Human Term Placental Tissue Research Articles

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue.

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.

Localization of type 1 17beta-hydroxysteroid dehydrogenase mRNA and protein in syncytiotrophoblasts and invasive cytotrophoblasts in the human term villi.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.

Secretory characteristics and viability of human term placental tissue after overnight cold preservation.

Collection of human term placentae for research purposes is generally limited during working hours. Preserving placental tissue overnight might help to postpone experiments and, by extent, to increase material availability. In this study, fragments from normal placentae were incubated at 37 degrees C either immediately after delivery or after preservation at 4 degrees C in a HEPES-buffered solution or in a Roswell Park Memorial Institute (RPMI) 1640 culture medium. Protein, human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and lactate dehydrogenase (LDH) contents within preserved explants were similar to those within freshly delivered ones. In contrast, HCG and HPL amounts released during incubation of preserved tissue were lower than with freshly delivered tissue. Differences were significant only during the first 3 h of incubation. Hormone releases were similarly Ca(2+)-stimulated, and Co(2+)- and low temperature-inhibited in preserved and freshly delivered tissues. After preservation, LDH leakage was also reduced. Furthermore, before and after 37 degrees C incubation during 6 h, preserved tissue was morphologically indistinguishable from freshly delivered tissue and showed neither higher incidence of DNA fragmentation, nor elevated caspase-3 activity, both of which are markers of apoptosis. This study validates an original, useful and rapid method to preserve placental tissue. Consequently, this preservation model may facilitate the study of physiological processes regulating placental hormone secretion in normal and pathological conditions.

The Effect of Dexamethasone on CRH and Prostanoid Production from Human Term Placenta

The human placenta at term produces large quantities of corticotropin releasing hormone (CRH) and prostanoids. These hormones play an important role in the maintenance of pregnancy, and the initiation and progress of labor; yet little is known of factors affecting their regulation and the interrelationship of CRH and prostanoid production. In these studies we have investigated the effect of dexamethasone on the production of CRH and prostanoids from fresh human term placental tissues. The basal release of prostaglandin E 2 (PGE 2), prostaglandin F 2α (PGF 2α), thromboxane B 2 (TxB 2) and 6-keto-prostaglandin F 1α (6-keto-PGF 1α) from human term placental explants increased from the fifth hour in culture, while the release of 13,14-dihydro-15-keto-PGE 2α (PGFM) was not significantly changed during this period. The addition of dexamethasone (10 −8 M) to the perifusing medium resulted in a rapid and dramatic inhibition of PGE 2, PGF 2α, PGFM, TxB 2 and 6-keto-PGF 1α release. On the other hand, CRH release was not significantly changed throughout the seven hours of incubation with dexamethasone. These data demonstrate that glucocorticoids at physiologic concentrations can inhibit human term placental prostanoid production, and thus glucocorticoid production may play an important role in the physiological regulation of placental prostanoid production in the human placenta. However, dexamethasone did not alter CRH release, demonstrating that the inhibition of placental prostanoids by dexamethasone is not a CRH mediated event.

Biochemical and biological characterization of a crude growth factor extract (EAP) from human term-placental tissue

A crude extract (EAP) is prepared on an industrial scale from human term placental tissue by acid extraction and alcohol fractionation. The major proteins [(gammaglobulins (11%), transferrin (3%), albumin (6%), peptides <20 kDa (65%)] of this extract were characterized by polyacrylamide gel electrophoresis. The concentrations of the different known proteins, particularly plasma proteins, lipoproteins, transport, and adhesion proteins, were determined by immunodiffusion. The lipids were identified by thin-layer chromatography. The extracted lipids were essentially composed of phospholipids (56%), free fatty acids (12%), and free cholesterol (29%). The steroid hormones were determined by RIA titrations and only low levels (under ng) could be detected. The EAP was mainly manufactured to get a growth factor extract. Its content in growth factors was estimated by specific techniques designed for each factor. The lowest levels were detected for IGF-2 (15–140 ng/ml), while EGF/TGF-alpha and bFGF concentrations were in the same range, i.e., 100–250 ng/ml. The highest levels were detected for IGF-1 (350–1300 ng/ml) and especially TGFβ (0.9–4.8 μg/ml). The growth promoting capacities of EAP were determined by cell culture assay. EAP stimulates proliferation, of various cells (MRC5 and various fibroblasts, corneal endothelial cells). The ED5O (half maximal stimulation) has been found to be in the range of 100–200 μg/ml. These results were obtained on three different pilot batches of EAP which ensure the reproducibility of the results. The growth factor content of this extract is of some interest in different fields as purification of isolated growth factors, serum replacement, and as a potentially useful pharmacological agent in wound healing.

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.

