, alpha hCG, and beta hCG levels in an additional 178 patients with nontrophoblastic tumors. Ectopic production of alpha hCG and beta hCG was rare (3%), and thus far, we have been unable to demonstrate the presence of hCG in such patients. Therefore, hCG and the free subunits appear not to be useful as serological markers for nontrophoblastic tumors.
The susceptibility of purified protein kinase C to oxidative inactivation by H2O2 was found to be increased by Ca2+ either alone at a high (5 mM) concentration or at a low (approximately 50 microM) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca2+. This suggested that the membrane-bound and/or catalytically active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic domains of protein kinase C were susceptible to oxidative inactivation, a selective modification of the regulatory domain was obtained under mild oxidative conditions by protecting the catalytic site with ATP/Mg2+. Under these conditions there was a loss of both phorbol ester binding and Ca2+/phospholipid-stimulated kinase activity. However, this modified form of enzyme exhibited an increase in Ca2+/phospholipid-independent kinase activity. This suggests that selective oxidative modification of the regulatory domain may negate the requirement for Ca2+ and lipids for activation. Treatment of intact C6 glioma or B16 melanoma cells with H2O2 resulted in a time- and temperature-dependent decrease in Ca2+/phospholipid-dependent protein kinase C activity along with a concomitant transient increase in an oxidatively modified isoform of protein kinase C that exhibited activity in the absence of Ca2+ and phospholipids. Since protein kinase C can initially be activated by mild oxidative modification and subsequently inactivated by further oxidation, this dual activation-inactivation of protein kinase C in response to H2O2 suggests an effective on/off signal mechanism to influence cellular events.
Since the chronically cyanotic tetralogy of Fallot (TOF) myocardium is more sensitive to reperfusion injury after cardiac surgery than the adult myocardium, we decided to study the regulation of myocardial superoxide dismutase (SOD), catalase and glutathione peroxidase by oxygen tension. TOF myocytes were cultured at a Po2 of 150 mmHg for 30 days to establish the culture. The cells were then cultured at Po2 of 150 and 40 mmHg and the myocyte antioxidant enzymes measured at days 3, 7, 14 and 21. On day 21 the myocytes cultured at Po2 of 40 mmHg were then cultured at 150 mmHg and SOD and catalase activities measured on days 28 and 35. Although there were no differences in the rates of incorporation of 35S-methionine into the myocytes at either Po2 on these days, the myocytes scavenger enzyme levels were significantly higher by day 14 when cultured at a Po2 of 150 mmHg than at a Po2 of 40 mmHg. With the increase in oxygen tension from 40 to 150 mmHg, SOD and catalase activities increased significantly by day 35. The myocytes cultured at Po2 40 mmHg were more sensitive by day 7 to an hypoxanthine-xanthine oxidase generated free radical injury than the Po2 150 mmHg cultured cells. The regulation of these enzyme activities by oxygen tension and the increased sensitivity to free radical injury of the myocytes cultured at an oxygen tension of 40 mmHg provide putative evidence that the chronically cyanotic myocardium may be less well protected than the normally perfused myocardium against oxygen-mediated free radical injury and be at higher risk for cardiovascular surgery.
To study the effects of macrophage and lymphocyte-derived factors on superoxide anion (O2-) generation and release from human umbilical vein endothelial cells (EC), cultured EC were stimulated by ultrapure interleukin 1 (IL 1) and recombinant interferon-gamma (IFN-gamma), and the O2- released into the supernatant was measured. Both of these cytokines enhanced O2- release in a dose and time-dependent manner. Addition of a combination of IL 1 and IFN-gamma, each in submaximal concentration, produced an additive effect on O2- release. It would appear from these findings that cytokines released by macrophages and lymphocytes during inflammatory reactions can promote O2- generation and release from human EC. O2- released from EC may alter the basement membrane of blood vessels and the surrounding connective tissue, and in this way promote the vascular injury and angiogenesis associated with local inflammation.
