Intact Placenta Research Articles

Equine retained placenta: Technique for and tolerance to umbilical artery injections of collagenase

Under laboratory conditions and in clinical experiments, bacterial collagenase has proven to be effective in hydrolyzing placenta and detaching cotyledon from caruncle in the bovine species. Laboratory studies in which placental samples were incubated with collagenase have also demonstrated that collagenase is 3.7 times more effective in hydrolyzing equine placenta than bovine placenta. This led to the hypothesis that collagenase may be a potential treatment for mares with retained placenta. However, that collagenase may hydrolyze the uterine wall and perforate the uterus was a concern. It was the purpose of this study thus to determine any adverse effects of collagenase on the equine uterus and to develop a method for intraplacental injection of collagenase. Three normally expelled intact placentas from Arabian mares, 10 cyclic mixed-breed mares, and 4 mares of various breeds with retained placenta were used. Fluoroscein dye and latex were used to study the placental vasculature and to determine a suitable dose of collagenase; placentas were hydrolyzed by collagenase solution in vitro. Bacterial collagenase solution (40,000 units, 200 ml) was infused into the uterine lumen of each cyclic mare. Uterine biopsies were obtained from the mares before collagenase infusion and again at 16 h and 26 d after infusion. In the mares with retained placenta, each placenta was infused via its umbilical cord vessels with 200,000 units of bacterial collagenase in 1 L of saline. Results showed that none of the uteri from cyclic mares were damaged by collagenase treatment. During a 4-wk period of monitoring (including endoscopy) mares with retained placenta did not show any abnormalities. Retained placentas were expelled in less than 6 h after collagenase treatment. It was concluded that intraplacental injections of collagenase are a safe and potentially effective treatment for retained placenta in mares.

Two molecular species of oxytocinase (L-cystine aminopeptidase) in human placenta: purification and characterization.

Two different forms of oxytocinase (L-cystine aminopeptidase, CAP; EC 3.4.11.3) were purified from the 9000 g and 105000 g precipitate fractions of human placenta homogenate by sequential chromatography on columns of hydroxyapatite, DE-32, nickel ion affinity, and Sephadex G-200. One species (CAP-I) purifed from the mitochondrial/lysosomal fraction migrated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with an apparent molecular mass of 61 kDa; the other (CAP-II) from the microsomal fraction was composed of two subunits with molecular masses of 56 and 40 kDa. The molecular masses of CAP-I and CAP-II estimated by gel filtration were 64 and 97 kDa, respectively. The specific activities of the two species for S-benzyl-L-cysteine p-nitroanilide increased by 357- (for CAP-I) and 139-fold (for CAP-II) compared with the starting preparations. The optimal pH values toward the artificial substrate were approx. 7.4-8.0 for CAP-I and 6.8-8.0 for CAP-II. The Km and Vmax values toward oxytocin were 5.6 microM and 23.4 micromol/h/mg protein for CAP-I, and 38 microM and 15.6 micromol/h/mg protein for CAP-II. Both enzymes were inhibited by the metal-chelating agents, EDTA and o-phenanthroline, whereas they were specifically activated by addition of Co2+: CAP-I was more sensitive to these reagents than CAP-II. L-Methionine strongly inhibited CAP-I, while CAP-II activity was only slightly affected. CAP-II was more sensitive to amastatin than CAP-I. Thus, the two enzymes are quite distinct in their molecular nature and biochemical properties. They may play a regulatory role in the metabolism of oxytocin and other biologically active peptides in intact placenta.

Transfer of Cl- across placenta of anesthetized rat.

The mechanisms of Cl- transfer across the rat placenta have been investigated. Clearance across the intact placenta from mother to fetus (m-->f) of 51Cr-EDTA (paracellular diffusion marker) and 36Cl- (Kmf) was 1.9 +/- 0.1 and 37.3 +/- 4.1 microliters/min, respectively (mean +/- SE, n = 10), the large difference indicating that most m-->f transfer of Cl- is transcellular. The clearance of 36Cl- across the dually perfused placenta in m-->f and fetal-to-maternal directions was symmetrical and highly sensitive to the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM). The Kmf of 36Cl- was not inhibited by anoxia and had a low temperature quotient (Q10 between 32 and 37 degrees C was 1.52). The m --> f transfer of Cl- seemed to be fully saturated at physiological concentrations of Cl-. 36Cl- could be displaced from the transporter on the maternal side by other anions with the following order of affinity: Cl- approximately NO3- > Br- > lactate- >> gluconate. It is concluded that most of the Cl- transfer across the rat placenta is effected by an anion exchanger.

