Classification of human placental villi

Summary The classification of human placental villi was reviewed on the basis of material prepared by means of special methods. The material from in situ normal-term placentae was biopsied by aspiration into glutaraldehyde. The classification was made on the basis of light-microscopic observations of semithin sections, reconstructions from serial sections, and scanning-electron micrographs. The peripheral villous tree is roughly divided into stem (ramuli), intermediate and terminal villi. The intermediate villi may be further subdivided as mature and immature types, which are found between the stem and terminal villi. Some of the terminal villi possess a local specialization described as the neck region. The histological characteristics and the branching pattern of each type are described, and the basis of the proposed classification is discussed.The authors wish to acknowledge the technical help of Mrs. Elke Böhm     

Classification of human placental villi

Villi of placental tissue obtained from normal term placentae at Caesarean section were embedded in Epon. Semithin sections were subjected to morphometry. The villi were first grouped according to the modified classification proposed in the foregoing paper. The parameters examined include villous numbers, size, vascularity, volume of trophoblast and connective tissue. The measured values differ markedly from those obtained from paraffin sections. Statistically significant differences exist between the different types of villi in various parameters, providing further evidence supporting the validity of the structural classification suggested.     

Androgen receptor in human placental villi

In studies with a synthetic androgen, R 1881, an androgen-binding component was found in the cytosol of human placental villi. Kinetic analysis indicated that the Kd value of this component was 1.4 nM at 0-4 degrees C and that binding of R 1881 amounted to 277 +/- 73 fmol/mg protein. glycerol density gradient ultracentrifugation showed a peak of binding activity in the 8S region in a medium of low ionic strength, but in the 4.5S region in a medium containing 9.5 M KCl. The R 1881-binding component was inactivated by mild heat- or trypsin-treatment, but not by treatment with DNase or RNase. Most of the R 1881-binding activity was sedimented at 20 to 40% saturation of ammonium sulfate. These findings indicate that the R 1881-binding component in human placental cytosol is quite similar in its characteristics to androgen receptors, which are present in various androgen-responsive organs. Testosterone was a more potent competitor of R 1881-binding than DHT or cyproterone acetate. Scatchard plots indicated that the binding site of testosterone was identical with that of R 1881. These findings suggest that the androgen receptor in placental cytosol is specific for testosterone. The Kd value for testosterone was calculated to be 3.2 nM.     

Secretion of lysosomal enzymes by human placental villi in vitro

Summary Chorionic villi from first trimester and term human placentas have been incubated in vitro and shown to release the lysosomal enzymes, -hexosaminidase, -glucosidase and -gluctlronidase. There was negligible release of the cytoplasmic enzyme, lactate dehydrogenase, under the same conditions. The first trimester villi released proportionally more of their lysosomal enzyme content than did the term villi. Extracellular levels of -hexosaminidase were raised and those of -glucosidase and, -glucuronidase were lowered when tissue was incubated with 1 M colchicine, suggesting that microtubules are involved in the control of lysosomal enzyme release from placental villi.     

Contractile properties of human placental anchoring villi

The presence of myofibroblasts arranged parallel to the longitudinal axes of anchoring villi of the placenta has previously been described. Furthermore, it has been suggested that intraplacental blood volume, and hence fetal-maternal oxygen-nutrient exchange, may in part be regulated through the longitudinal contraction of anchoring villi. We demonstrate here that anchoring villi have the ability to contract and relax longitudinally. Anchoring villi from normal term human placentae were dissected and suspended from force-displacement transducers to determine their longitudinal contractility in response to potassium chloride (KCl), N(omega)-nitro-l-arginine methyl ester (l-NAME) and the nitric oxide donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN). Treatment with both KCl and l-NAME resulted in up to a 62% and 74% increase, respectively, in longitudinal contraction over resting tone. In contrast, both SNP and GTN caused a dose-dependent relaxation of precontracted villi. Immunohistochemistry of longitudinal sections of villi confirmed the presence of alpha-actin-containing cells in the extravascular space. Histological staining with hemotoxylin and eosin confirm that the tissue used in these experiments were anchoring villi. These findings suggest that the contraction of anchoring villi may be an important mechanism whereby the placenta may regulate intraplacental volume.     