Human trophoblast differentiates by the fusion of cytotrophoblasts to form syncytiotrophoblast. To determine factors controlling this process, the effects of epidermal growth factor (EGF) on trophoblast differentiation were studied using long term serum-free culture of isolated trophoblast. Only trophoblast was present in the cultures, as demonstrated by positive immunoperoxidase staining with beta hCG, cytokeratin, and trophoblast-specific H315 monoclonal antisera and by the absence of contaminating endothelial cells, fibroblasts, and macrophages, as shown by negative staining with vimentin and OKM1 monoclonal antisera. EGF induced large sustained increases in hCG and human placental lactogen (hPL) secretion in a dose-dependent manner. The minimum effective dose was 0.1 ng/mL, and the maximum effective dose was 1 ng/mL. Light and electron microscopic studies showed EGF-induced differentiation of cytotrophoblast to form syncytiotrophoblast. DNA content and cell number did not change during the process. The formation of syncytia thus probably accounted for the increase in hCG and hPL secretion. We conclude that EGF causes morphological differentiation, but not cell proliferation, of trophoblasts, and the differentiation results in increased hCG and hPL secretion from the syncytia.
We examined the distribution of copper among four components of human serum separated by chromatography on Sephadex G-150 and Affi-gel blue. Analysis of copper by furnace atomic absorption indicated that normal adults have copper at an average of 600 ng/ml associated with ceruloplasmin; at 120 ng/ml with transcuprein, a new copper transport protein; at 150 ng/ml with albumin; and at 90 ng/ml with one to three components of low molecular weight (less than 30,000). Cancer patients had more total copper but similar proportions in the four serum fractions. In both groups, some individuals had very high levels of copper in transcuprein, albumin, and/or one or more components of the low-molecular-weight fraction. The results showed that, contrary to earlier conclusions, ceruloplasmin copper only comprised about 60% of the total in human serum; and not just ceruloplasmin but also other forms of serum copper may be elevated in cancer patients.
A procedure is described and evaluated for isolating leucocytes from 15 ml of whole blood using a modified dextran sedimentation procedure. The mean recovery of leucocytes is 62% without differential white cell loss, and there is no contamination from red cells, platelets or plasma. Digestion of leucocytes with nitric acid is performed prior to the determination by atomic-absorption spectrophotometry of zinc using flame atomisation and of copper by electrothermal atomisation. The relative standard deviations for the determinations range between 2.2 and 3.6%. The range reported for zinc in leucocytes is 68–246 pmol per 106 cells and 4.1–36.8 per 106 cells for copper. These results are compared with those from other studies.
The effect of labor on maternal serum copper levels was determined in normal and complicated pregnancies. The mean value +/- SD (3.16 +/- 0.48 micrograms/ml) in 82 clinically normal subjects at term during labor was compared with that (2.22 +/- 0.49 micrograms/ml) obtained from 50 controls matched for gestational age who were not in labor. Similarly, the mean value in labor (3.56 +/- 0.46 micrograms/ml) in 25 subjects with a complicated pregnancy was compared with that (2.87 +/- 0.43 micrograms/ml) obtained from 25 similar subjects prior to labor. A statistically significant difference (P less than .001) was observed in both comparisons. Copper levels in the corresponding fetal serum from the subjects in labor (normal and complicated) were compared with those of the maternal serum samples. The mean value of fetal serum samples in mothers with complications was higher than that in normal mothers, but the difference was not statistically significant. This trend of a rise in serum copper level during labor was further confirmed by analysis of the same subject during and before labor in normal (12 subjects) and complicated pregnancies (9 subjects). Moreover, maternal serum estriol and estetrol levels were determined from the same samples in the 4 groups to find a possible relationship with the corresponding copper levels. No statistically significant correlation was noted. A possible explanation for the rise of the serum copper level with the onset of labor and its clinical implications are also discussed.