Interconversion of cortisol and cortisone in the baboon placenta at midgestation: expression of 11beta-hydroxysteroid dehydrogenase type 1 messenger RNA.

At midgestation in the baboon, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalysed interconversion of cortisol (F) and cortisone (E) during transuterine passage favors the formation of F. Because the site(s) of oxidation/reduction of F and E are not clear, the present study compared F-E interconversion in placenta, decidua and chorion in vitro. In addition, because the reduction of E to F is catalysed only by the 11beta-HSD-1, we also determined whether the mRNA for this enzyme was expressed in baboon placenta, including placental syncytiotrophoblast cells; the site of fetal-maternal exchange. Placentas were obtained on day 100 of gestation (term = day 184) and villous tissue, decidua and chorion isolated, minced in HBSS and incubated (300-400 mg) in duplicate for 0.1-24 h in Medium 199 containing 10% fetal bovine serum and [3H]F and [14C]E. Radiolabelled F and E were purified from incubates and the percentage conversion of F to E and E to F was calculated. In decidua, mean (+/- SE; n = 3) conversion of E to F (69 +/- 2%) was greater than oxidation of F to E (26 +/- 2%). Conversion of E to F in placenta (50 +/- 1%) and chorion (39 +/- 9%) was also extensive and greater than or equal to that for the oxidation of F to E (39 +/- 4% and 32 +/- 4%, respectively). The apparent ratio of 11beta-HSD reductive/oxidative activity was maintained when respective tissues from four baboons were incubated for 18 h with or without 4.4 microM excess radioinert substrate. Expression of 11beta-HSD-1 mRNA was determined by Northern blot by hybridization of poly (A) + -enriched RNA from tissues obtained at midgestation with [32P]labelled human 11beta-HSD-1 cDNA. This cDNA hybridized to a single mRNA species of 1.6 kb in decidua, whole placental villous tissue and syncytiotrophoblast cells, but not to RNA isolated from fetal baboon kidney. The results of the present study demonstrate that at midgestation in the baboon, 11beta-HSD activity in intact placenta and decidua in vitro favored the formation of F from E, presumably catalysed by the 11beta-HSD-1 enzyme protein, the mRNA for which is present in placental syncytiotrophoblasts. Moreover, based on overall mass and extensive vascularity, we suggest that the net formation of F from E during transuterine passage in vivo at this stage of gestation results from the 11beta-HSD-1 in the syncytiotrophoblast cells of the baboon placenta.

Malignant Potential, Major Histocompatibility Complex Antigen Expression, and Genomic Imprinting of Rat Trophoblast Cell Lines1

Invasive growth, variation in major histocompatibility complex antigen expression, and genomic imprinting are important properties of both trophoblast cells and malignant tumors. This study, undertaken to address these three issues, used cultured trophoblast cell lines derived from Day 11/12 rat placentas of all mating combinations of the DA and WF inbred strains. In addition, genomic imprinting was also examined in intact rat placentas from Days 11-19. There was no correlation in trophoblast cells between class I antigen expression, DNA content, and cell ploidy on the one hand and oncogenic potential on the other hand. The constitutive suppression of class II antigens in the trophoblast cells could not be abrogated by treatment with interferon-gamma, whereas such treatment always maximally induced class I antigen expression regardless of the initial resting levels. The trophoblast cells at Day 11/12 expressed both maternal and paternal class I antigens, and studies in whole placental tissues showed that the imprinting of the maternal class I antigens was manifested by a decreased level of expression rather than an absence of expression. Thus, genomic imprinting in the rat placenta is a quantitative, rather than an all-or-none, phenomenon.