Fetal vasculogenesis and angiogenesis in human placental villi

Placental villi of 5 exactly defined early human specimens ranging from day 21 post conception (p.c.) until day 42 p.c. and from an additional 43 specimens from about 5 to 40 weeks menstrual age have been analyzed ultrastructurally with regard to fetal vasculogenesis and angiogenesis. The following results were obtained: The first cells differentiating at day 21 p.c., probably originating from mesenchymal precursors, are macrophage-like cells. At almost the same time, mesenchymal cells transform into haemangioblastic cell cords which are the forerunners of the capillary endothelium and haematopoietic stem cells. A third cell population related to the fetal circulatory system and derived from the mesenchymal cells are presumptive pericytes. Capillary formation takes place by the aggregation of haemangioblastic cells which are attached to each other by intercellular junctions. The lumen is formed by the dehiscence of the intercellular clefts. A capillary basal lamina cannot be detected earlier than in the last trimester. In this last period of gestation, fetal villous angiogenesis takes place by the proliferation of the existing endothelium and pericytes rather than via haemangioblastic cells.     

The organization of actin filaments in human placental villi

The surface of the syncytial trophoblast of the human placenta is covered by a microvillous (brush) border that is in direct contact with maternal blood. Because of this location, it is the site of a variety of transport, enzymatic and receptor activities vital to many placental functions. The organization of the brush border as well as other features of placental villus organization may well be influenced by the distribution of cytoplasmic actin filaments. In order to determine the distribution of actin filaments in human placenta, small pieces of villi were briefly fixed in glutaraldehyde, permeabilized with saponin, and incubated in solutions containing subfragment 1 of myosin (S1). After S1 decoration of actin filaments, tissue was fixed in glutaraldehyde containing tannic acid in order to better visualize the polarity of the filaments, and prepared for electron microscopic examination. The microvilli each contained a core of actin filaments running from the tip of the microvillus to the apical cytoplasm. Most of the actin filaments displayed a distinct polarity, with the S1 arrowheads pointing away from the microvillar tips. These filaments extended only a short distance into the apical cytoplasm. There appeared to be another group of actin filaments in a matlike arrangement in the apical cytoplasm. Coated pits and vesicles were often observed between the microvilli. There appeared to be no clear association between the coated pits and decorated actin filaments, but this was difficult to establish with certainty because of the close proximity of the microvilli. Bundles of actin filaments were sometimes observed near the basal cell surface of the syncytial trophoblast, and in pericytes and capillary endothelial cells in the cores of the villi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid receptors of human placental villi modulate acetylcholine release

The human placental villous tissue contains components of the cholinergic system and opioid receptors of the kappa type. In vitro stimulation of the villous tissue releases acetylcholine in organ baths. A selective kappa agonist, ethylketocyclazocine, inhibits the release of acetylcholine. This inhibition is reversed by the antagonist Mr 2266. The antagonist alone stimulates the release of acetylcholine 18-fold over control. These results demonstrate an interaction between the placental opioid receptors and the cholinergic system in a non-neural tissue. The modulation of acetylcholine release by endogenous opioid peptides could be one of the in vivo functions of placental opioid receptors.     

Kappa opioid receptors of human placental villi modulate acetylcholine release

Human placental villus tissue is non-innervated, yet it contains components of the opiate and cholinergic systems. We investigated whether opioids modulate a calcium dependent acetylcholine release from the villus tissue in a manner similar to that demonstrated by the parasympathetic nerve-smooth muscle junction. We reported that the kappa receptor agonist ethylketocyclazocine (EKC) inhibits acetylcholine release, and that the inhibition is reversed by the selective antagonist, Mr2266. Findings reported here substantiate the role of opioids as modulators of acetylcholine release from villus tissue. The nonselective agonist, morphine, also inhibits acetylcholine release. Inhibition caused by morphine is reversed by low concentrations of non-selective antagonists, naloxone and naltrexone. Naloxone at high concentrations potentiates the inhibition of acetylcholine release caused by morphine. In addition, the calcium channel blocker, diltiazem, was found to inhibit the release of acetylcholine. The combination of morphine and diltiazem resulted in a greater inhibition of acetylcholine release than by either alone. These results suggest that opiate cholinergic interactions occur in non-neural tissue with a mechanism similar to that known to occur at certain cholinergic synapses.     