Expression of the Pregnancy-Specific β1-Glycoprotein Gene in Cultured Human Trophoblasts

Human pregnancy-specific beta 1-glycoprotein (PS beta G), the major placental glycoprotein, shares strong sequence similarity with carcinoembryonic antigen and is a member of the immunoglobulin superfamily. To understand the role of PS beta G in placental ontogeny during pregnancy, we examined its synthesis and regulation in primary cultures of trophoblast cells. Freshly plated (12 h) cytotrophoblasts expressed little PS beta G transcripts; however, within 24 h of culture, PS beta G mRNAs became detectable. PS beta G synthesis and mRNA expression increased with time in culture, and maximal synthesis was achieved at 4 days, indicating that primary trophoblasts continue differentiating in vitro. Molecular cloning revealed that PS beta G is an extremely polymorphic protein. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity in the 5' (designated PSG-5') and coding regions, but differ in sequences at the 3' region. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three mRNAs of 2.3, 2.2, and 1.7 kilobases in primary trophoblasts and human term placental tissue. Ribonuclease protection analysis demonstrated that primary trophoblasts expressed most of the placental PS beta G transcripts. However, culturing in vitro altered PS beta G gene expression, and the level of PS beta G transcripts containing a PSG95-3' sequence was preferentially increased in primary trophoblasts. Moreover, primary trophoblasts synthesized a 64K PS beta G polypeptide in variable amounts and three PS beta Gs of 72K, 62K, and 54K in roughly equal amounts, whereas purified human term placental PS beta G consists of a major polypeptide of 72K and two minor ones of 64K and 54K. PS beta G gene expression in primary trophoblasts was slightly reduced by 8-bromo-cAMP, but was markedly inhibited by sodium butyrate.

Effects of Thromboxane Synthase Inhibition on Placental Prostacyclin and Thromboxane Production

Selective inhibition of thromboxane synthesis without affecting prostacyclin synthesis is being considered as a potential treatment for preeclampsia because of its imbalance of increased thromboxane/decreased prostacyclin production. Thromboxane production by the placenta is greatly increased in preeclampsia so effective therapy must inhibit placental, as well as maternal platelet, thromboxane production. The following study was done to evaluate the production rates of thromboxane and prostacyclin in placental tissues incubated with and without the throraboxane synthase inhibitor, U-63,557A. Fresh, human term placental tissues (350 mg) were incubated in a sterile manner in 7 ml Dulbecco's Modified Eagle's Medium for 48 hours at 37°C with 95% oxygen and 5% carbon dioxide in a metabolic shaker. Samples were collected at 0, 8, 20, 32 and 48 hours and analyzed for thromboxane A2 by radioimmunoassay of its stable metabolite, thromboxane B2, and for prostacyclin by radioimmunoassay of its stable metabolite, 6-k...

Developmental study of human fetal placental fibronectin: Alterations in carbohydrates of tissue fibronectin during gestation

Human term placental tissue fibronectin contains about twice the carbohydrate content of human adult plasma fibronectin or fetal plasma fibronectin. The chief difference is the presence of substantial amounts of large N-linked polylactosamine chains on the placental fibronectin. The large carbohydrate on placental tissue fibronectin weakens the binding of fibronectin to denatured collagen. To examine whether a developmental change takes place in the placental fibronectin during gestation, fibronectin was isolated from placentas of different developmental stages beginning with the first trimester and ending with term. Polylactosamine carbohydrate, as well as total quantity of carbohydrate, increased steadily during gestation, reaching a maximum at term of more than 9% carbohydrate. Weakened binding of fibronectin to collagen occurred near the end of gestation concomitant with an increase in the quantity of larger polylactosamine glycopeptides. Relationships among these developmental changes, the impending birth, and the end of the function of the placenta remain to be investigated.

Alkaline phosphatase and protein kinase(s) activities in free cytoplasmic mRNPs from human term placenta.

Free mRNPs isolated from human term placental tissue were examined for protein kinase and phosphoprotein-phosphatase activities. Free mRNPs incubated with [gamma-32P]ATP in a protein kinase standard buffer show self-phosphorylation in the absence of exogenous substrates. Treatment of phosphorylated products with alkali showed a significant phosphorylation of tyrosine residues within the mRNP-proteins. An alkaline-phosphatase activity was found to be tightly associated with the mRNPs. Both heat stable and heat labile alkaline phosphatase activities were found in the mRNPs. Heat labile alkaline phosphatase is the major isoenzyme form of the mRNPs. The existence of both protein kinase(s) and alkaline phosphatase activities in placental free cytoplasmic mRNPs might suggest that a balance between phosphorylation, specifically on tyrosine residues, and dephosphorylation states of some of the mRNP-proteins is relevant for their physiological functions, and may therefore play a role in the regulation of mRNPs' metabolism and, consequently, in mRNA translation.