A chloride channel from human placenta reconstituted into giant liposomes

OBJECTIVE: Ion channels play important roles in epithelial transport, but they are difficult to access for conventional electrophysiologic studies in intact placenta. The purpose of this work was to explore the suitability of purified trophoblast plasma membrane as a source of ion channels for reconstitution in artificial lipid membranes. STUDY DESIGN: Human placental brush border membranes were purified by differential and gradient centrifugation and fused with small liposomes. Giant liposomes were then generated by a cycle of dehydration and rehydration. These giant liposomes are suitable for electrophysiologic studies and were probed for the presence of active ion channels by the patch-clamp method. RESULTS: The results reported here indicate the presence of a high conductance chloride channel showing some similarities with “maxi” chloride channels described in secreting and absorbing epithelia. The channel had a slight outward rectification with conductances of 232 and 300 pS at negative and positive potentials, respectively. CONCLUSIONS: For the first time successful reconstitution of a human placental ion channel is achieved in a system suited for electrophysiologic studies. The chloride channel described might play a role in transplacental transport.

In Vitro Transformation of Rabbit Cytotrophoblast Cells into Syncytiotrophoblast: Stimulation of Hormone Secretion by Progesterone and Dibutyryl Cyclic 3′,5′-Adenosine Monophosphate1

Rabbit placenta syncytiotrophoblast cells exhibit immunostaining for the hormone relaxin. The objectives of this study were to evaluate the ability of cultured rabbit trophoblast cells to secrete immunoreactive (IR) relaxin and then to study the effects of progesterone, which is essential for maintenance of the placenta and of pregnancy in the rabbit, on that secretion. On Day 1, both treated and untreated trophoblast cell cultures consisted of 90% relaxin-negative mononuclear cells (cytotrophoblast and fibroblast) and 10% relaxin-positive multinuclear cells (syncytiotrophoblast). Media from untreated cultures, collected throughout 9 days of culture, contained low but constant levels of relaxin. Electron microscopy studies indicated that relaxin was localized in dense granules of the multinuclear cells and that these cells formed by fusion of mononuclear cytotrophoblast. Progesterone treatment (40 and 80 ng/ml) increased (p < 0.0001) media concentrations of relaxin, increased the number of desmosomes between cytotrophoblast cells (12 vs. 4 for controls on Day 5), and increased the percentage of multinuclear cells (73% of the cell population vs. 20% for controls on Day 7). Specificity of the progesterone effect was evaluated by treatment of cultures with dibutyryl cAMP (dbcAMP), insulin, hCG, estradiol-17 beta, prostaglandin E2, and prostaglandin F2 alpha. Only dbcAMP (2 mM and 4 mM) produced an increase (p < 0.0001) in media concentrations of relaxin. These results indicate that, like the intact placenta, cultured cytotrophoblast cells fuse to form syncytiotrophoblast and that the latter contain IR relaxin.

Mechanisms of the fetomaternal transfer of Na + across the dually perfused placenta of the rat

In order to investigate mechanisms of fetal-maternal (F-M) transfer of Na +, clearance of 22Na + and 51Cr-EDTA was measured simultaneously across the dually perfused placenta of the rat. In eight experiments clearance was measured successively in the F-M (K fm ) and in the maternal fetal (M-F; K mf ) directions. Clearance of 22Na + in the two directions was approximately equal (K mf = 11.6 ± 2.0 μl/ min; K fm = 11.1 ± 1.7 μl/min: mean ± s.d.) while K fm of 51Cr-EDTA (4.4 ± 0.7 μl/min) was nearly double K mf (2.4 ± 0.8 μl/min) for this tracer. Even greater asymmetry in the transfer of 51Cr-EDTA was found when measured across intact (non perfused) placenta. It is suggested that this asymmetry is caused by volume flow in the F-M direction. In other experiments transfer was measured in the F-M direction only. Ouabain (0.1 mM) on the maternal side and reduced concentration of Na + (25 mM) on the fetal side had no effect on the F-M transfer of the tracers. Reducing the temperature of the preparation by 5°C significantly decreased transfer of 22Na +. The transfer of 22Na was inversely related to the concentration of K + on the fetal side. These observations suggest that the F-M transfer of Na + has three components: diffusion through paracellular routes; convective flow by filtration through wide placental pores, and transcellular transport by a mechanism which is uncertain at present.