An integrative view on the physiology of human early placental villi

The placenta is an indispensable organ for intrauterine protection, development and growth of the embryo and fetus. It provides tight contact between mother and conceptus, enabling the exchange of gas, nutrients and waste products. The human placenta is discoidal in shape, and bears a hemo-monochorial interface as well as villous materno-fetal interdigitations. Since Peter Medawar's astonishment to the paradoxical nature of the mother–fetus relationship in 1953, substantial knowledge in the domain of placental physiology has been gathered. In the present essay, an attempt has been made to build an integrated understanding of morphological dynamics, cell biology, and functional aspects of genomic and proteomic expression of human early placental villous trophoblast cells followed by a commentary on the future directions of research in this field.     

Distribution of collagen types III and IV in human placental villi

Immunofluorescent examination showed more significant accumulation of interstitial collagen type III in the stroma of mature placenta compared with immature one. Localization of membrane collagen type IV was found neither in basal membranes of epithelium and villous vessels of mature term placenta, nor in their stroma. The described patterns of distribution of collagen types III and IV in human placenta villi were proved by immunoelectronmicroscopic method.     

Immunohistochemical localization of laminin and fibronectin isoforms in human placental villi.

We studied the localization of laminin alpha1, alpha2, alpha3, alpha5, beta1, beta2, and gamma1 chains and extradomain A- (EDA), EDB-, and oncofetal fibronectin by immunohistochemistry in human placental villi during placental development. The laminin alpha2, alpha5, beta1, beta2, and gamma1 chains were detected in the trophoblastic basement membrane (BM) at all stages of gestation, suggesting the presence of laminin-2, -4, -10, and -11 trimers. The laminin alpha1 chain was selectively found at sites where the villous BM was in contact with proliferating cells in trophoblastic islands or columns. EDA-Fn, but not other Fn isoforms, was found in the trophoblastic BM during the first trimester. The laminin alpha2, beta1, beta2, and gamma1 chains were detected in the villous stroma and capillaries throughout placental development, while the laminin alpha5 chain emerged distinctly during development. Extensive EDA-Fn immunoreactivity was found in first-trimester villous stroma, but distinctly fewer Fn isoforms were seen in the villous stroma during the later stages of gestation. Our results also suggest that, during the formation of new villi, laminins are not found in trophoblastic sprouts before the ingrowth of the villous mesenchyme. Rather, laminins may be deposited at the villous epithelial-mesenchymal interface. Furthermore, the results show that distinct changes occur in the localization of various laminin and Fn isoforms during the maturation of villous trophoblastic and capillary BMs.     

Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy

Summary Morphological changes in fixed stromal cells and Hofbauer cells were studied throughout pregnancy in different types of placental chorionic villi by scanning electron microscopy. In the mesenchymal villus the fixed stromal cells were characterized by thin cytoplasmic processes. Hofbauer cells exhibited blebs on their surface. Large sail-like processes with a crescent profile which surrounded well developed stromal channels and a small cell body typified the small reticulum cells of the immature intermediate villus. The Hofbauer cells here displayed blebs, microplicae and large lamellipodia. Short cytoplasmic expansions and a large cell body characterized the fibroblasts present inside the stem villus. Hofbauer cells were rare, having blebs or a few short lamellipodia. The mature intermediate villus contained small and large reticulum cells. The latter had a much larger cell body than the small ones and displayed a few short cytoplasmic processes partly delimiting narrow incomplete stromal channels. Occasional Hofbauer cells with small microplicae and/or blebs were present. The small reticulum cells and fibroblasts present in the terminal villus showed similar morphological features as above. However, the former exhibited less developed cytoplasmic extensions and therefore no stromal channels were observed. In the terminal villus, the morphology of the rare Hofbauer cells was similar to that found in the mature intermediate villus.     

Immunostaining of vascular, perivascular cells and stromal components in human placental villi.