In this paper we have studied copper (Cu) uptake by microvillar vesicles isolated from human term placenta. We have characterised Cu uptake from CuHis2 complexes and shown that ceruloplasmin (Cp) inhibits uptake. Inhibition is complex and variable; in one series of experiments, the Vmax for uptake drops from 31.3 +/- 1.2 nmol/min per mg vesicle protein without added Cp to 11.3 +/- 1 nmol/min per mg vesicle protein at 91 micrograms/ml Cp. Similarly, the K0.5 increases from 0.35 +/- 0.08 microM to 1.35 +/- 0.25 microM, while the n value (the Hill coefficient) falls from 1.9 +/- 0.23 in the absence of Cp to 1.1 +/- 0.13 In another series, Cp had no effect below concentrations of about 100 micrograms/ml and in a third series only increased K0.5. The variability in effect seems to be related to the specific activity of the ceruloplasmin, which in turn is related to the copper complexes of the protein. The effect is specific for Cp; apotransferrin and a2-macroglobulin have no effect. 67Cu-labelled ceruloplasmin binds specifically to vesicles of term placenta with an affinity of 2.8 microU/mg vesicle protein and a Bmax of 79 microU/mg vesicle protein. CuHis2, but not histidine alone, can block the uptake. The data can be reconciled by proposing that the binding site of the transporter is relatively small and recognises a Cu-dihistidine structure common to the low-molecular-weight complex and to the Type I and Type II coppers of ceruloplasmin. We have used these observations to develop an isolation method for the transporter and have identified it as a protein of M(r) 90,000 which is closely associated with alkaline phosphatase. There are also two proteins of M(r) 45,000 and 40,000 which may be breakdown products of the larger complex. Antibodies to the 45,000 protein block Cu binding and uptake from CuHis2 complexes, strongly implicating it as the copper transporter/ceruloplasmin receptor of human term placenta.
This paper has examined copper uptake from CuHis2 complexes by cytotrophoblast cells isolated from term human placenta. Uptake is time-dependent, reaching equilibrium after about 90 min, and saturable, with a calculated apparent Km of 0.174 +/- 0.061 microM and Vmax, measured over 30 min, of 0.721 +/- 0.092 pmol/min/micrograms DNA. To determine whether ATP was required for uptake, cells were incubated with inhibitors of glycolysis (iodoacetate) and the TCA cycle (sodium azide and cyanide). Iodoacetate and sodium azide had no effect on uptake, but cyanide decreased the initial rate of uptake. This effect was due to copper binding to the inhibitor and decreasing the effective substrate concentration rather than inhibition of uptake through ATP depletion. Ouabain and monensin had no effect, showing that neither the Na+ gradient nor endocytosis were involved in uptake. The monovalent ion chelator, bathocuproine sulphonate, had no effect on uptake but buthionine sulfoximine, an inhibitor of glutathione synthesis, did decrease both the rate of uptake and equilibrium copper levels, suggesting that copper may bind to glutathione within the cell. The data show that copper is taken up by a passive carrier-mediated transporter and, following uptake, binds to glutathione within the cell.
Our laboratory previously demonstrated that cytotrophoblasts and syncytiotrophoblasts in human placental tissue contain hCG/LH receptors. From this finding, we postulated that one role of hCG might be to promote the differentiation of cytotrophoblasts into syncytiotrophoblasts. To test this postulate, cytotrophoblasts were isolated from human term pregnancy placentas and cultured with and without increasing concentrations of highly purified hCG. The results showed that hCG had a biphasic effect on 1) the aggregation of cells without intervening plasma membranes; 2) the expression of cadherin, a cell adhesion receptor that facilitates cellular aggregation; 3) the expression of hCG/LH receptor gene; and 4) the expression of three different hormonal markers of differentiation. The hCG effects were time and dose dependent and hormone specific. The addition of excess polyclonal hCG antibody, but not normal rabbit serum or nonspecific antirabbit immunoglobulin G, decreased basal responses as well as those to exogenous hCG. The polyclonal hCG/LH receptor antibody increased differentiation and dramatically stimulated hCG secretion in the presence or absence of exogenous hCG. (Bu)2cAMP mimicked the actions of hCG. H-89, a protein kinase-A inhibitor, decreased basal as well as exogenous hCG responses. Calphostin, a protein kinase-C inhibitor and lavendustin, a tyrosine kinase inhibitor, on the other hand, had no effect. In summary, it is novel that hCG made in human placenta can regulate the differentiation of cytotrophoblasts, which make little hCG, into syncytiotrophoblasts, which make considerable amounts of hCG.