Rudimentary Horn Ectopic Pregnancy

Ultrasound is widely used for diagnosis of ectopic pregnancies, and is the accepted imaging modality to rule out ectopic pregnancies. Rudimentary horn pregnancy can be difficult to differentiate from an abdominal pregnancy. The visualization of an intact placenta with welldefined borders seems to be a key in the differential diagnosis. It is also helpful to obtain confirmation of a diagnosis such as this with another imaging modality. Magnetic resonance imaging (MRI) is the most helpful and widely recognized imaging modality for this purpose.

Evidence that human placenta is a site of sex hormone-binding globulin gene expression

The presence of an androgen-binding component in placenta was investigated in vitro using a tissue culture system of human placental explants. Explants of trophoblastic tissue from normal term placentas were kept in culture under appropriate conditions for at least 48 h in a serum-free medium. The existence of an androgen-binding protein was explored by binding assays, immunohistochemistry studies and Northern blot analyses of placental mRNA. Steady-state polyacrylamide gel electrophoresis and Scatchard plot analyses revealed the presence of a high affinity specific binding component for 5α-dihydrotestosterone in cultured placenta. Immunohistochemical studies performed on intact placenta and on Percoll-gradient purified trophoblastic cells demonstrated the presence of specific immunoreactivity in the cytoplasm of syncytial cells. Northern blot analyses of placental mRNA showed a single hybridizable 32P-labeled human sex hormone-binding globulin (SHBG) cDNA band of approx. 1.6 kb which was identical in size to that obtained with liver mRNA. The results strongly suggest the placenta as an origin of SHBG and point out this tissue as an additional site of SHBG synthesis during pregnancy.

Interleukin-1 is induced in the human placenta by endotoxin and isolation procedures for trophoblasts.

Although freshly obtained placenta contains little or no interleukin-1 (IL-1) mRNA, placenta and isolated trophoblast have been reported to produce significant quantities of bioactive IL-1 in vitro. The present study was designed to determine if endotoxin, a common contaminant of culture medium, and trophoblast isolation procedures could induce IL-1 expression in the placenta. Tissue-extractable IL-1 alpha and IL-1 beta immunoreactive proteins were readily detected in fresh placental membranes, but not placental villi. As little as 10 ng/mL endotoxin were found to induce the expression of IL-1 alpha and IL-1 beta mRNA in intact placental villi cultured in vitro. Intact placenta cultured in the presence of 1.0 microgram/mL endotoxin demonstrated expression of IL-1 alpha and IL-1 beta mRNA and cumulative production and release of immunoreactive IL-1 alpha and IL-1 beta into the medium during 24 h of culture. Placenta incubated in endotoxin-free medium, however, exhibited no detectable IL-1 alpha or IL-1 beta mRNA expression and little or no release of IL-1 alpha or IL-1 beta immunoreactive protein into the medium. When trophoblast cells were freshly isolated by enzymatic digestion, followed by Percoll separation at reduced temperatures to inhibit cell activation, no IL-1 alpha or IL-1 beta mRNA expression was initially detectable. However, IL-1 alpha and IL-1 beta mRNA in isolated trophoblast cells were induced after 30 min of culture in endotoxin-free medium, with maximal induction at 4 h. These results suggest that normally the placenta produces very little, if any, IL-1, and that endotoxin and trophoblast isolation procedures induce IL-1 expression in placental tissues cultured in vitro.

Regulation of chorionic gonadotropin-alpha and chorionic somatomammotropin messenger ribonucleic acid expression by 8-bromo-adenosine 3',5'-monophosphate and dexamethasone in cultured rhesus monkey syncytiotrophoblasts.