Immunoreactive oxytocin synthesis in human placental tissue.

The change in the amount of immunoreactive oxytocin in human term placental tissue due to cycloheximide, an inhibitory agent of protein biosynthesis, was observed by a tissue culture method. The immunoreactive oxytocin content per gram tissue was 1.81 ng/g and significantly decreased to 1.17-1.11 ng/g when the concentrations of cycloheximide were at 1-10 micrograms/ml in culture medium. The total quantity of immunoreactive oxytocin including medium content was 1.82 ng/g and significantly increased to 3.06 ng/g in the control group. However, those of the groups in which added cycloheximide was at 1-10 micrograms/ml, were 1.91-1.85 ng/g and showed no remarkable changes after incubation. The data suggest that immunoreactive placental oxytocin can be synthesized in the human placenta itself rather than in other tissues.

Uterine contractile effect of an oxytocin-like substance in the human placenta.

AbstractsThe bioactivity of an oxytocin‐like substance in human term placental tissue was examined using rat uterine muscle obtained during diestrus.The human term placental tissue extract contracted the rat uterine muscle on a Magnus apparatus. Placental tissue extract incubated with oxytocin antiserum did not induce significant contraction of the muscle, whereas placental tissue extract incubated with normal rabbit serum induced a significant effect on the rat uterine muscle. Synthetic oxytocin caused contraction similar to that caused by placental tissue extract in rat uterine muscle. This contraction was also inhibited when synthetic oxytocin was incubated with anti‐oxytocin serum for 24 hr at 4°. Normal rabbit serum and anti‐oxytocin serum alone did not cause contraction. These results suggest that the oxytocin‐like substance in the human placenta may possess uterine contractile activity.

The synthesis and secretion of human placental lactogen (hPL) in cultured term placenta

We studied the in vitro synthesis and secretion of hPL by human term placental tissue incubated in organ culture. Placental tissue maintains a constant pool of hPL. The synthesis of hPL may be the driving force for its secretion. The de novo synthesis and secretion rates of hPL were also investigated. The higher specific radioactivities of the secreted hPL than those found in the tissue may suggest that newly synthesized hPL is preferentially released. The intracellular distribution of hPL was also compared.

Microdetermination of adrenocortical steroids by double isotope method. IV. Determination of corticosteroids in human placenta.

Homogenate of the human term placenta was extracted consecutively with ethanol and methanol, and the extract was fractionated with ether-water system into free and conjugated corticosteroids. Fractions obtained by enzymic hydrolysis of conjugates and free fraction were submitted to reverse isotope dilution analysis and the kinds of corticosteroids present in each fraction were identified. Amount of these corticosteroids was determined by the double isotope derivative dilution method in which the carbonyl group in C-3 position alone is derived to thiosemicarbazone-35S. Presence of the following corticosteroids was proved per 1 g (wet weight) of human term placental tissue : 0.136-1.176μg of free and 0.014-0.099 μg of conjugated 11-deoxycorticosterone, 0.040-0.668 μg of free and 0.079-0.273 μg of conjugated 11-dehydrocorticosterone, 0.026-0.078 μg of free and 0.023-0.037 μg of conjugated corticosterone, 0.016-0.050 μg of free and 0.014-0.031 μg of conjugated cortisone, 0.004-0.045 μg of free and 0.009-0.015 μg of conjugated cortisol, and 0.014 μg of free and 0.011 μg of conjugated aldosterone, with a larger amount than the above of progesterone (0.731-2.370 μg of free and 0.235-0.964 μg of conjugated). The use of the present method of determination allows separatory determination of free and conjugated corticosteroids, using about one-half of the placenta (250-280g). Quantitative distribution of corticosteroids in three placenta analyzed here showed a marked individual difference but in all the placental tissues analyzed, the content of 11-deoxycorticosterone and 11-dehydrocorticosterone was higher than that of the three kinds of 11-oxo-17α-hydroxycorticosteroid.

The amine oxidases of human placenta and pregnancy plasma.

1. The purification of monoamine oxidase and diamine oxidase from normal human term placental tissue is described. 2. The properties of these enzymes are reported and compared with the properties of unpurified human pregnancy plasma. 3. This comparison shows that the amine oxidase of pregnancy plasma has properties corresponding to purified placental diamine oxidase, suggesting a placental origin for the plasma enzyme system. 4. Detailed kinetic study of the purified placental diamine oxidase suggests that it has a Ping Pong sequence, a mechanism of action and rate-limiting step similar to the diamine oxidase of pig kidney. 5. It is suggested that the enzyme system is important in protecting the foeto-placental unit from excesses of biogenic amines.