The placental syncytiotrophoblast (ST) is a terminally differentiated epithelial cell monolayer that constitutes the outermost boundary between fetal and maternal tissues and performs a variety of synthetic, secretory, and transport functions essential for the maintenance of pregnancy. Although it is known that the ST arises from the underlying germinal layer of mononuclear cytotrophoblasts (Langhans' cells) by a process of cell fusion, the molecular mechanisms involved in this process are unclear. In order to address this question, we have investigated the effects of macrophage colony-stimulating factor (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphohemopoietic cytokines implicated in mammalian placental development, on the in vitro morphological and functional differentiation of human trophoblast. Both CSF-1 and GM-CSF stimulated cytotrophoblast aggregation into large multinucleated structures composed of extensive patches of syncytium interspersed with mononuclear cells. Concomitant with this morphological differentiation was upregulation of the production of the placental hormones placental lactogen and chorionic gonadotrophin. Placental fibroblasts derived from the villous stroma that underlies the trophoblastic epithelium were found to produce both GM-CSF and CSF-1 under the control of the trophoblast-derived cytokines IL-1 and TNF alpha. These observations suggest that a network of interrelated cytokines operates within the basal (fetal) aspects of the villous stroma where they are situated to play a significant role in the morphological and functional development of the human placenta.
We have examined the tissue uptake of 67Cu from ceruloplasmin versus albumin and transcuprein, after its intravenous administration to pregnant rats, in the last 4 days of gestation. 67Cu infused as in vivo-labeled ceruloplasmin remained on ceruloplasmin in the maternal circulation over the 4- to 6-hr time period examined, as determined by gel chromatography and immunoreactivity. That infused as in vitro-labeled serum was initially on transcuprein and albumin but soon also with new ceruloplasmin. On the basis of percent dose as well as total actual Cu transferred (taking into account the sizes of the two plasma Cu pools), ceruloplasmin was the preferred source of Cu for most tissues. Total uptake of Cu from ceruloplasmin was seven times greater than that from albumin and transcuprein for the placenta, whole fetus, and fetal liver. It was 2- to 6-fold greater for other tissues (except liver and kidney). When synthesis of maternal 67Cu-ceruloplasmin (from 67Cu administered on albumin and transcuprein) was inhibited with cycloheximide, uptake by nonhepatic tissues was reduced markedly. In the fetal circulation, entering 67Cu was initially associated with transcuprein and alpha-fetoprotein (or albumin), but then also appeared with ceruloplasmin. Specific receptors for ceruloplasmin were detected on membranes from the placenta as well as fetal liver; mRNA for ceruloplasmin was detected on the endoplasmic reticulum-bound polyribosomes of placenta/yolk sac, and of fetal and maternal liver. We conclude that Cu destined for the fetus is delivered mainly or exclusively by ceruloplasmin. It may enter via placental receptors, arriving in fetal plasma in ionic form, for later incorporation into fetal ceruloplasmin. The importance of ceruloplasmin as a source of plasma Cu for nonhepatic organs is also confirmed.
Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific 125I-EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high-affinity binding sites (0.33 +/- 0.04 and 0.63 +/- 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 x 10(-10) mol/L). Affinity labeling of 125I-EGF in cultured trophoblast cells followed by SDS-PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF-dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR-kinase activity during syncytium formation. Northern blot analysis of EGFR-mRNA, followed by hybridization with a 32P-cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up-regulation of EGFR expression with cell differentiation.