We wished to establish an in vitro culture system to examine gene expression in the context of differentiated function with rhesus monkey syncytiotrophoblasts. Chorionic villous tissue from placentas obtained at cesarean section was dispersed with trypsin and DNase and fractionated on a 5-70% Percoll gradient. When placed in culture, cells from a mononuclear fraction demonstrated to be very highly enriched (95-97% pure) for cytotrophoblasts aggregated and began to form syncytia within 24 h in culture, reminiscent of placental syncytiotrophoblast formation. The migration and fusion of individual cytotrophoblasts to form multinuclear syncytia were documented with time-lapse video microscopy. Incorporation studies with tritiated thymidine supported the conclusion from videomicroscopy that syncytia form by the fusion of individual cells and the addition of mononuclear cells to existing syncytia, rather than by endomitosis. The syncytiotrophoblast marker pregnancy-specific beta 1-glycoprotein (SP1) was immunocytochemically identified in both intact placenta and cultured syncytiotrophoblast cells. With cells isolated from placentas obtained on day 28, 50, 70, or 140 of pregnancy, treatment with 8-bromo-cAMP increased both rhesus monkey CG alpha-subunit (mCG alpha) and chorionic somatomammotropin (mCS) mRNA levels by an average of 4-fold. Increases of up to 2.5-fold were seen with mCG alpha mRNA in as little as 2 h after treatment, with a statistically significant average response seen within 6 h. The response with mCS required at least 24 h before a significant effect was seen. Actin mRNA levels were generally unchanged or suppressed by this treatment, indicating that the effect of 8-bromo-cAMP is relatively specific for the hormone mRNAs. Treatment of syncytiotrophoblasts with dexamethasone, but not progesterone or androstenedione, resulted in an approximately 4-fold increase in mCG alpha mRNA levels within 6 h of treatment. Steroid treatment did not affect mCS mRNA levels. Treatment with 4.5-400 nM GnRH or 0.1 to 100 ng/ml basic fibroblast growth factor likewise had no effect on the level of either mRNA, suggesting that any actions of these factors on hormone secretion are not effected via changes in steady state mRNA. These results communicate that the expression of the mRNAs for rhesus monkey CG alpha and CS in syncytiotrophoblast are regulated by steroid hormone- and cAMP-mediated pathways.

Effect of chronic maternal dietary magnesium deficiency on placental calcium transport.

Metabolisms of calcium (Ca) and magnesium (Mg) are closely interrelated in the intestine, bone, and kidney. Interaction of Ca and Mg at the placental level, however, is not well defined. The occurrence of decreased bone mineral content and hypocalcemia in infants of hypomagnesemic mothers led us to test the hypothesis that chronic dietary maternal Mg deficiency decreases placental Ca transport. On day 10 of gestation, 20 Sprague-Dawley rats were randomized to a Mg-deficient diet (3.3 mg/day, n = 10) or to a control diet (70 mg/day, n = 10). On day 20 of gestation (term = 22 days), intact placentas were perfused in situ through the umbilical artery and perfusate was collected through the umbilical vein. Calcium 45 (45Ca) and chromium 51-EDTA (51Cr-EDTA) (a diffusional marker for placental membrane integrity) were injected to the dam and steady state maternofetal clearance (Kmf45Ca, microliter/min/g placenta) of both isotopes were calculated. There was no difference in the clearance of 45Ca and 51Cr in both groups (55 +/- 10 vs 57 +/- 16 and 3.2 +/- 0.4 vs 3.6 +/- 0.4, respectively, mean +/- SEM). We conclude that, in the rat, placental Ca transfer is unaffected by chronic maternal dietary Mg deficiency. We speculate that Ca and Mg cross the placenta by independent mechanisms.

Transport of calcium across the dually perfused placenta of the rat.