Fetal placental vessels develop and adapt in order to supply the fetus with nutrients. Immunostaining by antibodies against blood clotting factors, cell-cell and cell-matrix adhesion molecules, intermediate and contractile filaments, matrix components and enzymes give an overall view useful in assessing cell differentiation in placental villi. Endothelial cells stained positively for thrombomodulin, von Willebrand factor, CD34, CD31, cadherin-5, phalloidin and alpha 3-integrin. Trophoblastic cells were positive for cytokeratin, alpha 5 and alpha V integrins, L-prolyl hydroxylase and phalloidin. Myocytes from the media of stem villi exhibited positive vimentin, desmin, alpha-sm-actin and sm-myosin reactions but were CD26 negative. Myofibroblasts were vimentin, desmin, CD26, alpha-sm-actin and sm-myosin positive. Perivascular cells of intermediate and terminal villi were alpha-sm-actin, sm-myosin and anti-high molecular weight melanoma associated antigen (HMWMAA) positive. Trophoblastic and endothelial basement membranes were collagen IV positive. The most specific endothelial markers were cadherin-5, observed only at paracellular clefts, and von Willebrand factor. For perivascular cells, alpha-sm-actin, sm-myosin and HMWMAA provided a specific labeling. Differences in labeling intensity were noted along the cross section of the villous tree (vimentin, desmin, actin, myosin inward gradient). A continuity in the contractile function along the vessel length was indicated by alpha-sm-actin and sm-myosin positive cells, contrasting with the decreased von Willebrand reaction intensity. These data are discussed in relation to cell function and compared to cell culture results.     

Glucose metabolism by Lactobacillus divergens

Earlier studies on the fermentation of D-[1-14C]- and D-[3,4-14C]glucose by Lactobacillus divergens showed that lactate was the major fermentation product and that it was probably produced by glycolysis. It was therefore recommend that L. divergens be reclassified as a homofermentative organism. In the present investigation, products of D-[1-14C]-,D-[2-14C]- and D-[3,4-14C]glucose fermented by L. divergens were isolated, and their specific radioactivities and the distribution patterns of radioactivity in their C-atoms were determined. The positional labelling patterns of the fermentation products, their specific radioactivities and their concentrations confirmed that glucose is degraded via the glycolytic pathway. Some secondary decarboxylation/dissimilation of pyruvate to acetate, formate and CO2 was also observed. These results provide conclusive proof that L. divergens is indeed a homofermentative organism. Results obtained with D-[U-14C]glucose showed that approximately three-quarters of the lactate but less than 10% each of the formate and acetate were produced from glucose. The remainder was presumably derived to a varying degree from endogenous non-glucose sources such as fructose and/or amino acids.     

Trans-placental transport and metabolism of carbacyclin by perfused human placental in vitro

When carbacyclin (5E-6a-carba-prostaglandin I2) was added to the maternal afferent circulation of in vitro perfused placentae from normal term pregnancies, relatively little carbacyclin was found in either the maternal or fetal efferent circulations. When carbacyclin was added to the perfusate at 1.0 microM, the peak level in the maternal effluent was only 0.06 microM and in the fetal effluent, 0.026 microM. When infused at 10 microM, 0.77 microM carbacyclin was measured in the maternal effluent and 0.13 in the fetal effluent. These findings demonstrate that carbacyclin is transferred across the placenta from the maternal side to the fetal, but that the net transfer is small. The assay procedure employed HPLC resolution, followed by capillary gas chromatography and selected ion monitoring using PGB as an internal standard. The low levels of carbacyclin detected in the effluents did not result from poor recovery in the analyses. When carbacyclin was added to maternal or fetal effluents at 1 microM, the recovery averaged 85.4 +/- 14.1% (SD); at 10 microM recovery averaged 97.3 +/- 4.2%. Much of the loss of carbacyclin on passage through placental circulation resulted from metabolism. Extracts of both fetal and maternal effluents from placenta perfused with carbacyclin contained a component which on reverse phase HPLC appeared less polar than carbacyclin. When analyzed by GC/MS as the methyl ester-trimethylsilyl ether, this component had a mass spectrum expected for 15-dehydro-carbacyclin. When the presumed metabolite was further converted to the methoxime, the mass spectrum was identical to published spectra for that derivative of 15-dehydro-carbacyclin. When extracts of fetal effluents were analyzed for 15-dehydro-carbacyclin metabolite as well as carbacyclin, it appeared that the metabolite accounted for the majority of the carbacyclin recovered. Most of the metabolite was apparently not formed in the fetal circulation, since when carbacyclin was added to the fetal afferent circulation, little 15-dehydro-carbacyclin was observed in either efferent fluid, and most of the perfused carbacyclin was recovered unaltered in the fetal effluent.