During human pregnancy, the trophoblast develops from differentiation of cytotrophoblast cells into an endocrine active syncytiotrophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form a syncytium, reproducing the in vivo process. In this study, we examined the effect of low oxygen tension (approximately 9%, hypoxia) compared to standard conditions (approximately 19% oxygen, normoxia) on these cellular events. Under hypoxia, syncytial formation was less frequently observed, cell staining and electron microscopy revealed that cytotrophoblasts remain aggregated, with a positive proliferative cell nuclear antigen (PCNA) immunostaining. Desmoplakin and E-cadherin, both known to disappear with cytotrophoblast fusion, showed persistent expression in hypoxic cells after 3 days of culture. In contrast, the expression of actin and ezrin, two cytoskeletal proteins, was unchanged. hCG secretion and hPL expression were both decreased in hypoxic cells, reflecting a reduced syncytial formation. Thus, on day 3, the mean values for hCG secretion were 1,100 +/- 155 and 289 +/- 26 mlU/mL in normoxic and hypoxic conditions, respectively. The reduced cell fusion process as well as hCG secretion and hPL expression under hypoxia were reversed by reoxygenation of the cells. We conclude that under hypoxia, the formation of functional syncytiotrophoblast is impaired due to a defect in the cytotrophoblast fusion process. This may explain the observation of a higher number of cytotrophoblast cells and a reduced syncytial layer in placentas of some pathological pregnancies.
Antioxidant enzyme activities in fibroblasts and erythrocytes prepared from normal and psoriatic patients were measured and compared. The most significant differences were noted in superoxide dismutase (SOD) activities. A dramatic (5.2-fold) increase in Mn-SOD activity along with a lesser (1.8-fold) increase in CuZn-SOD activity was observed in fibroblasts from lesional and nonlesional psoriatic skin. The increase of Mn-SOD activity was correlated with an increase of both protein and mRNA. A slight (1.2-fold) increase in CuZn-SOD activity was also found in psoriatic as compared to normal red blood cells, while Mn-SOD activity was not present in these cells. In contrast, both glutathione peroxidase and catalase activities were only slightly (1.3-fold) increased in psoriatic fibroblasts, with no appreciable change noted in psoriatic erythrocytes. Likewise, glutathione levels were observed to be similar in normal and psoriatic cells. The increases in SOD activities did not appear to correlate with the severity of the disease as expressed by the Psoriatic Area Severity Index score or with plasma inflammatory markers. These results demonstrate that antioxidant enzyme activities, particularly Mn-SOD in fibroblasts and CuZn-SOD in erythrocytes, are significantly elevated in cells from psoriatic patients.
Copper (Cu) placental transport presents a steep downhill gradient from mother to fetus. This process could be altered by low-molecular-weight (LMW) ligands and maternal Cu deficiency. We compared the ratio of Cu transfer from dam-to-fetus in Cu-deficient (CuDf) and Cu-sufficient (CuSf) rats in the last day of gestation. Anaesthetized dams were iv injected 79 mumol/kg (5 mg/kg) of either Cu acetate [Cu (AcO)2]; Cu+L-histidine, 1:10. [Cu(His)10]; Cu-(glycyl-glycyl-L-histidine) [Cu(GGH)], or saline. Dam and fetal blood, as well as placentae were obtained at 0, 10, 20, 40 and 60 min. At time 0, CuDf dams had lower plasma Cu than CuSf dams (8.3 +/- 1.2 versus 26.7 +/- 1.1 mumol/l), but CuDf fetuses plasma Cu was unchanged. This resulted in a more favourable mean fetal: maternal plasma Cu ratio in the CuDf fetuses (0.61) than in the CuSf fetuses (0.21). Dam plasma Cu was unaffected by the chemical form of Cu injected. In CuDf fetuses lower plasma Cu was observed with Cu(GGH) and Cu (His)10 at 20 min than in the CuSf. In the presence of these LMW ligands CuDf placentae retained more Cu than those of the CuSf group. CuDf was associated ultrastructurally with extensive lipid deposition in dam hepatocytes and, to a lesser extent, in CuDf fetal liver. These results indicate that in CuDf, LMW ligands increase placental uptake of Cu, without improving placenta-fetus transport. Although the rat fetus is well adapted to intrauterine CuDf, it may also be susceptible to hepatic lipid infiltration when the dam is CuDf.