1. A rat placenta was dually perfused in situ with modified Krebs fluid. Perfusion was carried out through the femoral artery on the maternal side and through the umbilical artery on the fetal side. 2. Transfer of 45Ca2+ and [3H]L-glucose across the placenta was measured in the maternal-fetal direction. The transcellular component of the maternal-fetal transport of Ca2+, Jmf,tc, was estimated from transfer rates of the two tracers and from Ca2+ concentration in maternal perfusate, [Ca2+]m. 3. At [Ca2+]m of 1.1 mM (physiological concentration of Ca2+ in plasma) Jmf,tc was 92.4 +/- 13.7 nmol min-1 (mean +/- S.D.), which is about 90% of the transport expected in an intact placenta. The permeability-surface area product (PS) of the placenta to [3H]L-glucose was 13.8 +/- 3.9 microliters min-1, about 4 times higher than that expected in intact placenta. 4. Transport of 45Ca2+ changed rapidly when [Ca2+]m was varied. Kinetic constants of the transcellular transport of Ca2+ are the Michaelis constant, Km, = 0.45 mM and the maximum rate of transport, Vmax, = 116 nmol min-1. It follows from this that at physiological levels of Ca2+, transport of Ca2+ to the fetus is relatively independent of changes in [Ca2+]m. 5. Strontium and barium (SrCl2 and BaCl2, 1 mM) decreased Jmf,tc; the response was prompt and reversible. Magnesium (2 mM) had no effect. Maternal-fetal transport of 85Sr2+ and 133Ba2+ was decreased rapidly and reversibly by elevating [Ca2+]m from 0.35 to 2 mM. These observations suggest that Sr2+ and Ba2+ are transported across the placenta by the Ca2+ transport system. This means that the transport is not substrate specific. 6. Cadmium (1 mM-CdCl2) decreased Jmf,tc irreversibly with some latency. The slowness of the response suggests a non-competitive inhibition. Cadmium (0.02 mM-CdCl2) was without effect on Jmf,tc. 7. A Ca2+ channel blocker, nifedipine (10 microM), administered to the maternal side had no effect on Jmf,tc.

Epidermal growth factor receptors and human placenta

s: European Placenta Group/Rochester Trophoblast Joint Conference EPIDERMAL GROWTH FACTOR RECEPTORS AND HUMAN PLACENTA C. V. Rao & N. Chegini (University of Louisville School of Medicine, KY, USA) 47I Term human placenta is one of the richest sources of receptors for epidermal growth factor (EGF). EGF is present in maternal and fetal circulation and in amniotic fluid. However, the source of EGF in these compartments is not known. The EGF receptors are present primarily in syncytiotrophoblasts. By light microscope autoradiography, we have found that these receptors were high at mid pregnancy and decreased towards term. This is the opposite of biochemical binding measurements. The latter observations can be explained by the presence of more connective tissue in placental cotyledons at mid term compared with term pregnancy. The receptors are enriched in microvillous plasma membranes, which are exposed to maternal circulation. The receptors are also found in basolateral plasma membranes, which are in close proximity to fetal blood capillaries. Since EGF may not cross the placenta, maternal EGF may influence syncytiotrophoblast function by binding to receptors in microviilous plasma membranes, whereas fetal EGF may also influence syncytiotrophoblast function via receptors in basolateral plasma membranes. The EGF in amniotic fluid presumably interacts with receptors in fetal membranes and decidua. In addition to the two outer cell surface membranes of syncytiotrophoblasts, EGF binding and EGF responsive kinase activities were also found in lysosomes, rough and smooth endoplasmic reticulum and Golgi elements. Quantitative electron microscope autoradiographic studies revealed that 12SI-EGF internalizes and associates with all of the above intracellular organelles in addition to nuclei. While the placental 125I-EGF binding differed among some pregnancy states, fetal membrane 12SI-EGF binding was similar in all the pregnancy states. This suggested that placental and fetal membrane EGF receptors are regulated differently. PERMEABILITY OF THE IN SITU PERFUSED RAT PLACENTA N. Robinson, R. Jenkins, P. Thomas & C. Sibley (St Mary's Hospital, Manchester, UK) A method for the in situ perfusion of the rat placenta has recently been developed. This preparation has allowed simple measurement of the permeability of the rat placenta to a range of hydrophilic molecules as follows: At 2I + i days of gestation, animals were anaesthetized with sodium thiobutabarbital and, after laparotomy, the uterus was exposed and the fetal circulation of one of the placentae was perfused, via an umbilical artery cannula with a modified Krebs' Ringer, at o.5 ml/min. A range of radiolabelled hydrophilic molecules, of increasing molecular size, were injected as a bolus into the mother and three samples of maternal plasma were taken at to--min intervals for measurement of radioactivity whilst fetal side perfusate was collected at 4min intervals from a cannula in the umbilical vein. The permeability surface area product (PS) was calculated as: PS (/d/rain/g)= (venous perfusate radiolabel concentration • flow rate) (mean maternal plasma radiolabel concentration x placental weight). The ratio of PS/D, where D is the diffusion coefficient in water of the particular molecule, was independent of molecular size for 14C-mannitol, 51CrEDTA and 14C/3H-inulin, whilst the PS/D for ~25Ialbumin suggested steric hindrance of this molecule. By contrast, the PS/D of 22Na+, 15.06 + i.oocm/g suggested additional non-diffusional transport. These results are close to those obtained in the intact rat placenta, and therefore, unlike the guinea pig, perfusion in the rat does not appear to be associated with increased placental porosity.

Isolation and characterization of trophoblast from murine placenta

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.

Transport of calcium by the placenta of the rat.

Transport of 45Ca and of radioactively labelled inert saccharides across the intact or perfused placenta was measured in the rat on day 21 of pregnancy, the day after mating being day 1. The values of permeability--surface area product (PS) of the intact placenta to radioactive mannitol, sucrose, raffinose, and methoxyinulin were approximately proportional to their diffusion coefficients in water. This suggests that diffusion of inert hydrophilic molecules across the rat placenta takes place through wide aqueous channels. Net flux of Ca from mother to fetus, estimated from the increase of the fetal Ca content between day 20 and day 21 is 100 +/- 4 nmol min-1 (the limits here and below are S.E. of means). The unidirectional maternal-fetal flux of Ca (Jmf) in non-anaesthetized animals, estimated from the flux of 45Ca, is 100 +/- 7 nmol min-1. The similarity of the two values suggests that the fetal-maternal flux (Jfm) is small The umbilical vascular bed of the rat placenta was perfused in situ with Krebs-dextran fluid. Jmf estimated from the transfer of 45Ca from maternal plasma to perfusate was 81 +/- 4 nmol min-1. PS of the perfused placenta to radioactive sucrose was 2.6 +/- 0.3 microliter min-1. Jmf decreased reversibly when the placenta was perfused with 0.5 mM-dinitrophenol or 1 mM-CN-, which is consistent with the presumed active nature of the maternal-fetal transport of Ca. Jmf did not decrease when the placenta was perfused with Na-free fluids (substitution with Tris, Li or sucrose), indicating that Na-Ca exchange across the fetal border of the placental trophoblast is not involved in maternal-fetal transport of Ca. Transport of 45Ca to the perfusate was reduced to about 60% when maternal plasma concentration of Ca was doubled. This suggests that the affinity of the maternal-fetal transport system to Ca is high. Jmf did not change when the umbilical concentration of Ca was varied between 0.1 and 3 mM. There thus seems to be no rapid feed-back between umbilical concentration of Ca and transport of Ca from mother to fetus. Fetal-maternal transfer of Ca, estimated from the steady-state extraction of 45Ca from the umbilical perfusate, is only about 20% of the maternal-fetal transfer. Umbilical extraction of 45Ca changed only little when umbilical concentration of Ca was varied between 0.1 and 3 mM. This suggests that either most of the fetal-maternal transport of Ca is diffusional or the fetal-maternal transport system has a very low affinity to Ca.(ABSTRACT TRUNCATED AT 400 WORDS)

Inner-ring deiodination of 3,5,3'-triiodothyronine in the in situ perfused guinea pig placenta.