We have previously demonstrated that epidermal growth factor (EGF), colony stimulating factor-1 (CSF-I), and granulocyte-monocyte colony stimulating factor (GMCSF) stimulate, while transforming growth factor beta 1 (TGF beta 1) inhibits, cytotrophoblast differentiation. To identify genes mediating EGF induced differentiation, we constructed a subtracted cDNA library between undifferentiated cytotrophoblast and differentiating cytotrophoblast. We identified six novel genes and four known syncytial products alpha-human chorionic gonadotrophin (alpha hCG) pregnancy-specific beta 1-glycoprotein, 3 beta-hydroxysteroid dehydrogenase, and plasminogen activator inhibitor type 1 whose mRNAs increased during differentiation. Ten other genes were identified whose mRNAs increased during differentiation. Five of these (keratin 19, calcreticulin, heat shock protein 27, serum and glucocorticoid-regulated kinase and adrenomedullin) were not previously reported to be expressed in placenta. Five other genes known to be expressed in placenta were identified. keratin 8, fibronectin, mitochondrial ATP synthase, 1119, and cytosolic copper-zinc superoxide dismutase (SOD-1). Several of these genes may have regulatory functions in trophoblast differentiation.
The response to hypoxia of trophoblast isolated from term placenta and maintained in culture was studied. Trophoblast exposed to normoxic (PO2 120-130 mmHg) or hypoxic (PO2 12-14 mmHg) conditions were examined by electron microscopy. After 48 h, the cytoplasm of the hypoxic cells was more electron-dense with increased numbers of mitochondria, lysosomes and vacuoles. Compared to normoxic cells, the surface microvilli of the hypoxic cells were sparse, short and unevenly distributed. [3H]thymidine incorporation by both hypoxic and normoxic trophoblast fell rapidly and equivalently after 2 days in culture. The percentage of cells with the proliferation-associated nuclear antigen, Ki 67, also decreased, but remained higher in hypoxic cells suggesting that hypoxia retarded completion of the cell cycle (normoxia, 10.80 +/- 2.51 s.e.; hypoxia, 19.87 +/- 2.73, P < 0.01). Glucose consumption was elevated in hypoxia (3.73 +/- 1.07 s.e. mumol/10(6) cells/24 h) as compared to normoxia (1.46 +/- 0.83, P = 0.01). Although lactate production was consistently higher in hypoxia, the difference was not statistically significant (hypoxia 5.38 +/- 1.54 mumol/10(6) cells/24 h versus normoxia, 1.52 +/- 0.29, P = 0.07). After 48 h, uptake of [3H]2-deoxglucose ([3H]2DG) by hypoxic cells was reduced to 12 per cent +/- 4.3 s.e. of that in normoxic cells; return to normoxia resulted in recovery within 10 min. Lineweaver-Burk plots of [3H]2DG uptake indicated high affinity (KM 2.2 +/- 0.4 x 10(-4) M) and low affinity transporters (KM 4.5 +/- 1.6 x 10(-3) M). Northern blot analysis identified mRNA for GLUT1 and GLUT3. In hypoxia, steady-state GLUT1 and GLUT3 mRNA were approximately three- and 10-fold higher than in normoxia respectively. Inhibitors of oxidative metabolism of glucose increased the uptake of [3H]2DG within 2 h, whereas hypoxia reduced uptake. Hence, trophoblast in culture survives in extreme hypoxia, but manifests striking changes in morphology and in glucose metabolism and transport. Completion of cell cycle appears to be retarded.
This study investigated expression of the key antioxidant enzyme copper/zinc superoxide dismutase in the villous trophoblast of the human placenta at different gestational ages from 8 weeks (last menstrual period) to term. Immunostaining for the enzyme was observed in the cytotrophoblast cells at all stages. Staining was generally absent from the syncytiotrophoblast at 8 weeks, except for small isolated areas close to the basal surface. The size and location of these areas suggested they were the result of recent cytotrophoblastic fusion. By 10 weeks, examples were more frequent and diffuse staining throughout most of the syncytiotrophoblast was observed at 12 weeks. The intensity of the immunostaining within the syncytiotrophoblast continued to increase until 14 weeks, by which time it matched generally that within the cytotrophoblast cells. A similar pattern of staining was observed within term material. These results are entirely consistent with the hypothesis that the oxygen tension within the intervillous space is low throughout the first trimester of pregnancy. They support the idea that an effective maternal circulation to the human placenta is only established at the start of the second trimester.