Broken cell preparations of rat and human placentas contain an inner (tyrosyl)-ring iodothyronine deiodinase enzyme with greatest activity when the substrate is 3,5,3'-triiodothyronine (T3). This report describes the deiodination of T3 in the intact placenta and the effect of sodium iopanoate (IA) and propylthiouracil (PTU) on T3 deiodination. Under nembutal anesthesia, the placenta of 60-65-d-old pregnant guinea pigs was surgically exposed, a single umbilical artery and the umbilical vein were cannulated, and the fetus was removed. In a temperature-controlled chamber (37 degrees C), the fetal side of the placenta was perfused through the umbilical artery at a rate of 1 ml/min with 3% bovine serum albumin Krebs-Henseleit buffer containing 0.14 nM outer ring labeled [125I]T3. Placenta effluent fractions were collected at timed intervals from the umbilical vein cannula throughout a 120-min perfusion period. The contents of the perfusion buffer and the various effluent fractions were analyzed for their iodothyronine content by high pressure liquid chromatography. In five experiments, the percent composition of 125I-labeled iodothyronines in the perfusion buffer and placenta effluent was 95.3 +/- 1.0 (mean +/- SE) and 70.2 +/- 2.1 for T3 (P less than 0.01), 2.5 +/- 0.7 and 20.1 +/- 1.8 for 3,3'-T2 (P less than 0.01), and 0 and 8.2 +/- 0.9 for 3'-T1. There was no difference between the percent [125I]iodide in the perfusion buffer and in the placenta effluents. When placentas were perfused with IA and [125I]T3, after perfusion with [125I]T3 alone, there was a significant increase (P less than 0.01) in the percent [125I]T3 in the placenta effluents, and a significant decrease in [125I]3,3'-T2 (P less than 0.01) and [125I]3'-T1 (P less than 0.01). In contrast, PTU did not affect the composition of labeled iodothyronines in the placenta effluents, despite the fact that the addition of PTU significantly (P less than 0.001) inhibits the inner-ring deiodination of [125I]T3 in human or guinea pig placenta microsomes in the presence of low (0.25 mM) concentrations of dithiothreitol. The present studies demonstrate that T3 is actively deiodinated in the inner ring to 3,3'-T2 by the intact guinea pig placenta. A portion of 3,3'-T2 is further deiodinated in the inner ring to generate 3'-T1. No outer ring deiodination of T3 was seen under the conditions employed. IA, but not PTU, inhibits T3 deiodination in the placenta perfused in situ. We conclude that the placenta is probably a site for fetal T3 metabolism.

Fetal iron balance in the rat

Maternal and fetal iron balance through pregnancy was examined in the rat. The 20th day was selected for detailed study because of the peak iron requirements at that time. The standard diet provided a borderline iron supply to the fetus due to the limited availability of its iron for absorption. When a more available form of iron was used, normal fetal development occurred over a range of dietary iron content from 16 to 2500 mg/kg. At a level of 5 to 8 mg/kg, there was attrition of placental tissues with frequent fetal death and resorption. When the iron-deficient pregnancy was sustained, both maternal and fetal iron deficiency were present. At progressively higher levels of dietary iron, feto-placental iron content was constant despite a progressive increase in maternal iron stores. Fetal iron supply appeared to be determined primarily by plasma iron concentration, and, at normal levels, about 25% of transferrin iron passing through the uterine vasculature, was removed by the intact placentas. Low levels of plasma iron resulted in damage to fetal tissues and reduced the capacity of placental tissues to take up iron. At high levels of plasma iron, plasma iron turnover initially increased 5-fold over basal levels in nonpregnant animals due to increased placental uptake. However, with continued hyperferremia, uptake was regulated so as to maintain fetal iron at a normal level. A comparison of these data with human iron requirements explained the occurrence of both maternal and fetal iron deficiency in the rat, but only maternal iron deficiency in the human.

Physical abuse of abandoned children in India

Child abuse and neglect, though existing in India, has not come to light because of underlying social constraints like poverty, malnutrition and infection. With an intention to study the problem of physical abuse in neglected children, patients admitted to the pediatric wards of our institution, predominantly with some form of physical abuse were specifically looked for from January 1976 onwards. Over a period of 4 1 2 years, 18 such cases were encountered. Most of the babies were less than four weeks of age, were predominantly females, were quite often premature or malnourished, and rarely had a physical deformity. These babies were deserted and later were found by police or social workers in gutters, dustbins, garbage tins, railway platforms, etc. The spectrum of abuse and injuries included newborn babies with intact placenta, at times lacerated, strangling marks on the throat, multiple bruises, bleeding from the umbilicus, avulsion of the scalp with severe bleeding, shock as a result of environmental stress (i.e., hypothermia), evidence of infection, rat bite marks, and fractures. The morbidity was very high, ultimately resulting in 88.9% mortality over varying periods of time, maximum survival being 22 days. The motive behind this abuse always was an unwanted baby.